Improved Cognitive Performance and Reduced Monocyte Activation in Virally Suppressed Chronic HIV Following Dual CCR2 and CCR5 Antagonism.

PubMed

D'Antoni, Michelle L; Paul, Robert H; Mitchell, Brooks I; Kohorn, Lindsay; Fischer, Laurent; Lefebvre, Eric; Seyedkazemi, Star; Nakamoto, Beau K; Walker, Maegen; Kallianpur, Kalpana J; Ogata-Arakaki, Debra; Ndhlovu, Lishomwa C; Shikuma, Cecilia

2018-05-16

To evaluate changes in neuropsychological (NP) performance and in plasma and cell surface markers of peripheral monocyte activation/migration following treatment with cenicriviroc (CVC), a dual C-C chemokine receptor type 2 (CCR2) and type 5 (CCR5) antagonist, in treatment-experienced, HIV-infected individuals. Single-arm, 24-week, open-label clinical trial. HIV-infected individuals on antiretroviral therapy (ART) >1 year with plasma HIV RNA <50 copies/ml and below-normal cognitive performance [defined as age, gender and education-adjusted NP performance (NPZ) <-0.5 in a single cognitive domain or in global performance] were enrolled. Changes over 24 weeks were assessed for global and domain-specific NPZ scores, plasma markers of monocyte/macrophage activation [neopterin, soluble (s)CD14 and sCD163] quantified by ELISA, and CCR2 and CCR5 expression on monocytes and T cells measured by flow cytometry. Seventeen of 20 enrolled participants completed the study. Improvements over 24 weeks were observed in global NPZ [median change (Δ)=0.24; p=0.008], and in cognitive domains of attention (Δ0.23; p=0.011) and working memory (Δ0.44; p=0.017). Plasma levels of sCD163, sCD14, and neopterin decreased significantly (p's<0.01). CCR2 and CCR5 monocyte expression remained unchanged; however, CCR5 levels on CD4 and CD8 T cells and CCR2 expression on CD4 T cells increased (p's<0.01). CVC given over 24 weeks was associated with improved NP test performance and decreased plasma markers of monocyte immune activation in virally-suppressed, HIV-infected participants. These data potentially link changes in monocyte activation to cognitive performance. Further study of CVC for HIV cognitive impairment in a randomized controlled study is warranted.

Resistance to human immunodeficiency virus type 1 (HIV-1) generated by lentivirus vector-mediated delivery of the CCR5Δ32 gene despite detectable expression of the HIV-1 co-receptors

PubMed Central

Jin, Qingwen; Marsh, Jon; Cornetta, Kenneth; Alkhatib, Ghalib

2009-01-01

It has previously been demonstrated that there are two distinct mechanisms for genetic resistance to human immunodeficiency virus type 1 (HIV-1) conferred by the CCR5Δ32 gene: the loss of wild-type CCR5 surface expression and the generation of CCR5Δ32 protein, which interacts with CXCR4. To analyse the protective effects of long-term expression of the CCR5Δ32 protein, recombinant lentiviral vectors were used to deliver the CCR5Δ32 gene into human cell lines and primary peripheral blood mononuclear cells that had been immortalized by human T-cell leukemia virus type 1. Blasticidin S-resistant cell lines expressing the lentivirus-encoded CCR5Δ32 showed a significant reduction in HIV-1 Env-mediated fusion assays. It was shown that CD4+ T lymphocytes expressing the lentivirus-encoded CCR5Δ32 gene were highly resistant to infection by a primary but not by a laboratory-adapted X4 strain, suggesting different infectivity requirements. In contrast to previous studies that analysed the CCR5Δ32 protective effects in a transient expression system, this study showed that long-term expression of CCR5Δ32 conferred resistance to HIV-1 despite cell-surface expression of the HIV co-receptors. The results suggest an additional unknown mechanism for generating the CCR5Δ32 resistance phenotype and support the hypothesis that the CCR5Δ32 protein acts as an HIV-suppressive factor by altering the stoichiometry of the molecules involved in HIV-1 entry. The lentiviral-CCR5Δ32 vectors offer a method of generating HIV-resistant cells by delivery of the CCR5Δ32 gene that may be useful for stem cell- or T-cell-based gene therapy for HIV-1 infection. PMID:18796731

Altered expression of glial markers, chemokines, and opioid receptors in the spinal cord of type 2 diabetic monkeys.

PubMed

Kiguchi, Norikazu; Ding, Huiping; Peters, Christopher M; Kock, Nancy D; Kishioka, Shiroh; Cline, J Mark; Wagner, Janice D; Ko, Mei-Chuan

2017-01-01

Neuroinflammation is a pathological condition that underlies diabetes and affects sensory processing. Given the high prevalence of pain in diabetic patients and crosstalk between chemokines and opioids, it is pivotal to know whether neuroinflammation-associated mediators are dysregulated in the central nervous system of diabetic primates. Therefore, the aim of this study was to investigate whether mRNA expression levels of glial markers, chemokines, and opioid receptors are altered in the spinal cord and thalamus of naturally occurring type 2 diabetic monkeys (n=7) compared with age-matched non-diabetic monkeys (n=6). By using RT-qPCR, we found that mRNA expression levels of both GFAP and IBA1 were up-regulated in the spinal dorsal horn (SDH) of diabetic monkeys compared with non-diabetic monkeys. Among all chemokines, expression levels of three chemokine ligand-receptor systems, i.e., CCL2-CCR2, CCL3-CCR1/5, and CCL4-CCR5, were up-regulated in the SDH of diabetic monkeys. Moreover, in the SDH, seven additional chemokine receptors, i.e., CCR4, CCR6, CCR8, CCR10, CXCR3, CXCR5, and CXCR6, were also up-regulated in diabetic monkeys. In contrast, expression levels of MOP, KOP, and DOP, but not NOP receptors, were down-regulated in the SDH of diabetic monkeys, and the thalamus had fewer changes in the glial markers, chemokines and opioids. These findings indicate that neuroinflammation, manifested as glial activation and simultaneous up-regulation of multiple chemokine ligands and receptors, seems to be permanent in type 2 diabetic monkeys. As chemokines and opioids are important pain modulators, this first-in-primate study provides a translational bridge for determining the functional efficacy of spinal drugs targeting their signaling cascades. Copyright © 2016 Elsevier B.V. All rights reserved.

Increased levels of CCR7(lo)PD-1(hi) CXCR5+ CD4+ T cells, and associated factors Bcl-6, CXCR5, IL-21 and IL-6 contribute to repeated implantation failure.

PubMed

Gong, Qiaoqiao; Zhu, Yuejie; Pang, Nannan; Ai, Haiquan; Gong, Xiaoyun; La, Xiaolin; Ding, Jianbing

2017-12-01

In vitro fertilization-embryo transfer (IVF-ET) can be used by infertile couples to assist with reproduction; however, failure of the embryo to implant into the endometrial lining results in failure of the IVF treatment. The present study investigated the expression of chemokine receptor 7 (CCR7)(lo) programmed death-1(PD-1)(hi) chemokine receptor type 5 (CXCR5) + cluster of differentiation 4 (CD4) + T cells and associated factors in patients with repeated implantation failure (RIF). A total of 30 females with RIF and 30 healthy females were enrolled in the current study. Flow cytometry was used to detect the proportion of CCR7(lo)PD-1(hi) CXCR5 + CD4 + T cells in the peripheral blood. Cytokine bead arrays were performed to detect the levels of interleukin (IL)-6, -4 and -2 in the serum. ELISAs were used to detect the level of IL-21 in the serum. Quantitative real time polymerase chain reaction analysis and immunohistochemistry were used to investigate the expression of B-cell lymphoma 6 (Bcl-6), chemokine receptor type 5 (CXCR5) and IL-21 in the endometrium. The results revealed that the percentage of CCR7(lo)PD-1(hi) CXCR5 + CD4 + T cells was increased in the RIF group compared with the control group during the mid luteal phase. The mRNA and protein levels of Bcl-6, IL-21 and CXCR5 in the endometrium and the concentrations of IL-21 and IL-6 in the serum were significantly increased in the RIF group; however, no significant difference was observed between the two groups in regards to the expression of IL-4 and IL-2. Furthermore, a significant positive correlation was identified between the percentage of CCR7(lo)PD-1(hi) CXCR5 + CD4 + T cells and IL-21 and IL-6 levels. The expression of IL-21 also had a positive correlation with Bcl-6 and CXCR5 expression in the RIF group. These results suggest that increased levels of CCR7(lo)PD-1(hi) CXCR5 + CD4 + T cells and associated factors contribute to RIF and could therefore be a potential therapeutic target.

A Maraviroc-Resistant HIV-1 with Narrow Cross-Resistance to Other CCR5 Antagonists Depends on both N-Terminal and Extracellular Loop Domains of Drug-Bound CCR5▿

PubMed Central

Tilton, John C.; Wilen, Craig B.; Didigu, Chukwuka A.; Sinha, Rohini; Harrison, Jessamina E.; Agrawal-Gamse, Caroline; Henning, Elizabeth A.; Bushman, Frederick D.; Martin, Jeffrey N.; Deeks, Steven G.; Doms, Robert W.

2010-01-01

CCR5 antagonists inhibit HIV entry by binding to a coreceptor and inducing changes in the extracellular loops (ECLs) of CCR5. In this study, we analyzed viruses from 11 treatment-experienced patients who experienced virologic failure on treatment regimens containing the CCR5 antagonist maraviroc (MVC). Viruses from one patient developed high-level resistance to MVC during the course of treatment. Although resistance to one CCR5 antagonist is often associated with broad cross-resistance to other agents, these viruses remained sensitive to most other CCR5 antagonists, including vicriviroc and aplaviroc. MVC resistance was dependent upon mutations within the V3 loop of the viral envelope (Env) protein and was modulated by additional mutations in the V4 loop. Deep sequencing of pretreatment plasma viral RNA indicated that resistance appears to have occurred by evolution of drug-bound CCR5 use, despite the presence of viral sequences predictive of CXCR4 use. Envs obtained from this patient before and during MVC treatment were able to infect cells expressing very low CCR5 levels, indicating highly efficient use of a coreceptor. In contrast to previous reports in which CCR5 antagonist-resistant viruses interact predominantly with the N terminus of CCR5, these MVC-resistant Envs were also dependent upon the drug-modified ECLs of CCR5 for entry. Our results suggest a model of CCR5 cross-resistance whereby viruses that predominantly utilize the N terminus are broadly cross-resistant to multiple CCR5 antagonists, whereas viruses that require both the N terminus and antagonist-specific ECL changes demonstrate a narrow cross-resistance profile. PMID:20702642

Antibody-Mediated Rejection of Single Class I MHC-Disparate Cardiac Allografts

PubMed Central

Hattori, Yusuke; Bucy, R. Pat; Kubota, Yoshinobu; Baldwin, William M.; Fairchild, Robert L.

2012-01-01

Murine CCR5−/− recipients produce high titers of antibody to complete MHC-mismatched heart and renal allografts. To study mechanisms of class I MHC antibody-mediated allograft injury, we tested the rejection of heart allografts transgenically expressing a single class I MHC disparity in wild-type C57BL/6 (H-2b) and B6.CCR5−/− recipients. Donor-specific antibody titers in CCR5−/− recipients were 30-fold higher than in wild-type recipients. B6.Kd allografts survived longer than 60 days in wild-type recipients whereas CCR5−/− recipients rejected all allografts within 14 days. Rejection was accompanied by infiltration of CD8 T cells, neutrophils, and macrophages and C4d deposition in the graft capillaries. B6.Kd allografts were rejected by CD8−/−/CCR5−/−, but not μMT−/−/CCR5−/−, recipients indicating the need for antibody but not CD8 T cells. Grafts retrieved at day 10 from CCR5−/− and CD8−/−/CCR5−/− recipients and from RAG-1−/− allograft recipients injected with anti-Kd antibodies expressed high levels of perforin, myeloperoxidase and CCL5 mRNA. These studies indicate that the continual production of anti-donor class I MHC antibody can mediate allograft rejection, that donor-reactive CD8 T cells synergize with the antibody to contribute to rejection, and that expression of three biomarkers during rejection can occur in the absence of this CD8 T cell activity. PMID:22578247

Constitutive and Stress-induced Expression of CCL5 Machinery in Rodent Retina

PubMed Central

Duncan, D'Anne S.; McLaughlin, William M.; Vasilakes, Noah; Echevarria, Franklin D.; Formichella, Cathryn R.; Sappington, Rebecca M.

2017-01-01

Signaling by inflammatory cytokines and chemokines is associated with neurodegeneration in disease and injury. Here we examined expression of the β-chemokine CCL5 and its receptors in the mouse retina and evaluated its relevance in glaucoma, a common optic neuropathy associated with sensitivity to intraocular pressure (IOP). Using quantitative PCR, fluorescent in situ hybridization, immunohistochemistry and quantitative image analysis, we found CCL5 mRNA and protein was constitutively expressed in the inner retina and synaptic layers. CCL5 appeared to associate with Müller cells and RGCs as well as synaptic connections between horizontal cells and bipolar cells in the OPL and amacrine cells, bipolar cells and RGCs in the IPL. Although all three high-affinity receptors (CCR5, CCR3, CCR1) for CCL5 were expressed constitutively, CCR5 expression was significantly higher than CCR3, which was also markedly greater than CCR1. Localization patterns for constitutive CCR5, CCR3 and CCR1 expression differed, particularly with respect to expression in inner retinal neurons. Stress-related expression of CCL5 was primarily altered in aged DBA/2 mice with elevated IOP. In contrast, changes in expression and localization of both CCR3 and CCR5 were evident not only in aged DBA/2 mice, but also in age-matched control mice and young DBA/2 mice. These groups do not exhibit elevated IOP, but possess either the aging stress (control mice) or the genetic predisposition to glaucoma (DBA/2 mice). Together, these data indicate that CCL5 and its high-affinity receptors are constitutively expressed in murine retina and differentially induced by stressors associated with glaucomatous optic neuropathy. Localization patterns further indicate that CCL5 signaling may be relevant for modulation of synapses in both health and disease, particularly in the inner plexiform layer. PMID:28936366

Simvastatin Inhibits IL-5-Induced Chemotaxis and CCR3 Expression of HL-60-Derived and Human Primary Eosinophils.

PubMed

Fu, Chia-Hsiang; Tsai, Wan-Chun; Lee, Ta-Jen; Huang, Chi-Che; Chang, Po-Hung; Su Pang, Jong-Hwei

2016-01-01

IL-5-induced chemotaxis of eosinophils is an important feature of allergic airway inflammatory diseases. Simvastatin, a lipid lowering agent, has been shown to exhibit anti-inflammatory and anti-allergic effects. Our aim was to investigate the effect of simvastatin on IL-5-induced eosinophil chemotaxis and its regulatory mechanisms. Eosinophils were derived by treating HL-60 clone 15 (HC15) cells with butyric acid (BA) in an alkaline condition or through direct isolation from human peripheral blood. The expressions of CC chemokine receptor 3 (CCR3) and interleukin (IL)-5 receptors (IL5Rα and β) were analyzed using RT/real-time PCR. The granular proteins were stained using fast green. Eotaxin-induced chemotaxis was measured using a transwell migration assay. CCR3 protein expression was revealed by immunocytochemistry. An animal model of allergic rhinitis was established by challenging Sprague-Dawley® rats repeatedly with ovalbumin. Butyric acid significantly increased the expression of IL5Rα and IL5Rβ, CCR3 and granular proteins in HC15 cells, indicating the maturation of eosinophils (BA-E cells). IL-5 further enhanced the CCR3 expression at both the mRNA and protein levels and the eotaxin-induced chemotaxis of BA-E cells. Simvastatin inhibited the effects of IL-5 on BA-E cells, but not in the presence of mevalonate. Similar results were also exhibited in human primary eosinophils. In vivo animal studies further confirmed that oral simvastatin could significantly suppress the infiltration of eosinophils into turbinate tissues of allergic rats. Therefore, simvastatin was demonstrated to inhibit IL-5-induced CCR3 expression and chemotaxis of eosinophils mediated via the mevalonate pathway. We confirmed that simvastatin also reduced eosinophilic infiltration in allergic rhinitis.

α4+β7hiCD4+ Memory T cells Harbor Most Th-17 cells and are Preferentially Infected During Acute SIV Infection

PubMed Central

Kader, Muhamuda; Wang, Xiaolei; Piatak, Michael; Lifson, Jeffrey; Roederer, Mario; Veazey, Ronald; Mattapallil, Joseph J.

2009-01-01

HIV/SIV are thought to infect minimally activated CD4+ T cells after viral entry. Not much is known about why SIV selectively targets these cells. Here we show that CD4+ T cells that express high levels of the α4β7 heterodimer are preferentially infected very early during the course of SIV infection. At day 2–4 post infection, α4+β7hiCD4+ T cells had ∼ 5x more SIV-gag DNA than β7−CD4+ T cells. α4+β7hiCD4+ T cells displayed a predominantly central memory (CD45RA−CD28+CCR7+) and resting (CD25−CD69−HLA-DR−Ki-67−) phenotype. Though the expression of detectable CCR5 was variable on α4+β7hi and β7−CD4+ T cells, both CCR5+ and CCR5− subsets of α4+β7hi and β7−CD4+ T cells were found to express sufficient levels of CCR5 mRNA suggesting that both these subsets could be efficiently infected by SIV. In line with this, we found similar levels of SIV infection in β7−CD4+CCR5+ and β7−CD4+CCR5− T cells. α4β7hiCD4+ T cells were found to harbor most Th-17 cells that were significantly depleted during acute SIV infection. Taken together, our results show that resting memory α4+β7hiCD4+ T cells in blood are preferentially depleted during acute SIV infection, and the loss of these cells alters the balance between Th-17 and Th-1 responses thereby contributing to disease pathogenesis. PMID:19571800

T lymphocyte recruitment into renal cell carcinoma tissue: a role for chemokine receptors CXCR3, CXCR6, CCR5, and CCR6.

PubMed

Oldham, Kimberley A; Parsonage, Greg; Bhatt, Rupesh I; Wallace, D Michael A; Deshmukh, Nayneeta; Chaudhri, Shalini; Adams, David H; Lee, Steven P

2012-02-01

Evidence suggests that some patients with renal cell carcinoma (RCC) respond to immunomodulatory therapies that activate T lymphocytes. A prerequisite for effective T cell therapy is efficient targeting of effector T cells to the tumour site, yet the molecular basis of T cell recruitment to RCC is unknown. Furthermore, some T cells that naturally infiltrate this cancer are regulatory T cells (Tregs) that may suppress antitumour immune responses. Determine the mechanisms of effector and regulatory T cell recruitment to RCC to allow targeted therapy that promotes local anti-tumour immunity. Tumour-infiltrating and peripheral blood T cells were collected from 70 patients undergoing nephrectomy for RCC. T cells were analysed by multicolour flow cytometry for expression of 19 chemokine receptors and 7 adhesion molecules. Receptors that were expressed at higher levels on tumour-infiltrating lymphocytes (TILs) compared with matched peripheral blood lymphocytes (PBLs) were analysed further for their ability to mediate migration responses in TILs and for expression of corresponding ligands in tumour tissue. Three chemokine receptors-CCR5, CXCR3, and CXCR6-were significantly overexpressed on TILs compared with matched PBLs (n=16 cases) and were capable of promoting migration in vitro. Their corresponding ligands CCL4-5, CXCL9-11, and CXCL16 were all detected in RCC tissue. However, since they were present in all cases studied, it was not possible to correlate ligand expression with levels of T cell infiltration. Foxp3(+) Tregs were enriched within TILs compared with matched PBLs and expressed high levels of CCR5, CXCR3, and CXCR6, as well as CCR6, the ligand for which (CCL20) was detectable in RCC tissue. Our data support a role for CCR5, CXCR3, and CXCR6 in the selective recruitment of T cells into RCC tissue and, together with CCR6, in the recruitment of Tregs. Copyright © 2011 European Association of Urology. Published by Elsevier B.V. All rights reserved.

High Expression of CCR7 Predicts Lymph Node Metastasis and Good Prognosis in Triple Negative Breast Cancer.

PubMed

Li, Xuelu; Sun, Siwen; Li, Ning; Gao, Jiyue; Yu, Jing; Zhao, Jinbo; Li, Man; Zhao, Zuowei

2017-01-01

Previous preclinical and clinical studies have reported a positive correlation between the expression of the C-C chemokine receptor 7 (CCR7) and the incidence of lymph node metastasis in breast cancer. However, the prognostic relevance of CCR7 expression in breast cancer remains contradictory till now. The aim of this study is to assess the correlation of the CCR7 expression with other clinicopathological features and prognosis in breast cancer. The CCR7 gene amplification and mRNA expression levels from approximately 3,000 patients were retrieved from human breast cancer databases and analyzed. Furthermore, a total of 188 primary triple negative breast cancer patients were enrolled in this study (diagnosed since January 2009 to January 2013 from the Second Hospital of Dalian Medical University). The protein levels of CCR7 were examined by immunohistochemistry using paraffin-embedded tumor tissues. The analysis of gene amplification and mRNA levels showed the expression of CCR7 in breast cancer correlated with better prognosis. When we compared the CCR7 expressions in different subtypes, the basal-like group showed the highest expression of CCR7 and exhibited a better prognosis. Consistently, Kaplan-Meier analysis of 188 triple negative breast cancer patients showed that the prognosis of patients with positive CCR7 expression was significantly better than those with negative expression (HR=0.642, p=0.0275). Additionally, we also observed a positive correlation between lymph node metastasis and the CCR7 expression (p=0.0096). Our results indicated that elevated CCR7 expression as a marker for increased lymph node metastasis, in addition to serve as an independent prognostic indicator for better overall survival in triple negative breast cancer patients. © 2017 The Author(s). Published by S. Karger AG, Basel.

Immune response genes receptors expression and polymorphisms in relation to multiple sclerosis susceptibility and response to INF-β therapy.

PubMed

Karam, Rehab A; Rezk, Noha A; Amer, Mona M; Fathy, Hala A

2016-09-01

Interferon (IFN)-β is one of the disease modifying drugs used in the treatment of multiple sclerosis. A predictive marker that indicates good or poor response to the treatment is highly desirable. We aimed to investigate the relation between the immune response genes receptors (IFNAR1, IFNAR2, and CCR5) expression and their polymorhic variants and multiple sclerosis (MS) susceptibility as well as the response to IFN-β therapy. The immune response genes receptors expression and genotyping were analyzed in 80 patients with MS, treated with IFN-β and in 110 healthy controls. There was a significant decrease of IFNAR1 and IFNAR2 mRNA expression and a significant increase of CCR5 mRNA expression in MS patients compared with the control group. Also, the level of IFNAR1, IFNAR2, and CCR5 mRNA expression was found to be significantly lower in the responders than nonresponders. Carriers of IFNAR1 18417 C/C genotype and C allele had an increased risk of developing MS. There was a significant relation between CCR5 Δ32 allele and IFN-β treatment response in MS patients. Our results highlighted the significance of IFNAR and CCR5 genes in multiple sclerosis risk and the response to IFN-β therapy. © 2016 IUBMB Life, 68(9):727-734, 2016. © 2016 International Union of Biochemistry and Molecular Biology.

Genetic diversity and prevalence of CCR2-CCR5 gene polymorphisms in the Omani population

PubMed Central

Al-Mahruqi, Samira H.; Zadjali, Fahad; Beja-Pereira, Albano; Koh, Crystal Y.; Balkhair, Abdullah; Al-Jabri, Ali A.

2014-01-01

Polymorphisms in the regulatory region of the CCR5 gene affect protein expression and modulate the progress of HIV-1 disease. Because of this prominent role, variations in this gene have been under differential pressure and their frequencies vary among human populations. The CCR2V64I mutation is tightly linked to certain polymorphisms in the CCR5 gene. The current Omani population is genetically diverse, a reflection of their history as traders who ruled extensive regions around the Indian Ocean. In this study, we examined the CCR2-CCR5 haplotypes in Omanis and compared the patterns of genetic diversity with those of other populations. Blood samples were collected from 115 Omani adults and genomic DNA was screened to identify the polymorphic sites in the CCR5 gene and the CCR2V64I mutation. Four minor alleles were common: CCR5-2554T and CCR5-2086G showed frequencies of 49% and 46%, respectively, whereas CCR5-2459A and CCR5-2135C both had a frequency of 36%. These alleles showed moderate levels of heterozygosity, indicating that they were under balancing selection. However, the well-known allele CCR5Δ32 was relatively rare. Eleven haplotypes were identified, four of which were common: HHC (46%), HHE (20%), HHA (14%) and HHF*2 (12%). PMID:24688285

CXCR3/CCR5 pathways in metastatic melanoma patients treated with adoptive therapy and interleukin-2

PubMed Central

Bedognetti, D; Spivey, T L; Zhao, Y; Uccellini, L; Tomei, S; Dudley, M E; Ascierto, M L; De Giorgi, V; Liu, Q; Delogu, L G; Sommariva, M; Sertoli, M R; Simon, R; Wang, E; Rosenberg, S A; Marincola, F M

2013-01-01

Background: Adoptive therapy with tumour-infiltrating lymphocytes (TILs) induces durable complete responses (CR) in ∼20% of patients with metastatic melanoma. The recruitment of T cells through CXCR3/CCR5 chemokine ligands is critical for immune-mediated rejection. We postulated that polymorphisms and/or expression of CXCR3/CCR5 in TILs and the expression of their ligands in tumour influence the migration of TILs to tumours and tumour regression. Methods: Tumour-infiltrating lymphocytes from 142 metastatic melanoma patients enrolled in adoptive therapy trials were genotyped for CXCR3 rs2280964 and CCR5-Δ32 deletion, which encodes a protein not expressed on the cell surface. Expression of CXCR3/CCR5 in TILs and CXCR3/CCR5 and ligand genes in 113 available parental tumours was also assessed. Tumour-infiltrating lymphocyte data were validated by flow cytometry (N=50). Results: The full gene expression/polymorphism model, which includes CXCR3 and CCR5 expression data, CCR5-Δ32 polymorphism data and their interaction, was significantly associated with both CR and overall response (OR; P=0.0009, and P=0.007, respectively). More in detail, the predicted underexpression of both CXCR3 and CCR5 according to gene expression and polymorphism data (protein prediction model, PPM) was associated with response to therapy (odds ratio=6.16 and 2.32, for CR and OR, respectively). Flow cytometric analysis confirmed the PPM. Coordinate upregulation of CXCL9, CXCL10, CXCL11, and CCL5 in pretreatment tumour biopsies was associated with OR. Conclusion: Coordinate overexpression of CXCL9, CXCL10, CXCL11, and CCL5 in pretreatment tumours was associated with responsiveness to treatment. Conversely, CCR5-Δ32 polymorphism and CXCR3/CCR5 underexpression influence downregulation of the corresponding receptors in TILs and were associated with likelihood and degree of response. PMID:24129241

The CCL5/CCR5 Chemotactic Pathway Promotes Perineural Invasion in Salivary Adenoid Cystic Carcinoma.

PubMed

Gao, Tao; Shen, Zhiyuan; Ma, Chao; Li, Yun; Kang, Xiangfeng; Sun, Moyi

2018-02-20

Perineural invasion (PNI) is a hallmark of salivary adenoid cystic carcinoma (SACC) and represents an important risk factor for local recurrence and poor survival. However, the mechanism of PNI has yet to be explored. We sought to examine the CCL5-CCR5 ligand-receptor interaction between nerves and SACC cells. CCL5/CCR5 expression was determined by immunohistochemistry in SACC tissue specimens. The correlations between CCL5/CCR5 expression and clinicopathologic features were investigated. Dorsal root ganglia (DRG) and SACC cells cocultured in vitro were used to evaluate the effects of CCL5/CCR5 on PNI progression and pathogenesis. CCR5 expression was significantly elevated in SACC tissues and associated with distant metastasis, PNI, and TNM grade (P < .05). DRG and SACC cells cocultured in vitro showed that the activation of the CCL5/CCR5 axis significantly increased SACC cell invasion and promoted the outgrowth of the DRG. SACC cell lines expressing CCR5 migrated in response to CCL5 derived from DRG, eventually leading to PNI. More importantly, further study showed that blocking of CCL5 or CCR5 effectively inhibited the invasive capacity and PNI activity of SACC cells (P < .05). Our results suggest a pivotal role of CCL5/CCR5 axis in tumor-nerve interactions during PNI of SACC. The CCL5/CCR5 pathway might prove to be an attractive new target for the treatment of SACC with PNI. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

The Isolation of Novel Phage Display-Derived Human Recombinant Antibodies Against CCR5, the Major Co-Receptor of HIV

PubMed Central

Shimoni, Moria; Herschhorn, Alon; Britan-Rosich, Yelena; Kotler, Moshe; Benhar, Itai

2013-01-01

Abstract Selecting for antibodies against specific cell-surface proteins is a difficult task due to many unrelated proteins that are expressed on the cell surface. Here, we describe a method to screen antibody-presenting phage libraries against native cell-surface proteins. We applied this method to isolate antibodies that selectively recognize CCR5, which is the major co-receptor for HIV entry (consequently, playing a pivotal role in HIV transmission and pathogenesis). We employed a phage screening strategy by using cells that co-express GFP and CCR5, along with an excess of control cells that do not express these proteins (and are otherwise identical to the CCR5-expressing cells). These control cells are intended to remove most of the phages that bind the cells nonspecifically; thus leading to an enrichment of the phages presenting anti-CCR5-specific antibodies. Subsequently, the CCR5-presenting cells were quantitatively sorted by flow cytometry, and the bound phages were eluted, amplified, and used for further successive selection rounds. Several different clones of human single-chain Fv antibodies that interact with CCR5-expressing cells were identified. The most specific monoclonal antibody was converted to a full-length IgG and bound the second extracellular loop of CCR5. The experimental approach presented herein for screening for CCR5-specific antibodies can be applicable to screen antibody-presenting phage libraries against any cell-surface expressed protein of interest. PMID:23941674

DOE Office of Scientific and Technical Information (OSTI.GOV)

Murooka, Thomas T.; Rahbar, Ramtin; Department of Immunology, University of Toronto, Ont.

The proliferative capacity of cancer cells is regulated by factors intrinsic to cancer cells and by secreted factors in the microenvironment. Here, we investigated the proto-oncogenic potential of the chemokine receptor, CCR5, in MCF-7 breast cancer cell lines. At physiological levels, CCL5, a ligand for CCR5, enhanced MCF-7.CCR5 proliferation. Treatment with the mTOR inhibitor, rapamycin, inhibited this CCL5-inducible proliferation. Because mTOR directly modulates mRNA translation, we investigated whether CCL5 activation of CCR5 leads to increased translation. CCL5 induced the formation of the eIF4F translation initiation complex through an mTOR-dependent process. Indeed, CCL5 initiated mRNA translation, shown by an increase inmore » high-molecular-weight polysomes. Specifically, we show that CCL5 mediated a rapid up-regulation of protein expression for cyclin D1, c-Myc and Dad-1, without affecting their mRNA levels. Taken together, we describe a mechanism by which CCL5 influences translation of rapamycin-sensitive mRNAs, thereby providing CCR5-positive breast cancer cells with a proliferative advantage.« less

Chemokine Receptor Expression on Normal Blood CD56+ NK-Cells Elucidates Cell Partners That Comigrate during the Innate and Adaptive Immune Responses and Identifies a Transitional NK-Cell Population

PubMed Central

Queirós, Maria Luís; Gonçalves, Marta; Fonseca, Sónia; Moura, João

2015-01-01

Studies of chemokine receptors (CKR) in natural killer- (NK-) cells have already been published, but only a few gave detailed information on its differential expression on blood NK-cell subsets. We report on the expression of the inflammatory and homeostatic CKR on normal blood CD56+low CD16+ and CD56+high  CD16−/+low NK-cells. Conventional CD56+low and CD56+high NK-cells present in the normal PB do express CKR for inflammatory cytokines, although with different patterns CD56+low NK-cells are mainly CXCR1/CXCR2+ and CXCR3/CCR5−/+, whereas mostly CD56+high NK-cells are CXCR1/CXCR2− and CXCR3/CCR5+. Both NK-cell subsets have variable CXCR4 expression and are CCR4− and CCR6−. The CKR repertoire of the CD56+low NK-cells approaches to that of neutrophils, whereas the CKR repertoire of the CD56+high NK-cells mimics that of Th1+ T cells, suggesting that these cells are prepared to migrate into inflamed tissues at different phases of the immune response. In addition, we describe a subpopulation of NK-cells with intermediate levels of CD56 expression, which we named CD56+int NK-cells. These NK-cells are CXCR3/CCR5+, they have intermediate levels of expression of CD16, CD62L, CD94, and CD122, and they are CD57− and CD158a−. In view of their phenotypic features, we hypothesize that they correspond to a transitional stage, between the well-known CD56+high and CD56+low NK-cells populations. PMID:26543875

A Novel CCR5 Mutation Common in Sooty Mangabeys Reveals SIVsmm Infection of CCR5-Null Natural Hosts and Efficient Alternative Coreceptor Use In Vivo

PubMed Central

Riddick, Nadeene E.; Hermann, Emilia A.; Loftin, Lamorris M.; Elliott, Sarah T.; Wey, Winston C.; Cervasi, Barbara; Taaffe, Jessica; Engram, Jessica C.; Li, Bing; Else, James G.; Li, Yingying; Hahn, Beatrice H.; Derdeyn, Cynthia A.; Sodora, Donald L.; Apetrei, Cristian; Paiardini, Mirko; Silvestri, Guido; Collman, Ronald G.

2010-01-01

In contrast to HIV infection in humans and SIV in macaques, SIV infection of natural hosts including sooty mangabeys (SM) is non-pathogenic despite robust virus replication. We identified a novel SM CCR5 allele containing a two base pair deletion (Δ2) encoding a truncated molecule that is not expressed on the cell surface and does not support SIV entry in vitro. The allele was present at a 26% frequency in a large SM colony, along with 3% for a CCR5Δ24 deletion allele that also abrogates surface expression. Overall, 8% of animals were homozygous for defective CCR5 alleles and 41% were heterozygous. The mutant allele was also present in wild SM in West Africa. CD8+ and CD4+ T cells displayed a gradient of CCR5 expression across genotype groups, which was highly significant for CD8+ cells. Remarkably, the prevalence of natural SIVsmm infection was not significantly different in animals lacking functional CCR5 compared to heterozygous and homozygous wild-type animals. Furthermore, animals lacking functional CCR5 had robust plasma viral loads, which were only modestly lower than wild-type animals. SIVsmm primary isolates infected both homozygous mutant and wild-type PBMC in a CCR5-independent manner in vitro, and Envs from both CCR5-null and wild-type infected animals used CXCR6, GPR15 and GPR1 in addition to CCR5 in transfected cells. These data clearly indicate that SIVsmm relies on CCR5-independent entry pathways in SM that are homozygous for defective CCR5 alleles and, while the extent of alternative coreceptor use in SM with CCR5 wild type alleles is uncertain, strongly suggest that SIVsmm tropism and host cell targeting in vivo is defined by the distribution and use of alternative entry pathways in addition to CCR5. SIVsmm entry through alternative pathways in vivo raises the possibility of novel CCR5-negative target cells that may be more expendable than CCR5+ cells and enable the virus to replicate efficiently without causing disease in the face of extremely restricted CCR5 expression seen in SM and several other natural host species. PMID:20865163

A novel CCR5 mutation common in sooty mangabeys reveals SIVsmm infection of CCR5-null natural hosts and efficient alternative coreceptor use in vivo.

PubMed

Riddick, Nadeene E; Hermann, Emilia A; Loftin, Lamorris M; Elliott, Sarah T; Wey, Winston C; Cervasi, Barbara; Taaffe, Jessica; Engram, Jessica C; Li, Bing; Else, James G; Li, Yingying; Hahn, Beatrice H; Derdeyn, Cynthia A; Sodora, Donald L; Apetrei, Cristian; Paiardini, Mirko; Silvestri, Guido; Collman, Ronald G

2010-08-26

In contrast to HIV infection in humans and SIV in macaques, SIV infection of natural hosts including sooty mangabeys (SM) is non-pathogenic despite robust virus replication. We identified a novel SM CCR5 allele containing a two base pair deletion (Δ2) encoding a truncated molecule that is not expressed on the cell surface and does not support SIV entry in vitro. The allele was present at a 26% frequency in a large SM colony, along with 3% for a CCR5Δ24 deletion allele that also abrogates surface expression. Overall, 8% of animals were homozygous for defective CCR5 alleles and 41% were heterozygous. The mutant allele was also present in wild SM in West Africa. CD8+ and CD4+ T cells displayed a gradient of CCR5 expression across genotype groups, which was highly significant for CD8+ cells. Remarkably, the prevalence of natural SIVsmm infection was not significantly different in animals lacking functional CCR5 compared to heterozygous and homozygous wild-type animals. Furthermore, animals lacking functional CCR5 had robust plasma viral loads, which were only modestly lower than wild-type animals. SIVsmm primary isolates infected both homozygous mutant and wild-type PBMC in a CCR5-independent manner in vitro, and Envs from both CCR5-null and wild-type infected animals used CXCR6, GPR15 and GPR1 in addition to CCR5 in transfected cells. These data clearly indicate that SIVsmm relies on CCR5-independent entry pathways in SM that are homozygous for defective CCR5 alleles and, while the extent of alternative coreceptor use in SM with CCR5 wild type alleles is uncertain, strongly suggest that SIVsmm tropism and host cell targeting in vivo is defined by the distribution and use of alternative entry pathways in addition to CCR5. SIVsmm entry through alternative pathways in vivo raises the possibility of novel CCR5-negative target cells that may be more expendable than CCR5+ cells and enable the virus to replicate efficiently without causing disease in the face of extremely restricted CCR5 expression seen in SM and several other natural host species.

CCR5 Controls Immune and Metabolic Functions during Toxoplasma gondii Infection

PubMed Central

Bonfá, Giuliano; Benevides, Luciana; Souza, Maria do Carmo; Fonseca, Denise Morais; Mineo, Tiago Wilson Patriarca; Rossi, Marcos Antônio; Silva, Neide Maria; Silva, João Santana; de Barros Cardoso, Cristina Ribeiro

2014-01-01

CCR5, an important receptor related to cell recruitment and inflammation, is expressed during experimental Toxoplasma gondii infection. However, its role in the immunopathology of toxoplasmosis is not clearly defined yet. Thus, we inoculated WT and CCR5-/- mice with a sub lethal dose of the parasite by oral route. CCR5-/- mice were extremely susceptible to infection, presenting higher parasite load and lower tissue expression of IL-12p40, IFN-γ, TNF, IL-6, iNOS, Foxp3, T-bet, GATA-3 and PPARα. Although both groups presented inflammation in the liver with prominent neutrophil infiltration, CCR5-/- mice had extensive tissue damage with hepatocyte vacuolization, steatosis, elevated serum triglycerides and transaminases. PPARα agonist Gemfibrozil improved the vacuolization but did not rescue CCR5-/- infected mice from high serum triglycerides levels and enhanced mortality. We also found intense inflammation in the ileum of CCR5-/- infected mice, with epithelial ulceration, augmented CD4 and decreased frequency of NK cells in the gut lamina propria. Most interestingly, these findings were accompanied by an outstanding accumulation of neutrophils in the ileum, which seemed to be involved in the gut immunopathology, once the depletion of these cells was accompanied by reduced local damage. Altogether, these data demonstrated that CCR5 is essential to the control of T. gondii infection and to maintain the metabolic, hepatic and intestinal integrity. These findings add novel information on the disease pathogenesis and may be relevant for directing future approaches to the treatment of multi-deregulated diseases. PMID:25119429

[Differential regulation of CCR5 expression on T lymphocytes in healthy donors after mobilization with rhG-CSF and its correlation with aGVHD].

PubMed

Wang, Meng; Ma, Xiang-Juan; Dong, Yu-Jun; Qiu, Zhi-Xiang; Liu, Wei; Li, Yuan; Wang, Mang-Ju; Sun, Yu-Hua; Ren, Han-Yun

2013-08-01

This study was to investigate the differential regulation of CCR5 expression on T cells in healthy donors after mobilization with recombinant human granulocyte colony-stimulating factor (rhG-CSF) and analyze its correlation with acute graft-versus-host disease (aGVHD) so as to understand the possible mechanisms underlying rhG-CSF-induced immune tolerance. Sixty-eight related healthy donor and their corresponding recipient for allogeneic hematopoietic stem cell transplantation (allo-HSCT) were enrolled in this study. The expression of CCR5 on CD4(+) and CD8(+) T cells in the peripheral blood (PB) before and after mobilization were detected by using flow cytometry (FCM) respectively. According to the changes of CCR5 expression on CD4(+) and CD8(+) T cells, the Sixty-two evaluable donors were divided into the downregulated and unchanged/upregulated (non-downregulated) groups, and the incidence of grades II to IV aGVHD in two groups were compared. The results showed that the mean value of CCR5 expression on CD4(+) and CD8(+) T cells in PB was not different significantly after mobilization (P > 0.05). Apparent inconsistency was showed among different individuals. Thirty-four (50%) donors displayed downregulation of CCR5 expression, while 34 (50%) donors manifested unchanged or upregulated CCR5 expression on CD4(+) T cells. CCR5 expression on CD8(+) T cells was downregulated in 42 (61.8%), unchanged or upregulated in 26 (38.3%) donors. The cumulative incidence of grades II to IV aGVHD in the downregulated and non-downregulated groups for CD4(+) T cells were 16.1% and 41.9% (P = 0.032), and recipients with CCR5 downregulation on CD8(+) T cells showed an increased tendency of developing aGVHD (37.8% vs 16.0%, P = 0.065). In conclusion, rhG-CSF mobilization could lead to differential regulation of CCR5 expression on T cells, which might influence the migration of T cells in vivo, decrease T cell trafficking towards GVHD target organs, and thus reduce the incidence of aGVHD after transplantation.

Role of CXCL13 and CCL20 in the recruitment of B cells to inflammatory foci in chronic arthritis.

PubMed

Armas-González, Estefanía; Domínguez-Luis, María Jesús; Díaz-Martín, Ana; Arce-Franco, Mayte; Castro-Hernández, Javier; Danelon, Gabriela; Hernández-Hernández, Vanesa; Bustabad-Reyes, Sagrario; Cantabrana, Alberto; Uguccioni, Mariagrazia; Díaz-González, Federico

2018-06-07

B cells exert their pathogenic action in rheumatoid arthritis (RA) locally in the synovium. This study was undertaken to elucidate the chemokines responsible for the recruitment of B cells in the inflamed synovium, taking into account that the rich chemokine milieu present in the synovial tissue can fine-tune modulate discrete chemokine receptors. Expression levels of chemokine receptors from the CC and CXC family, as well as CD27, were assessed by flow cytometry in CD20 + mononuclear cells isolated from the peripheral blood (PB) and synovial fluid (SF) of RA and psoriatic arthritis patients. Transwell experiments were used to study migration of B cells in response to a chemokine or in the presence of multiple chemokines. B cells from the SF of arthritis patients showed a significant increase in the surface expression of CCR1, CCR2, CCR4, CCR5 and CXCR4 with respect to PB. Conversely, SF B cells expressed consistently lower amounts of CXCR5, CXCR7 and CCR6, independent of CD27 expression. Analysis of permeabilized B cells suggested internalization of CXCR5 and CCR6 in SF B cells. In Transwell experiments, CCL20 and CXCL13, ligands of CCR6 and CXCR5, respectively, caused a significantly higher migration of B cells from PB than of those from SF of RA patients. Together, these two chemokines synergistically increased B-cell migration from PB, but not from SF. These results suggest that CXCL13 and CCL20 might play major roles in RA pathogenesis by acting singly on their selective receptors and synergistically in the accumulation of B cells within the inflamed synovium.

Increased expression of RANTES, CCR3 and CCR5 in the lesional skin of patients with atopic eczema.

PubMed

Kato, Yoko; Pawankar, Ruby; Kimura, Yoichi; Kawana, Seiji

2006-01-01

Atopic eczema (AE) is a relapsing inflammatory disease based on IgE sensitization and characterized by peripheral blood eosinophilia and eosinophil infiltration into the lesional skin. In the patch test reaction of AE by allergens, an increased infiltration of activated eosinophils has been demonstrated peaking at 24-48 h. Regulated on activation normal T cell expressed and secreted (RANTES/CCL5) is a chemokine that induces eosinophil migration, and CCR3 and CCR5 are the receptors of RANTES. In order to further clarify the pathomechanisms of eosinophil infiltration in ongoing chronic inflammation in the skin of patients with AE and its relation to disease severity, we examined the expression of RANTES and its receptors CCR3 and CCR5 in challenged and unchallenged lesional skin of AE. We examined the number of RANTES+ cells, CCR3+ cells, CCR5+cells, activated (EG2+) eosinophils and CD3+ T cells in normal skin of healthy volunteers, and in challenged lesional skin (24 h after mite patch test) as well as unchallenged lesional skin of AE patients by immunohistochemistry. The cellular source of RANTES, CCR3 and CCR5 was analyzed by double immunohistochemistry using specific antibodies to RANTES, CCR3 or CCR5, and antibodies to ECP (EG2) or CD3. The numbers of RANTES+ cells, CCR3+ cells, CCR5+ cells, EG2+ cells and CD3+ cells were all significantly increased in challenged (mite patch-tested) lesional skin of AE patients as compared to those in unchallenged lesional skin and normal skin. The numbers of these cells in unchallenged lesional skin were greater than those in normal skin. The number of EG2+ cells in the unchallenged lesional skin correlated with both the peripheral blood eosinophil count and the SCORAD index. The number of EG2+ cells in challenged lesional skin correlated with the number of CCR5+ cells. Activated eosinophils and T cells expressed RANTES and various proportions of these cells were CCR3+ and CCR5+ in both challenged and unchallenged lesional skin. Taken together, these results suggest that RANTES as well as its receptors CCR3 and CCR5 may play important roles in the orchestration of eosinophil infiltration in ongoing chronic inflammation in AE, and also reflect the severity of disease. Copyright (c) 2006 S. Karger AG, Basel.

Critical roles of chemokine receptor CCR5 in regulating glioblastoma proliferation and invasion.

PubMed

Zhao, Lanfu; Wang, Yuan; Xue, Yafei; Lv, Wenhai; Zhang, Yufu; He, Shiming

2015-11-01

Glioblastoma (GBM) is the most prevalent malignant primary brain tumor in adults and exhibits a spectrum of aberrantly aggressive phenotype. Tumor cell proliferation and invasion are critically regulated by chemokines and their receptors. Recent studies have shown that the chemokine CCL5 and its receptor CCR5 play important roles in tumor invasion and metastasis. Nonetheless, the roles of the CCR5 in GBM still remain unclear. The present study provides the evidence that the chemokine receptor CCR5 is highly expressed and associated with poor prognosis in human GBM. Mechanistically, CCL5-CCR5 mediates activation of Akt, and subsequently induces proliferation and invasive responses in U87 and U251 cells. Moreover, down-regulation of CCR5 significantly inhibited the growth of glioma in U87 tumor xenograft mouse model. Finally, high CCR5 expression in GBM is correlated with increased p-Akt expression in patient samples. Together, these findings suggest that the CCR5 is a critical molecular event associated with gliomagenesis. © The Author 2015. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

Distribution of simian immunodeficiency virus target cells in vaginal tissues of normal rhesus macaques: implications for virus transmission.

PubMed

Poonia, Bhawna; Wang, Xiaolei; Veazey, Ronald S

2006-12-01

Most new cases of HIV-1 infection occur as the result of vaginal transmission. Identifying the phenotype and distribution of potential viral target cells in the vagina is important for understanding events in viral transmission and for developing effective prevention strategies. For example, compounds that prevent CD4 or CCR5 binding have been demonstrated recently to prevent vaginal transmission in rhesus macaques, but the expression and distribution of CCR5 has not been examined in the macaque vagina. The objective of this study was to examine the distribution and phenotype of cells and molecules in the vagina of rhesus macaques that may be involved in HIV transmission, including CCR5, CD3, CD4, CD8, CD1a, CD28, CD95, CD123 and HLA-DR. Normal juvenile and adult female rhesus macaques were examined by multicolor immunohistochemistry and flow cytometry. Although both CD4 and CCR5 were observed in the lamina propria, essentially no CD4 or CCR5 expression was detected within the squamous or keratinized layers of the vaginal epithelium. CCR5 expression was higher in the vaginal lamina propria of mature macaques compared to 1-3-year-old juveniles. The vast majority of CD4(+)CCR5(+) lymphocytes in the vagina had a central memory (CD95(+)CD28(+)) phenotype. Numerous CCR5-expressing dendritic cells (CD123(+)) or macrophages (CD68(+)) were observed in the lamina propria, but no CCR5, CD4 or DC-SIGN expression was detectable in the epithelium. Thus, the multiple layers of squamous epithelium normally covering the vaginal mucosa may provide an effective barrier against vaginal HIV-1 transmission. Microbicides that block CD4 or CCR5 expression may act within the deeper layers of the vaginal epithelium rather than on the epithelial surface.

Paucity of CD4+CCR5+ T cells is a typical feature of natural SIV hosts

PubMed Central

Pandrea, Ivona; Apetrei, Cristian; Gordon, Shari; Barbercheck, Joseph; Dufour, Jason; Bohm, Rudolf; Sumpter, Beth; Roques, Pierre; Marx, Preston A.; Hirsch, Vanessa M.; Kaur, Amitinder; Lackner, Andrew A.; Veazey, Ronald S.; Silvestri, Guido

2007-01-01

In contrast to lentiviral infections of humans and macaques, simian immunodeficiency virus (SIV) infection of natural hosts is nonpathogenic despite high levels of viral replication. However, the mechanisms underlying this absence of disease are unknown. Here we report that natural hosts for SIV infection express remarkably low levels of CCR5 on CD4+ T cells isolated from blood, lymph nodes, and mucosal tissues. Given that this immunologic feature is found in 5 different species of natural SIV hosts (sooty mangabeys, African green monkeys, mandrills, sun-tailed monkeys, and chimpanzees) but is absent in 5 nonnatural/recent hosts (humans, rhesus, pigtail, cynomolgus macaques, and baboons), it may represent a key feature of the coevolution between the virus and its natural hosts that led to a nonpathogenic infection. Beneficial effects of low CCR5 expression on CD4+ T cells may include the reduction of target cells for viral replication and a decreased homing of activated CD4+ T cells to inflamed tissue. PMID:17003371

CCR5 Knockout Prevents Neuronal Injury and Behavioral Impairment Induced in a Transgenic Mouse Model by a CXCR4-using HIV-1 Glycoprotein 1201

PubMed Central

Maung, Ricky; Hoefer, Melanie M.; Sanchez, Ana B.; Sejbuk, Natalia E.; Medders, Kathryn E.; Desai, Maya K.; Catalan, Irene C.; Dowling, Cari C.; de Rozieres, Cyrus M.; Garden, Gwenn A.; Russo, Rossella; Roberts, Amanda J.; Williams, Roy; Kaul, Marcus

2014-01-01

The innate immune system has been implicated in several neurodegenerative diseases, including human immunodeficiency virus (HIV)-1 associated dementia. Here we show that genetic ablation of CCR5 prevents microglial activation and neuronal damage in a transgenic model of HIV-associated brain injury induced by a CXCR4-utilizing viral envelope gp120. The CCR5 knockout (KO) also rescues spatial learning and memory in gp120-transgenic (tg) mice. However, the CCR5KO does not abrogate astrocytosis, indicating it can occur independently from neuronal injury and behavioral impairment. To further characterize the neuroprotective effect of CCR5-deficiency we performed a genome –wide gene expression analysis of brains from HIVgp120tg mice expressing or lacking CCR5 and non-transgenic controls. Comparison with a human brain microarray study reveals that brains of HIVgp120tg mice and HIV patients with neurocognitive impairment share numerous differentially regulated genes. Furthermore, brains of CCR5 wild-type (WT) and CCR5KO gp120tg mice express markers of an innate immune response. One of the most significantly up-regulated factors is the acute phase protein lipocalin-2 (LCN2). Using cerebrocortical cell cultures, we find that LCN2 is neurotoxic in a CCR5-dependent fashion while inhibition of CCR5 alone is not sufficient to abrogate neurotoxicity of a CXCR4-utilizing gp120. However, the combination of pharmacological CCR5 blockade and LCN2 protects neurons from toxicity of a CXCR4-utilizing gp120 thus recapitulating the finding in CCR5-deficient gp120tg mouse brain. Altogether, our study provides evidence for an indirect pathological role of CCR5 and a novel protective effect of LCN2 in combination with inhibition of CCR5 in HIV-associated brain injury. PMID:25031461

1,25-dihydroxyvitamin D3 induces CCR10 expression in terminally differentiating human B cells.

PubMed

Shirakawa, Aiko-Konno; Nagakubo, Daisuke; Hieshima, Kunio; Nakayama, Takashi; Jin, Zhe; Yoshie, Osamu

2008-03-01

In the B cell lineage, CCR10 is known to be selectively expressed by plasma cells, especially those secreting IgA. In this study, we examined the regulation of CCR10 expression in terminally differentiating human B cells. As reported previously, IL-21 efficiently induced the differentiation of activated human CD19+ B cells into IgD-CD38+ plasma cells in vitro. A minor proportion of the resulting CD19+IgD-CD38+ cells expressed CCR10 at low levels. 1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3), the active metabolite of vitamine D3, dramatically increased the proportion of CD19+IgD-CD38+ cells expressing high levels of CCR10. The 1,25-(OH)2D3 also increased the number of CCR10+ cells expressing surface IgA, although the majority of CCR10+ cells remained negative for surface IgA. Thus, 1,25-(OH)2D3 alone may not be sufficient for the induction of IgA expression in terminally differentiating human B cells. To further determine whether 1,25-(OH)2D3 directly induces CCR10 expression in terminally differentiating B cells, we next performed the analysis on the human CCR10 promoter. We identified a proximal Ets-1 site and an upstream potential vitamin D response element to be critical for the inducible expression of CCR10 by 1,25-(OH)2D3. We confirmed the specific binding of Ets-1 and 1,25-(OH)2D3-activated vitamin D receptor to the respective sites. In conclusion, 1,25-(OH)2D3 efficiently induces CCR10 expression in terminally differentiating human B cells in vitro. Furthermore, the human CCR10 promoter is cooperatively activated by Ets-1 and vitamin D receptor in the presence of 1,25-(OH)2D3.

CD4-CCR5 interaction in intracellular compartments contributes to receptor expression at the cell surface

PubMed Central

Achour, Lamia; Scott, Mark G.H.; Shirvani, Hamasseh; Thuret, Alain; Bismuth, Georges; Labbé-Jullié, Catherine; Marullo, Stefano

2009-01-01

The association of CD4, a glycoprotein involved in T cell development and antigen recognition, and CCR5, a chemotactic G protein-coupled receptor, which regulates trafficking and effector functions of immune cells, forms the main receptor for the human immunodeficiency virus HIV. We observed that the vast majority of CCR5 is maintained within the intracellular compartments of primary T lymphocytes and in a monocytic cell line, contrasting with its relative low density at the cell surface. The CCR5-CD4 association, which occurs in the endoplasmic reticulum, enhanced CCR5 export to the plasma membrane in a concentration–dependent manner, whereas inhibition of endogenous CD4 with small interfering RNAs decreased cell surface expression of endogenous CCR5. This effect was specific for CCR5, as CD4 did not affect cell distribution of CXCR4, the other HIV co-receptor. These results reveal a previously unappreciated role of CD4, which contributes to regulate CCR5 export to the plasma membrane. PMID:19064722

Characteristics of HIV target CD4 T cells collected using different sampling methods from the genital tract of HIV seronegative women.

PubMed

Iyer, Smita S; Sabula, Michael J; Mehta, C Christina; Haddad, Lisa B; Brown, Nakita L; Amara, Rama R; Ofotokun, Igho; Sheth, Anandi N

2017-01-01

Understanding the immune profile of CD4 T cells, the primary targets for HIV, in the female genital tract (FGT) is critical for evaluating and developing effective biomedical HIV prevention strategies in women. However, longitudinal investigation of HIV susceptibility markers expressed by FGT CD4 T cells has been hindered by low cellular yield and risk of sampling-associated trauma. We investigated three minimally invasive FGT sampling methods to characterize and compare CD4 T cell yield and phenotype with the goal of establishing feasible sampling strategies for immune profiling of mucosal CD4 T cells. FGT samples were collected bimonthly from 12 healthy HIV negative women of reproductive age in the following order: 1) Cervicovaginal lavage (CVL), 2) two sequential endocervical flocked swabs (FS), and 3) two sequential endocervical cytobrushes (CB1, CB2). Cells were isolated and phentoyped via flow cytometry. CD4 T cell recovery was highest from each individual CB compared to either CVL or FS (p < 0.0001). The majority of CD4 T cells within the FGT, regardless of sampling method, expressed CCR5 relative to peripheral blood (p < 0.01). Within the CB, CCR5+ CD4 T cells expressed significantly higher levels of α4β7, CD69, and low levels of CD27 relative to CCR5- CD4 T cells (all p < 0.001). We also identified CD4 Treg lineage cells expressing CCR5 among CB samples. Using three different mucosal sampling methods collected longitudinally we demonstrate that CD4 T cells within the FGT express CCR5 and α4β7 and are highly activated, attributes which could act in concert to facilitate HIV acquisition. FS and CB sampling methods can allow for investigation of strategies to reduce HIV target cells in the FGT and could inform the design and interpretation microbicide and vaccine studies in women.

Myocardial Chemokine Expression and Intensity of Myocarditis in Chagas Cardiomyopathy Are Controlled by Polymorphisms in CXCL9 and CXCL10

PubMed Central

Nogueira, Luciana Gabriel; Santos, Ronaldo Honorato Barros; Ianni, Barbara Maria; Fiorelli, Alfredo Inácio; Mairena, Eliane Conti; Benvenuti, Luiz Alberto; Frade, Amanda; Donadi, Eduardo; Dias, Fabrício; Saba, Bruno; Wang, Hui-Tzu Lin; Fragata, Abilio; Sampaio, Marcelo; Hirata, Mario Hiroyuki; Buck, Paula; Mady, Charles; Bocchi, Edimar Alcides; Stolf, Noedir Antonio; Kalil, Jorge; Cunha-Neto, Edecio

2012-01-01

Background Chronic Chagas cardiomyopathy (CCC), a life-threatening inflammatory dilated cardiomyopathy, affects 30% of the approximately 8 million patients infected by Trypanosoma cruzi. Even though the Th1 T cell-rich myocarditis plays a pivotal role in CCC pathogenesis, little is known about the factors controlling inflammatory cell migration to CCC myocardium. Methods and Results Using confocal immunofluorescence and quantitative PCR, we studied cell surface staining and gene expression of the CXCR3, CCR4, CCR5, CCR7, CCR8 receptors and their chemokine ligands in myocardial samples from end-stage CCC patients. CCR5+, CXCR3+, CCR4+, CCL5+ and CXCL9+ mononuclear cells were observed in CCC myocardium. mRNA expression of the chemokines CCL5, CXCL9, CXCL10, CCL17, CCL19 and their receptors was upregulated in CCC myocardium. CXCL9 mRNA expression directly correlated with the intensity of myocarditis, as well as with mRNA expression of CXCR3, CCR4, CCR5, CCR7, CCR8 and their ligands. We also analyzed single-nucleotide polymorphisms for genes encoding the most highly expressed chemokines and receptors in a cohort of Chagas disease patients. CCC patients with ventricular dysfunction displayed reduced genotypic frequencies of CXCL9 rs10336 CC, CXCL10 rs3921 GG, and increased CCR5 rs1799988CC as compared to those without dysfunction. Significantly, myocardial samples from CCC patients carrying the CXCL9/CXCL10 genotypes associated to a lower risk displayed a 2–6 fold reduction in mRNA expression of CXCL9, CXCL10, and other chemokines and receptors, along with reduced intensity of myocarditis, as compared to those with other CXCL9/CXCL10 genotypes. Conclusions Results may indicate that genotypes associated to reduced risk in closely linked CXCL9 and CXCL10 genes may modulate local expression of the chemokines themselves, and simultaneously affect myocardial expression of other key chemokines as well as intensity of myocarditis. Taken together our results may suggest that CXCL9 and CXCL10 are master regulators of myocardial inflammatory cell migration, perhaps affecting clinical progression to the life-threatening form of CCC. PMID:23150742

Novel AgoshRNA molecules for silencing of the CCR5 co-receptor for HIV-1 infection

PubMed Central

Herrera-Carrillo, Elena

2017-01-01

Allogeneic transplantation of blood stem cells from a CCR5-Δ32 homozygous donor to an HIV-infected individual, the “Berlin patient”, led to a cure. Since then there has been a search for approaches that mimic this intervention in a gene therapy setting. RNA interference (RNAi) has evolved as a powerful tool to regulate gene expression in a sequence-specific manner and can be used to inactivate the CCR5 mRNA. Short hairpin RNA (shRNA) molecules can impair CCR5 expression, but these molecules may cause unintended side effects and they will not be processed in cells that lack Dicer, such as monocytes. Dicer-independent RNAi pathways have opened opportunities for new AgoshRNA designs that rely exclusively on Ago2 for maturation. Furthermore, AgoshRNA processing yields a single active guide RNA, thus reducing off-target effects. In this study, we tested different AgoshRNA designs against CCR5. We selected AgoshRNAs that potently downregulated CCR5 expression on human T cells and peripheral blood mononuclear cells (PBMC) and that had no apparent adverse effect on T cell development as assessed in a competitive cell growth assay. CCR5 knockdown significantly protected T cells from CCR5 tropic HIV-1 infection. PMID:28542329

RANK Expression and Osteoclastogenesis in Human Monocytes in Peripheral Blood from Rheumatoid Arthritis Patients.

PubMed

Nanke, Yuki; Kobashigawa, Tsuyoshi; Yago, Toru; Kawamoto, Manabu; Yamanaka, Hisashi; Kotake, Shigeru

2016-01-01

Rheumatoid arthritis (RA) appears as inflammation of synovial tissue and joint destruction. Receptor activator of NF- κ B (RANK) is a member of the TNF receptor superfamily and a receptor for the RANK ligand (RANKL). In this study, we examined the expression of RANK high and CCR6 on CD14 + monocytes from patients with RA and healthy volunteers. Peripheral blood samples were obtained from both the RA patients and the healthy volunteers. Osteoclastogenesis from monocytes was induced by RANKL and M-CSF in vitro . To study the expression of RANK high and CCR6 on CD14 + monocytes, two-color flow cytometry was performed. Levels of expression of RANK on monocytes were significantly correlated with the level of osteoclastogenesis in the healthy volunteers. The expression of RANK high on CD14 + monocyte in RA patients without treatment was elevated and that in those receiving treatment was decreased. In addition, the high-level expression of RANK on CD14 + monocytes was correlated with the high-level expression of CCR6 in healthy volunteers. Monocytes expressing both RANK and CCR6 differentiate into osteoclasts. The expression of CD14 + RANK high in untreated RA patients was elevated. RANK and CCR6 expressed on monocytes may be novel targets for the regulation of bone resorption in RA and osteoporosis.

RANK Expression and Osteoclastogenesis in Human Monocytes in Peripheral Blood from Rheumatoid Arthritis Patients

PubMed Central

Kobashigawa, Tsuyoshi

2016-01-01

Rheumatoid arthritis (RA) appears as inflammation of synovial tissue and joint destruction. Receptor activator of NF-κB (RANK) is a member of the TNF receptor superfamily and a receptor for the RANK ligand (RANKL). In this study, we examined the expression of RANKhigh and CCR6 on CD14+ monocytes from patients with RA and healthy volunteers. Peripheral blood samples were obtained from both the RA patients and the healthy volunteers. Osteoclastogenesis from monocytes was induced by RANKL and M-CSF in vitro. To study the expression of RANKhigh and CCR6 on CD14+ monocytes, two-color flow cytometry was performed. Levels of expression of RANK on monocytes were significantly correlated with the level of osteoclastogenesis in the healthy volunteers. The expression of RANKhigh on CD14+ monocyte in RA patients without treatment was elevated and that in those receiving treatment was decreased. In addition, the high-level expression of RANK on CD14+ monocytes was correlated with the high-level expression of CCR6 in healthy volunteers. Monocytes expressing both RANK and CCR6 differentiate into osteoclasts. The expression of CD14+RANKhigh in untreated RA patients was elevated. RANK and CCR6 expressed on monocytes may be novel targets for the regulation of bone resorption in RA and osteoporosis. PMID:27822475

The Effects of the Recombinant CCR5 T4 Lysozyme Fusion Protein on HIV-1 Infection.

PubMed

Jin, Qingwen; Chen, Hong; Wang, Xingxia; Zhao, Liandong; Xu, Qingchen; Wang, Huijuan; Li, Guanyu; Yang, Xiaofan; Ma, Hongming; Wu, Haoquan; Ji, Xiaohui

2015-01-01

Insertion of T4 lysozyme (T4L) into the GPCR successfully enhanced GPCR protein stability and solubilization. However, the biological functions of the recombinant GPCR protein have not been analyzed. We engineered the CCR5-T4L mutant and expressed and purified the soluble recombinant protein using an E.coli expression system. The antiviral effects of this recombinant protein in THP-1 cell lines, primary human macrophages, and PBMCs from different donors were investigated. We also explored the possible mechanisms underlying the observed antiviral effects. Our data showed the biphasic inhibitory and promotion effects of different concentrations of soluble recombinant CCR5-T4L protein on R5 tropic human immunodeficiency virus-1 (HIV-1) infection in THP-1 cell lines, human macrophages, and PBMCs from clinical isolates. We demonstrated that soluble recombinant CCR5-T4L acts as a HIV-1 co-receptor, interacts with wild type CCR5, down-regulates the surface CCR5 expression in human macrophages, and interacts with CCL5 to inhibit macrophage migration. Using binding assays, we further determined that recombinant CCR5-T4L and [125I]-CCL5 compete for the same binding site on wild type CCR5. Our results suggest that recombinant CCR5-T4L protein marginally promotes HIV-1 infection at low concentrations and markedly inhibits infection at higher concentrations. This recombinant protein may be helpful in the future development of anti-HIV-1 therapeutic agents.

Human Beta Defensin 2 Selectively Inhibits HIV-1 in Highly Permissive CCR6⁺CD4⁺ T Cells.

PubMed

Lafferty, Mark K; Sun, Lingling; Christensen-Quick, Aaron; Lu, Wuyuan; Garzino-Demo, Alfredo

2017-05-16

Chemokine receptor type 6 (CCR6)⁺CD4⁺ T cells are preferentially infected and depleted during HIV disease progression, but are preserved in non-progressors. CCR6 is expressed on a heterogeneous population of memory CD4⁺ T cells that are critical to mucosal immunity. Preferential infection of these cells is associated, in part, with high surface expression of CCR5, CXCR4, and α4β7. In addition, CCR6⁺CD4⁺ T cells harbor elevated levels of integrated viral DNA and high levels of proliferation markers. We have previously shown that the CCR6 ligands MIP-3α and human beta defensins inhibit HIV replication. The inhibition required CCR6 and the induction of APOBEC3G. Here, we further characterize the induction of apolipoprotein B mRNA editing enzyme (APOBEC3G) by human beta defensin 2. Human beta defensin 2 rapidly induces transcriptional induction of APOBEC3G that involves extracellular signal-regulated kinases 1/2 (ERK1/2) activation and the transcription factors NFATc2, NFATc1, and IRF4. We demonstrate that human beta defensin 2 selectively protects primary CCR6⁺CD4⁺ T cells infected with HIV-1. The selective protection of CCR6⁺CD4⁺ T cell subsets may be critical in maintaining mucosal immune function and preventing disease progression.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Sun-Mi, E-mail: lala1647@hanmail.net; Kim, Bo-Young, E-mail: kimboyoung@pusan.ac.kr; Lee, Sae-A, E-mail: saeah486@nate.com

Th1 lymphocytes are predominant in atherosclerotic lesions. However, mechanisms involved in the Th1 predominance are unknown. We have investigated the possibility of Th1 lymphocyte recruitment in a cholesterol-rich milieu. A high cholesterol diet resulted in enhanced expression of CCR5 ligands, including CCL3 and CCL4, but not of proatherogenic CXCR3 ligands, in atherosclerotic arteries of ApoE{sup −/−} mice. 27-Hydroxycholesterol and 7α-hydroxycholesterol, cholesterol oxides (oxysterols) detected in abundance in atherosclerotic lesions, greatly induced the transcription of CCL3 and CCL4 genes in addition to enhancing secretion of corresponding proteins by THP-1 monocytic cells. However, an identical or even higher concentration of cholesterol, 7β-hydroxycholesterol,more » and 7-ketocholsterol did not influence expression of these chemokines. Conditioned media containing the CCR5 ligands secreted from THP-1 cells induced migration of Jurkat T cells expressing CCR5, a characteristic chemokine receptor of Th1 cells, but not of Jurkat T cells that do not express CCR5. The migration of CCR5-expressing Jurkat T cells was abrogated in the presence of a CCR5-neutralizing antibody. 27-Hydroxycholesterol and 7α-hydroxycholesterol enhanced phosphorylation of Akt. Pharmacological inhibitors of phosphoinositide-3-kinase/Akt pathways blocked transcription as well as secretion of CCL3 and CCL4 in conjunction with attenuated migration of CCR5-expressing Jurkat T cells. This is the first report on the involvement of cholesterol oxides in migration of distinct subtype of T cells. We propose that 27-hydroxycholesterol and 7α-hydroxycholesterol can trigger a sequence of events that leads to recruitment of Th1 lymphocytes and phosphoinositide-3-kinase/Akt pathways play a major role in the process. - Graphical abstract: Th1 lymphocytes are predominant in atherosclerotic lesions. However, mechanisms involved in the Th1 predominance are unknown. We have investigated the possibility of Th1 lymphocyte recruitment in a cholesterol-rich milieu. We propose a model via which 27OHChol and 7αOHChol contribute to the predominance of Th1 cells in atherosclerotic lesions on the basis of our results and previous findings. Cholesterol deposited in the artery undergoes oxidative modification to oxysterols. Exposure of monocytic cells to 27OHChol or 7αOHChol results in increased transcription and secretion of CCR5 ligands, like CCL3 and CCL4, which leads to a concentration gradient of the chemokines. Among the lymphocytes attached to cell adhesion molecules expressed on endothelial cells, Th1 cells that express CCR5 recognize the gradient and follow the signal of increasing chemokine concentration towards the source of the chemokines, whereas other subtypes of T cells that do not express CCR5 (Tregs and Th2 cells) do not respond. The preferential infiltration of Th1 cells leads to predominance of Th1 cells. Since oxidized LDL (oxLDL) enhances the expression of cell adhesion molecules on endothelial cells, existence of oxLDL will accelerate the recruitment of Th1 lymphocytes into atherosclerotic lesions in response to the oxysterols. - Highlights: • High-cholesterol diet induces CCR5L expression, like CCL3 and CCL4, in ApoE{sup −/−} mice. • 27OHChol and 7αOHChol enhance secretion of CCL3 and CCL4 by monocytic cells. • The secreted CCR5 ligands promote migration of CCR5-expressing Th1 cells. • We report a mechanism underlying Th1 cell recruitment into atherosclerotic lesions.« less

Human Eosinophils Express Functional CCR7

PubMed Central

Ueki, Shigeharu; Estanislau, Jessica; Weller, Peter F.

2013-01-01

Human eosinophils display directed chemotactic activity toward an array of soluble chemokines. Eosinophils have been observed to migrate to draining lymph nodes in experimental models of allergic inflammation, yet it is unknown whether eosinophils express CCR7, a key chemokine receptor in coordinating leukocyte trafficking to lymph nodes. The purpose of this study is to demonstrate expression of CCR7 by human eosinophils and functional responses to CCL19 and CCL21, the known ligands of CCR7. Human eosinophils were purified by negative selection from healthy donors. CCR7 expression of freshly purified, unstimulated eosinophils and of IL-5–primed eosinophils was determined by flow cytometry and Western blot. Chemotaxis to CCL19 and CCL21 was measured in transwell assays. Shape changes to CCL19 and CCL21 were analyzed by flow cytometry and microscopy. Calcium fluxes of fluo-4 AM–loaded eosinophils were recorded by flow cytometry after chemokine stimulation. ERK phosphorylation of CCL19- and CCL21-stimulated eosinophils was measured by Western blot and Luminex assay. Human eosinophils expressed CCR7 as demonstrated by flow cytometry and Western blots. Eosinophils exhibited detectable cell surface expression of CCR7. IL-5–primed eosinophils exhibited chemotaxis toward CCL19 and CCL21 in a dose-dependent fashion. Upon stimulation with CCL19 or CCL21, IL-5–primed eosinophils demonstrated dose-dependent shape changes with polarization of F-actin and exhibited calcium influxes. Finally, primed eosinophils stimulated with CCL19 or CCL21 exhibited increased phosphorylation of ERK in response to both CCR7 ligands. We demonstrate that human eosinophils express CCR7 and have multipotent responses to the known ligands of CCR7. PMID:23449735

CXCR6-Mediated Simian Immunodeficiency Virus SIVagmSab Entry into Sabaeus African Green Monkey Lymphocytes Implicates Widespread Use of Non-CCR5 Pathways in Natural Host Infections

PubMed Central

Wetzel, Katherine S.; Yi, Yanjie; Elliott, Sarah T. C.; Romero, Dino; Jacquelin, Beatrice; Hahn, Beatrice H.; Muller-Trutwin, Michaela; Apetrei, Cristian; Pandrea, Ivona

2016-01-01

ABSTRACT African green monkeys (AGM) and sooty mangabeys (SM) are well-studied natural hosts of simian immunodeficiency virus (SIV) that do not progress to AIDS when infected with their species-specific viruses. Natural hosts of SIV express very low levels of the canonical entry coreceptor CCR5, and recent studies have shown that CCR5 is dispensable for SIV infection of SM in vivo and that blocking of CCR5 does not prevent ex vivo infection of peripheral blood mononuclear cells (PBMC) from SM or vervet AGM. In both hosts, CXCR6 is an efficient entry pathway in vitro. Here we investigated the use of species-matched CXCR6 and other alternative coreceptors by SIVagmSab, which infects sabaeus AGM. We cloned sabaeus CD4 and 10 candidate coreceptors. Species-matched CXCR6, CCR5, and GPR15 mediated robust entry into transfected cells by pseudotypes carrying SIVagmSab92018ivTF Env, with lower-level entry through GPR1 and APJ. We cloned genetically divergent env genes from the plasma of two wild-infected sabaeus AGM and found similar patterns of coreceptor use. Titration experiments showed that CXCR6 and CCR5 were more efficient than other coreceptors when tested at limiting CD4/coreceptor levels. Finally, blocking of CXCR6 with its ligand CXCL16 significantly inhibited SIVagmSab replication in sabaeus PBMC and had a greater impact than did the CCR5 blocker maraviroc, confirming the use of CXCR6 in primary lymphocyte infection. These data suggest a new paradigm for SIV infection of natural host species, whereby a shared outcome of virus-host coevolution is the use of CXCR6 or other alternative coreceptors for entry, which may direct SIV toward CD4+ T cell subsets and anatomical sites that support viral replication without disrupting immune homeostasis and function. IMPORTANCE Natural hosts of SIV do not progress to AIDS, in stark contrast to pathogenic human immunodeficiency virus type 1 (HIV-1)-human and SIVmac-macaque infections. Identifying how natural hosts avoid immunodeficiency can elucidate key mechanisms of pathogenesis. It is known that despite high viral loads, natural hosts have a low frequency of CD4+ cells expressing the SIV coreceptor CCR5. In this study, we demonstrate the efficient use of the coreceptor CXCR6 by SIVagmSab to infect sabaeus African green monkey lymphocytes. In conjunction with studies of SIVsmm, which infects sooty mangabeys, and SIVagmVer, which infects vervet monkeys, our data suggest a unifying model whereby in natural hosts, in which the CCR5 expression level is low, the use of CXCR6 or other coreceptors to mediate infection may target SIV toward distinct cell populations that are able to support high-level viral replication without causing a loss of CD4+ T cell homeostasis and lymphoid tissue damage that lead to AIDS in HIV-1 and SIVmac infections. PMID:27903799

CXCR6-Mediated Simian Immunodeficiency Virus SIVagmSab Entry into Sabaeus African Green Monkey Lymphocytes Implicates Widespread Use of Non-CCR5 Pathways in Natural Host Infections.

PubMed

Wetzel, Katherine S; Yi, Yanjie; Elliott, Sarah T C; Romero, Dino; Jacquelin, Beatrice; Hahn, Beatrice H; Muller-Trutwin, Michaela; Apetrei, Cristian; Pandrea, Ivona; Collman, Ronald G

2017-02-15

African green monkeys (AGM) and sooty mangabeys (SM) are well-studied natural hosts of simian immunodeficiency virus (SIV) that do not progress to AIDS when infected with their species-specific viruses. Natural hosts of SIV express very low levels of the canonical entry coreceptor CCR5, and recent studies have shown that CCR5 is dispensable for SIV infection of SM in vivo and that blocking of CCR5 does not prevent ex vivo infection of peripheral blood mononuclear cells (PBMC) from SM or vervet AGM. In both hosts, CXCR6 is an efficient entry pathway in vitro Here we investigated the use of species-matched CXCR6 and other alternative coreceptors by SIVagmSab, which infects sabaeus AGM. We cloned sabaeus CD4 and 10 candidate coreceptors. Species-matched CXCR6, CCR5, and GPR15 mediated robust entry into transfected cells by pseudotypes carrying SIVagmSab92018ivTF Env, with lower-level entry through GPR1 and APJ. We cloned genetically divergent env genes from the plasma of two wild-infected sabaeus AGM and found similar patterns of coreceptor use. Titration experiments showed that CXCR6 and CCR5 were more efficient than other coreceptors when tested at limiting CD4/coreceptor levels. Finally, blocking of CXCR6 with its ligand CXCL16 significantly inhibited SIVagmSab replication in sabaeus PBMC and had a greater impact than did the CCR5 blocker maraviroc, confirming the use of CXCR6 in primary lymphocyte infection. These data suggest a new paradigm for SIV infection of natural host species, whereby a shared outcome of virus-host coevolution is the use of CXCR6 or other alternative coreceptors for entry, which may direct SIV toward CD4 + T cell subsets and anatomical sites that support viral replication without disrupting immune homeostasis and function. Natural hosts of SIV do not progress to AIDS, in stark contrast to pathogenic human immunodeficiency virus type 1 (HIV-1)-human and SIVmac-macaque infections. Identifying how natural hosts avoid immunodeficiency can elucidate key mechanisms of pathogenesis. It is known that despite high viral loads, natural hosts have a low frequency of CD4 + cells expressing the SIV coreceptor CCR5. In this study, we demonstrate the efficient use of the coreceptor CXCR6 by SIVagmSab to infect sabaeus African green monkey lymphocytes. In conjunction with studies of SIVsmm, which infects sooty mangabeys, and SIVagmVer, which infects vervet monkeys, our data suggest a unifying model whereby in natural hosts, in which the CCR5 expression level is low, the use of CXCR6 or other coreceptors to mediate infection may target SIV toward distinct cell populations that are able to support high-level viral replication without causing a loss of CD4 + T cell homeostasis and lymphoid tissue damage that lead to AIDS in HIV-1 and SIVmac infections. Copyright © 2017 American Society for Microbiology.

Lack of CCR5 modifies glial phenotypes and population of the nigral dopaminergic neurons, but not MPTP-induced dopaminergic neurodegeneration.

PubMed

Choi, Dong-Young; Lee, Myung Koo; Hong, Jin Tae

2013-01-01

Constitutive expression of C-C chemokine receptor (CCR) 5 has been detected in astrocytes, microglia and neurons, but its physiological roles in the central nervous system are obscure. The bidirectional interactions between neuron and glial cells through CCR5 and its ligands were thought to be crucial for maintaining normal neuronal activities. No study has described function of CCR5 in the dopaminergic neurodegeneration in Parkinson's disease. In order to examine effects of CCR5 on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic neurodegeneration, we employed CCR5 wild type (WT) and knockout (KO) mice. Immunostainings for tyrosine hydroxylase (TH) exhibited that CCR5 KO mice had lower number of TH-positive neurons even in the absence of MPTP. Difference in MPTP (15mg/kg×4 times, 2hr interval)-mediated loss of TH-positive neurons was subtle between CCR5 WT and KO mice, but there was larger dopamine depletion, behavioral impairments and microglial activation in CCR5 deficient mice. Intriguingly, CCR5 KO brains contained higher immunoreactivity for monoamine oxidase (MAO) B which was mainly localized within astrocytes. In agreement with upregulation of MAO B, concentration of MPP+ was higher in the substantia nigra and striatum of CCR5 KO mice after MPTP injection. We found remarkable activation of p38 MAPK in CCR5 deficient mice, which positively regulates MAO B expression. These results indicate that CCR5 deficiency modifies the nigrostriatal dopaminergic neuronal system and bidirectional interaction between neurons and glial cells via CCR5 might be important for dopaminergic neuronal survival. Copyright © 2012 Elsevier Inc. All rights reserved.

CCR4 is expressed on infiltrating cells in lesional skin of early mycosis fungoides and atopic dermatitis.

PubMed

Sugaya, Makoto; Morimura, Sohshi; Suga, Hiraku; Kawaguchi, Makiko; Miyagaki, Tomomitsu; Ohmatsu, Hanako; Fujita, Hideki; Sato, Shinichi

2015-06-01

CCR4 is expressed on tumor cells of mycosis fungoides (MF) and Sézary syndrome (SS). In MF, most infiltrating cells in patches and plaques express CXCR3, while tumor cells express CCR4 in advanced stages. Poteligeo Test IHC (CCR4 staining kit) is a newly developed staining kit that can examine the presence of CCR4 expressed on tumor cells of adult T-cell leukemia/lymphoma, peripheral T-cell lymphoma and cutaneous T-cell lymphoma before treatment of anti-CCR4 antibody using paraffin-embedded samples. In this study, we analyzed CCR4 expression in lesional skin of MF, SS, atopic dermatitis (AD) and psoriasis with this new kit. CCR4 was expressed on infiltrating cells in lesional skin of patch, plaque, tumor MF and SS, and the number of positive cells increased as the disease progressed. Immunohistochemistry with frozen sections also showed some positive cells scattered in the dermis, although the quality was not high enough to quantify positive cells. There were significant positive correlations between CCR4(+) cells and serum lactate dehydrogenase levels. Interestingly, CCR4(+) cells were also detected in AD skin, whose number was larger than that in psoriatic skin. Previous studies showed only scattered CCR4(+) cells in skin samples by standard immunohistochemical staining. The new, sensitive CCR4 staining kit has revealed that CCR4 is expressed on infiltrating cells in lesional skin of early MF and AD as well as advanced MF and SS. These cells can be therapeutic targets for patients who are resistant to standard treatments. © 2015 Japanese Dermatological Association.

Targeting both viral and host determinants of human immunodeficiency virus entry, using a new lentiviral vector coexpressing the T20 fusion inhibitor and a selective CCL5 intrakine.

PubMed

Petit, Nicolas; Dorgham, Karim; Levacher, Béatrice; Burlion, Aude; Gorochov, Guy; Marodon, Gilles

2014-08-01

Numerous strategies targeting early and late steps of the HIV life cycle have been proposed for gene therapy. However, targeting viral and host determinants of HIV entry is the only strategy that would prevent viral DNA-mediated CD4(+) cell death while diminishing the possibility for the virus to escape. To this end, we devised a bicistronic lentiviral vector expressing the membrane-bound form of the T20 fusion inhibitor, referred to as the C46 peptide, and a CCR5 superagonist, modified to sequester CCR5 away from the cell surface, referred to as the P2-CCL5 intrakine. We tested the effects of the vector on HIV infection and replication, using the human CEMR5 cell line expressing CD4 and CCR5, and primary human T cells. Transduced cells expressed the C46 peptide, detected with the 2F5 monoclonal antibody by flow cytometry. Expression of the P2-CCL5 intrakine correlates with lower levels of cell surface CCR5. Complete protection against HIV infection could be observed in cells expressing the protective transgenes. Importantly, we show that the combination of the transgenes was more potent than either transgene alone, showing the interest of expressing two entry inhibitors to inhibit HIV infection. Last, genetically modified cells possessed a selective advantage over nonmodified cells on HIV challenge in vitro, showing that modified cells were protected from HIV-induced cell death. Our results demonstrate that lentiviral vectors coexpressing the T20 fusion inhibitor and the P2-CCL5 intrakine represent promising tools for HIV gene therapy.

Abnormal immune response of CCR5-deficient mice to ocular infection with herpes simplex virus type 1

PubMed Central

Carr, Daniel J.J.; Ash, John; Lane, Thomas E.; Kuziel, William A.

2006-01-01

Summary Ocular herpes simplex virus type 1 (HSV-1) infection elicits a strong inflammatory response that is associated with production of the β chemokines CCL3 and CCL5, which share a common receptor, CCR5. To gain insight into the role of these molecules in ocular immune responses, we infected the corneas of WT and CCR5-deficient (CCR5-/-) mice with HSV-1 and measured inflammatory parameters. In the absence of CCR5, the early infiltration of neutrophils into the cornea was diminished. Associated with this aberrant leukocyte recruitment, neutrophils in CCR5-/- mice were restricted to the stroma whereas in wild type mice these cells trafficked to the stroma and epithelial layers of the infected cornea. Virus titers and cytokine/chemokine levels in the infected tissue of these mice were similar for the first 5 days after infection. However, by day 7 post-infection, the CCR5-/- mice showed a significant elevation in the chemokines CCL2, CCL5, CXCL9, and CXCL10 in the trigeminal ganglion and brain stem as well as a significant increase in viral burden. The increase in chemokine expression was associated with an increase in the infiltration of CD4 and/or CD8 T cells into the trigeminal ganglion and brain stem of CCR5-/- mice. Surprisingly, even though infected CCR5-/- mice were less efficient at controlling the progression of virus replication, there was no difference in mortality. These results suggest that, although CCR5 plays a role in regulating leukocyte trafficking and control of virus burden, compensatory mechanisms are involved in preventing mortality following HSV-1 infection. PMID:16476970

Chemokine CCL28 induces apoptosis of decidual stromal cells via binding CCR3/CCR10 in human spontaneous abortion.

PubMed

Sun, Chan; Zhang, Yuan-Yuan; Tang, Chuan-Ling; Wang, Song-Cun; Piao, Hai-Lan; Tao, Yu; Zhu, Rui; Du, Mei-Rong; Li, Da-Jin

2013-10-01

Spontaneous abortion is the most common complication of pregnancy. Immune activation and the subsequent inflammation-induced tissue injury are often observed at the maternal-fetal interface as the final pathological assault in recurrent spontaneous abortion. However, the precise mechanisms responsible for spontaneous abortion involving inflammation are not fully understood. Chemokine CCL28 and its receptors CCR3 and CCR10 are important regulators in inflammatory process. Here, we examined the expression of CCL28 and its receptors in decidual stromal cells (DSCs) by immunochemistry and flow cytometry (FCM), and compared their expression level in DSCs from normal pregnancy versus spontaneous abortion, and their relationship to inflammatory cytokines production by DSCs. We further analyzed regulation of the pro-inflammatory cytokines on CCL28 expression in DSCs by real-time polymerase chain reaction, In-cell Western and FCM. The effects of CCL28-CCR3/CCR10 interaction on DSC apoptosis was investigated by Annexin V staining and FCM analysis or DAPI staining and nuclear morphology. Higher levels of the inflammatory cytokines interleukin (IL)-1β, IL-17A and tumor necrosis factor-α, and increased CCR3/CCR10 expression were observed in DSCs from spontaneous abortion compared with normal pregnancy. Treatment with inflammatory cytokines differently affected CCL28 and CCR3/CCR10 expression in DSCs. Human recombinant CCL28 promoted DSC apoptosis, which was eliminated by pretreatment with neutralizing antibodies against CCR3/CCR10 and CCL28. However, CCL28 did not affect DSC growth. These results suggest that the inflammation-promoted up-regulation of CCL28 and its receptors interaction in DSCs is involved in human spontaneous abortion via inducing DSC apoptosis.

Dualtropic CXCR6/CCR5 Simian Immunodeficiency Virus (SIV) Infection of Sooty Mangabey Primary Lymphocytes: Distinct Coreceptor Use in Natural versus Pathogenic Hosts of SIV.

PubMed

Elliott, Sarah T C; Wetzel, Katherine S; Francella, Nicholas; Bryan, Steven; Romero, Dino C; Riddick, Nadeene E; Shaheen, Farida; Vanderford, Thomas; Derdeyn, Cynthia A; Silvestri, Guido; Paiardini, Mirko; Collman, Ronald G

2015-09-01

Natural-host sooty mangabeys (SM) infected with simian immunodeficiency virus (SIV) exhibit high viral loads but do not develop disease, whereas infection of rhesus macaques (RM) causes CD4(+) T cell loss and AIDS. Several mechanisms have been proposed to explain these divergent outcomes, including differences in cell targeting, which have been linked to low expression of the canonical SIV entry receptor CCR5 on CD4(+) T cells of SM and other natural hosts. We previously showed that infection and high-level viremia occur even in a subset of SM that genetically lack functional CCR5, which indicates that alternative entry coreceptors are used by SIV in vivo in these animals. We also showed that SM CXCR6 is a robust coreceptor for SIVsmm in vitro. Here we identify CXCR6 as a principal entry pathway for SIV in SM primary lymphocytes. We show that ex vivo SIV infection of lymphocytes from CCR5 wild-type SM is mediated by both CXCR6 and CCR5. In contrast, infection of RM lymphocytes is fully dependent on CCR5. These data raise the possibility that CXCR6-directed tropism in CCR5-low natural hosts may alter CD4(+) T cell subset targeting compared with that in nonnatural hosts, enabling SIV to maintain high-level replication without leading to widespread CD4(+) T cell loss. Natural hosts of SIV, such as sooty mangabeys, sustain high viral loads but do not develop disease, while nonnatural hosts, like rhesus macaques, develop AIDS. Understanding this difference may help elucidate mechanisms of pathogenesis. Natural hosts have very low levels of the SIV entry coreceptor CCR5, suggesting that restricted entry may limit infection of certain target cells, although it is unclear how the virus replicates so robustly. Here we show that in sooty mangabey lymphocytes, infection is mediated by the alternative entry coreceptor CXCR6, as well as CCR5. In rhesus macaque lymphocytes, however, infection occurs entirely through CCR5. The use of CXCR6 for entry, combined with very low CCR5 levels, may redirect the virus to different cell targets in natural hosts. It is possible that differential targeting may favor infection of nonessential cells and limit infection of critical cells in natural hosts, thus contributing to benign outcome of infection. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Dualtropic CXCR6/CCR5 Simian Immunodeficiency Virus (SIV) Infection of Sooty Mangabey Primary Lymphocytes: Distinct Coreceptor Use in Natural versus Pathogenic Hosts of SIV

PubMed Central

Elliott, Sarah T. C.; Wetzel, Katherine S.; Francella, Nicholas; Bryan, Steven; Romero, Dino C.; Riddick, Nadeene E.; Shaheen, Farida; Vanderford, Thomas; Derdeyn, Cynthia A.; Silvestri, Guido; Paiardini, Mirko

2015-01-01

ABSTRACT Natural-host sooty mangabeys (SM) infected with simian immunodeficiency virus (SIV) exhibit high viral loads but do not develop disease, whereas infection of rhesus macaques (RM) causes CD4+ T cell loss and AIDS. Several mechanisms have been proposed to explain these divergent outcomes, including differences in cell targeting, which have been linked to low expression of the canonical SIV entry receptor CCR5 on CD4+ T cells of SM and other natural hosts. We previously showed that infection and high-level viremia occur even in a subset of SM that genetically lack functional CCR5, which indicates that alternative entry coreceptors are used by SIV in vivo in these animals. We also showed that SM CXCR6 is a robust coreceptor for SIVsmm in vitro. Here we identify CXCR6 as a principal entry pathway for SIV in SM primary lymphocytes. We show that ex vivo SIV infection of lymphocytes from CCR5 wild-type SM is mediated by both CXCR6 and CCR5. In contrast, infection of RM lymphocytes is fully dependent on CCR5. These data raise the possibility that CXCR6-directed tropism in CCR5-low natural hosts may alter CD4+ T cell subset targeting compared with that in nonnatural hosts, enabling SIV to maintain high-level replication without leading to widespread CD4+ T cell loss. IMPORTANCE Natural hosts of SIV, such as sooty mangabeys, sustain high viral loads but do not develop disease, while nonnatural hosts, like rhesus macaques, develop AIDS. Understanding this difference may help elucidate mechanisms of pathogenesis. Natural hosts have very low levels of the SIV entry coreceptor CCR5, suggesting that restricted entry may limit infection of certain target cells, although it is unclear how the virus replicates so robustly. Here we show that in sooty mangabey lymphocytes, infection is mediated by the alternative entry coreceptor CXCR6, as well as CCR5. In rhesus macaque lymphocytes, however, infection occurs entirely through CCR5. The use of CXCR6 for entry, combined with very low CCR5 levels, may redirect the virus to different cell targets in natural hosts. It is possible that differential targeting may favor infection of nonessential cells and limit infection of critical cells in natural hosts, thus contributing to benign outcome of infection. PMID:26109719

Blood expression levels of chemokine receptor CCR3 and chemokine CCL11 in age-related macular degeneration: a case–control study

PubMed Central

2014-01-01

Background Dysregulation of the CCR3/CCL11 pathway has been implicated in the pathogenesis of choroidal neovascularisation, a common feature of late age-related macular degeneration (AMD). The aim of this study was to investigate the expression of CCR3 and its ligand CCL11 in peripheral blood in patients with neovascular AMD. Methods Patients with neovascular AMD and healthy controls were included. Blood samples were obtained and prepared for flow cytometry to investigate the expression of CCR3. Levels of CCL11 were measured in plasma using Cytometric Bead Array. Differences between the groups were tested using Kruskal-Wallis test and Mann–Whitney U test. Results Patients (n = 83) with neovascular AMD and healthy control persons (n = 114) were included in the study. No significant difference in the expression of CCR3 was found on CD9+ granulocytes when comparing patients suffering from neovascular AMD with any of the control groups. We did not find any alteration in CCL11 levels in patients among the age matched groups. There was no correlation between expression of CCR3/CCL11 and clinical response to treatment with anti-vascular endothelial growth factor (VEGF). Conclusion Our results do not suggest a systemic alteration of the CCR3/CCL11 receptor/ligand complex in patients with neovascular AMD. PMID:24575855

Blood expression levels of chemokine receptor CCR3 and chemokine CCL11 in age-related macular degeneration: a case-control study.

PubMed

Falk, Mads Krüger; Singh, Amardeep; Faber, Carsten; Nissen, Mogens Holst; Hviid, Thomas; Sørensen, Torben Lykke

2014-02-27

Dysregulation of the CCR3/CCL11 pathway has been implicated in the pathogenesis of choroidal neovascularisation, a common feature of late age-related macular degeneration (AMD). The aim of this study was to investigate the expression of CCR3 and its ligand CCL11 in peripheral blood in patients with neovascular AMD. Patients with neovascular AMD and healthy controls were included. Blood samples were obtained and prepared for flow cytometry to investigate the expression of CCR3. Levels of CCL11 were measured in plasma using Cytometric Bead Array. Differences between the groups were tested using Kruskal-Wallis test and Mann-Whitney U test. Patients (n = 83) with neovascular AMD and healthy control persons (n = 114) were included in the study. No significant difference in the expression of CCR3 was found on CD9+ granulocytes when comparing patients suffering from neovascular AMD with any of the control groups. We did not find any alteration in CCL11 levels in patients among the age matched groups. There was no correlation between expression of CCR3/CCL11 and clinical response to treatment with anti-vascular endothelial growth factor (VEGF). Our results do not suggest a systemic alteration of the CCR3/CCL11 receptor/ligand complex in patients with neovascular AMD.

Downregulation of CXCR6 and CXCR3 in lymphocytes from birch-allergic patients.

PubMed

Casas, R; Lindau, C; Zetterström, O; Duchén, K

2008-09-01

Preferential expression of chemokine receptors on Th1 or Th2 T-helper cells has mostly been studied in cell lines generated in vitro or in animal models; however, results are less well characterized in humans. We determined T-cell responses through chemokine receptor expression on lymphocytes, and cytokine secretion in plasma from birch-allergic and healthy subjects. The expression of CCR2, CCR3, CCR4, CCR5, CCR7, CXCR3, CXCR4, CXCR6, IL-12 and IL-18R receptors was studied on CD4(+) and CD8(+) cells from birch-allergic (n = 14) and healthy (n = 14) subjects by flow cytometry. The concentration of IL-4, IL-5, IL-10, IL-12, IFN-gamma and TNF-alpha cytokines was measured in plasma from the same individuals using a cytometric bead array human cytokines kit. The similar expression of CCR4 in T cells from atopic and healthy individuals argues against the use of the receptor as an in vivo marker of Th2 immune responses. Reduced percentages of CD4(+) cells expressing IL-18R, CXCR6 and CXCR3 were found in the same group of samples. TNF-alpha, IFN-gamma, IL-10, IL-5, IL-4 and IL-12 cytokines were elevated in samples from allergic individuals. Reduced expression of Th1-associated chemokine receptors together with higher levels of Th1, Th2 and anti-inflammatory cytokines in samples from allergic patients indicate that immune responses in peripheral blood in atopic diseases are complex and cannot be simplified to the Th1/Th2 paradigm. Not only the clinical picture of atopic diseases but also the clinical state at different time points of the disease might influence the results of studies including immunological markers associated with Th1- or Th2-type immune responses.

Disruption of CCR5-dependent homing of regulatory T cells inhibits tumor growth in a murine model of pancreatic cancer

PubMed Central

Tan, Marcus C. B.; Goedegebuure, Peter S.; Belt, Brian A.; Flaherty, Brian; Sankpal, Narendra; Gillanders, William E.; Eberlein, Timothy J.; Hsieh, Chyi-Song; Linehan, David C.

2013-01-01

Tumors evade immune destruction by actively inducing immune tolerance through the recruitment of CD4+CD25+Foxp3+ regulatory T cells (Treg). We have previously described increased prevalence of these cells in pancreatic adenocarcinoma, but it remains unclear what mechanisms are involved in recruiting Treg into the tumor microenvironment. Here, we postulated that chemokines might direct Treg homing to tumor. We show, in both human pancreatic adenocarcinoma and a murine pancreatic tumor model (Pan02), that tumor cells produce increased levels of ligands for the CCR5 chemokine receptor, and, reciprocally, that CD4+ Foxp3+ Treg, compared with CD4+ Foxp3− effector T cells, preferentially express CCR5. When CCR5/CCL5 signaling is disrupted, either by reducing CCL5 production by tumor cells or by systemic administration of a CCR5 inhibitor (TAK-779), Treg migration to tumors is reduced and tumors are smaller than in control mice. Thus, this study demonstrates the importance of Treg in immune evasion by tumors, how blockade of Treg migration may inhibit tumor growth, and, specifically in pancreatic adenocarcinoma, the role of CCR5 in the homing of tumor-associated Treg. Selective targeting of CCR5/CCL5 signaling may represent a novel immunomodulatory strategy for the treatment of cancer. PMID:19155524

Mucosal CCR1 gene expression as a marker of molecular activity in Crohn's disease: preliminary data.

PubMed

Dobre, Maria; Mănuc, Teodora Ecaterina; Milanesi, Elena; Pleşea, Iancu Emil; Ţieranu, Eugen Nicolae; Popa, Caterina; Mănuc, Mircea; Preda, Carmen Monica; Ţieranu, Ioana; Diculescu, Mihai Mircea; Ionescu, Elena Mirela; Becheanu, Gabriel

2017-01-01

A series of mechanisms of immune response, inflammation and apoptosis have been demonstrated to contribute to the appearance and evolution of Crohn's disease (CD) through the overexpression of several cytokines and chemokines in a susceptible host. The aim of this study was to identify the differences in gene expression profiles analyzing a panel of candidate genes in the mucosa from patients with active CD (CD-A), patients in remission (CD-R), and normal controls. Nine individuals were enrolled in the study: six CD patients (three with active lesions, three with mucosal healing) and three controls without inflammatory bowel disease (IBD) seen on endoscopy. All the individuals underwent mucosal biopsy during colonoscopy. Gene expression levels of 84 genes previously associated with CD were evaluated by polymerase chain reaction (PCR) array. Ten genes out of 84 were found significantly differentially expressed in CD-A (CCL11, CCL25, DEFA5, GCG, IL17A, LCN2, REG1A, STAT3, MUC1, CCR1) and eight genes in CD-R (CASP1, IL23A, STAT1, STAT3, TNF, CCR1, CCL5, and HSP90B1) when compared to controls. A quantitative gene expression analysis revealed that CCR1 gene was more expressed in CD-A than in CD-R. Our data suggest that CCR1 gene may be a putative marker of molecular activity of Crohn's disease. Following these preliminary data, a confirmation in larger cohort studies could represent a useful method in order to identify new therapeutic targets.

Induced Pluripotent Stem Cell‐Derived Endothelial Cells Overexpressing Interleukin‐8 Receptors A/B and/or C‐C Chemokine Receptors 2/5 Inhibit Vascular Injury Response

PubMed Central

Giordano, Samantha; Zhao, Xiangmin; Chen, Yiu‐Fai; Litovsky, Silvio H.; Hage, Fadi G.; Townes, Tim M.; Sun, Chiao‐Wang; Wu, Li‐Chen; Oparil, Suzanne

2017-01-01

Abstract Recruitment of neutrophils and monocytes/macrophages to the site of vascular injury is mediated by binding of chemoattractants to interleukin (IL) 8 receptors RA and RB (IL8RA/B) C‐C chemokine receptors (CCR) 2 and 5 expressed on neutrophil and monocyte/macrophage membranes. Endothelial cells (ECs) derived from rat‐induced pluripotent stem cells (RiPS) were transduced with adenovirus containing cDNA of IL8RA/B and/or CCR2/5. We hypothesized that RiPS‐ECs overexpressing IL8RA/B (RiPS‐IL8RA/B‐ECs), CCR2/5 (RiPS‐CCR2/5‐ECs), or both receptors (RiPS‐IL8RA/B+CCR2/5‐ECs) will inhibit inflammatory responses and neointima formation in balloon‐injured rat carotid artery. Twelve‐week‐old male Sprague‐Dawley rats underwent balloon injury of the right carotid artery and intravenous infusion of (a) saline vehicle, (b) control RiPS‐Null‐ECs (ECs transduced with empty virus), (c) RiPS‐IL8RA/B‐ECs, (d) RiPS‐CCR2/5‐ECs, or (e) RiPS‐IL8RA/B+CCR2/5‐ECs. Inflammatory mediator expression and leukocyte infiltration were measured in injured and uninjured arteries at 24 hours postinjury by enzyme‐linked immunosorbent assay (ELISA) and immunohistochemistry, respectively. Neointima formation was assessed at 14 days postinjury. RiPS‐ECs expressing the IL8RA/B or CCR2/5 homing device targeted the injured arteries and decreased injury‐induced inflammatory cytokine expression, neutrophil/macrophage infiltration, and neointima formation. Transfused RiPS‐ECs overexpressing IL8RA/B and/or CCR2/5 prevented inflammatory responses and neointima formation after vascular injury. Targeted delivery of iPS‐ECs with a homing device to inflammatory mediators in injured arteries provides a novel strategy for the treatment of cardiovascular diseases. Stem Cells Translational Medicine 2017;6:1168–1177 PMID:28233474

CCR8 Signaling Influences Toll-Like Receptor 4 Responses in Human Macrophages in Inflammatory Diseases ▿

PubMed Central

Kvist Reimer, Martina; Brange, Charlotte; Rosendahl, Alexander

2011-01-01

CCR8 immunity is generally associated with Th2 responses in allergic diseases. In this study, we demonstrate for the first time a pronounced attenuated influx of macrophages in ovalbumin (OVA)-challenged CCR8 knockout mice. To explore whether macrophages in human inflamed lung tissue also were CCR8 positive, human lung tissue from patients with chronic obstructive pulmonary disease (COPD) was evaluated. Indeed, CCR8 expression was pronounced in invading monocytes/macrophages from lungs of patients with Global Initiative for Obstructive Lung Disease (GOLD) stage IV COPD. Given this expression pattern, the functional role of CCR8 on human macrophages was evaluated in vitro. Human peripheral blood monocytes expressed low levels of CCR8, while macrophage colony-stimulating factor (M-CSF)-derived human macrophages expressed significantly elevated surface levels of CCR8. Importantly, CCL1 directly regulated the expression of CD18 and CD49b and hence influenced the adhesion capacity of human macrophages. CCL1 drives chemotaxis in M-CSF-derived macrophages, and this could be completely inhibited by lipopolysaccharide (LPS). Whereas both CCL1 and LPS monotreatment inhibited spontaneous superoxide release in macrophages, CCL1 significantly induced superoxide release in the presence of LPS in a dose-dependent manner. Finally, CCL1 induced production of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) and could inhibit LPS-induced cytokine production in a dose-dependent manner. Our data demonstrate, for the first time, the presence of CCR8 on inflammatory macrophages in human COPD lung tissue. Importantly, the functional data from human macrophages suggest a potential cross talk between the CCR8 and the Toll-like receptor 4 (TLR4) pathways, both of which are present in COPD patients. PMID:21976223

CCR8 signaling influences Toll-like receptor 4 responses in human macrophages in inflammatory diseases.

PubMed

Reimer, Martina Kvist; Brange, Charlotte; Rosendahl, Alexander

2011-12-01

CCR8 immunity is generally associated with Th2 responses in allergic diseases. In this study, we demonstrate for the first time a pronounced attenuated influx of macrophages in ovalbumin (OVA)-challenged CCR8 knockout mice. To explore whether macrophages in human inflamed lung tissue also were CCR8 positive, human lung tissue from patients with chronic obstructive pulmonary disease (COPD) was evaluated. Indeed, CCR8 expression was pronounced in invading monocytes/macrophages from lungs of patients with Global Initiative for Obstructive Lung Disease (GOLD) stage IV COPD. Given this expression pattern, the functional role of CCR8 on human macrophages was evaluated in vitro. Human peripheral blood monocytes expressed low levels of CCR8, while macrophage colony-stimulating factor (M-CSF)-derived human macrophages expressed significantly elevated surface levels of CCR8. Importantly, CCL1 directly regulated the expression of CD18 and CD49b and hence influenced the adhesion capacity of human macrophages. CCL1 drives chemotaxis in M-CSF-derived macrophages, and this could be completely inhibited by lipopolysaccharide (LPS). Whereas both CCL1 and LPS monotreatment inhibited spontaneous superoxide release in macrophages, CCL1 significantly induced superoxide release in the presence of LPS in a dose-dependent manner. Finally, CCL1 induced production of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) and could inhibit LPS-induced cytokine production in a dose-dependent manner. Our data demonstrate, for the first time, the presence of CCR8 on inflammatory macrophages in human COPD lung tissue. Importantly, the functional data from human macrophages suggest a potential cross talk between the CCR8 and the Toll-like receptor 4 (TLR4) pathways, both of which are present in COPD patients.

AMP-activated protein kinase activation mediates CCL3-induced cell migration and matrix metalloproteinase-2 expression in human chondrosarcoma

PubMed Central

2013-01-01

Chemokine (C-C motif) ligand 3 (CCL3), also known as macrophage inflammatory protein-1α, is a cytokine involved in inflammation and activation of polymorphonuclear leukocytes. CCL3 has been detected in infiltrating cells and tumor cells. Chondrosarcoma is a highly malignant tumor that causes distant metastasis. However, the effect of CCL3 on human chondrosarcoma metastasis is still unknown. Here, we found that CCL3 increased cellular migration and expression of matrix metalloproteinase (MMP)-2 in human chondrosarcoma cells. Pre-treatment of cells with the MMP-2 inhibitor or transfection with MMP-2 specific siRNA abolished CCL3-induced cell migration. CCL3 has been reported to exert its effects through activation of its specific receptor, CC chemokine receptor 5 (CCR5). The CCR5 and AMP-activated protein kinase (AMPK) inhibitor or siRNA also attenuated CCL3-upregulated cell motility and MMP-2 expression. CCL3-induced expression of MMP-2 and migration were also inhibited by specific inhibitors, and inactive mutants of AMPK, p38 mitogen activated protein kinase (p38 or p38-MAPK), and nuclear factor κB (NF-κB) cascades. On the other hand, CCL3 treatment demonstrably activated AMPK, p38, and NF-κB signaling pathways. Furthermore, the expression levels of CCL3, CCR5, and MMP-2 were correlated in human chondrosarcoma specimens. Taken together, our results indicate that CCL3 enhances the migratory ability of human chondrosarcoma cells by increasing MMP-2 expression via the CCR5, AMPK, p38, and NF-κB pathways. PMID:24047437

Efficient Modification of the CCR5 Locus in Primary Human T Cells With megaTAL Nuclease Establishes HIV-1 Resistance

PubMed Central

Romano Ibarra, Guillermo S; Paul, Biswajit; Sather, Blythe D; Younan, Patrick M; Sommer, Karen; Kowalski, John P; Hale, Malika; Stoddard, Barry; Jarjour, Jordan; Astrakhan, Alexander; Kiem, Hans-Peter; Rawlings, David J

2016-01-01

A naturally occurring 32-base pair deletion of the HIV-1 co-receptor CCR5 has demonstrated protection against HIV infection of human CD4+ T cells. Recent genetic engineering approaches using engineered nucleases to disrupt the gene and mimic this mutation show promise for HIV therapy. We developed a megaTAL nuclease targeting the third extracellular loop of CCR5 that we delivered to primary human T cells by mRNA transfection. The CCR5 megaTAL nuclease established resistance to HIV in cell lines and disrupted the expression of CCR5 on primary human CD4+ T cells with a high efficiency, achieving up to 80% modification of the locus in primary cells as measured by molecular analysis. Gene-modified cells engrafted at levels equivalent to unmodified cells when transplanted into immunodeficient mice. Furthermore, genetically modified CD4+ cells were preferentially expanded during HIV-1 infection in vivo in an immunodeficient mouse model. Our results demonstrate the feasibility of targeting CCR5 in primary T cells using an engineered megaTAL nuclease, and the potential to use gene-modified cells to reconstitute a patient's immune system and provide protection from HIV infection. PMID:27741222

SIV Coreceptor Specificity in Natural and Non-Natural Host Infection: Implications for Cell Targeting and Differential Outcomes from Infection.

PubMed

Wetzel, Katherine S; Elliott, Sarah T C; Collman, Ronald G

2018-01-01

Pathogenic HIV-1 infection of humans and SIVmac infection of macaques are the result of zoonotic transfer of primate immunodeficiency viruses from their natural hosts into non-natural host species. Natural host infections do not result in pathogenesis despite high levels of virus replication, and evidence suggests that differences in anatomical location and specific subsets of CD4+ T cells infected may underlie distinct outcomes from infection. The coreceptor CCR5 has long been considered the sole pathway for SIV entry and the key determinant of CD4+ cell targeting, but it has also been known that natural hosts express exceedingly low levels of CCR5 despite maintaining high levels of virus replication. This review details emerging data indicating that in multiple natural host species, CCR5 is dispensable for SIV infection ex vivo and/or in vivo and, contrary to the established dogma, alternative coreceptors, particularly CXCR6, play a central role in infection and cell targeting. Infections of non-natural hosts, however, are characterized by CCR5-exclusive entry. These findings suggest that alternative coreceptor-mediated cell targeting in natural hosts, combined with low CCR5 expression, may direct the virus to distinct populations of cells that are dispensable for immune homeostasis, particularly extralymphoid and more differentiated CD4+ T cells. In contrast, CCR5-mediated entry in non-natural hosts results in targeting of CD4+ T cells that are located in lymphoid tissues, critical for immune homeostasis, or necessary for gut barrier integrity. Thus, fundamental differences in viral entry coreceptor use may be central determinants of infection outcome. These findings redefine the normal SIV/host relationship in natural host species, shed new light on key features linked to zoonotic immunodeficiency virus transfer, and highlight important questions regarding how and why this coreceptor bottleneck occurs and the coevolutionary equilibrium is lost following cross-species transfer that results in AIDS. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

ccrABEnt serine recombinase genes are widely distributed in the Enterococcus faecium and Enterococcus casseliflavus species groups and are expressed in E. faecium

PubMed Central

Bjørkeng, Eva Katrin; Tessema, Girum Tadesse; Lundblad, Eirik Wasmuth; Butaye, Patrick; Willems, Rob; Sollid, Johanna Ericsson; Sundsfjord, Arnfinn; Hegstad, Kristin

2010-01-01

The presence, distribution and expression of cassette chromosome recombinase (ccr) genes, which are homologous to the staphylococcal ccrAB genes and are designated ccrABEnt genes, were examined in enterococcal isolates (n=421) representing 13 different species. A total of 118 (28 %) isolates were positive for ccrABEnt genes by PCR, and a number of these were confirmed by Southern hybridization with a ccrAEnt probe (n=76) and partial DNA sequencing of ccrAEnt and ccrBEnt genes (n=38). ccrABEnt genes were present in Enterococcus faecium (58/216, 27 %), Enterococcus durans (31/38, 82 %), Enterococcus hirae (27/52, 50 %), Enterococcus casseliflavus (1/4, 25 %) and Enterococcus gallinarum (1/2, 50 %). In the eight other species tested, including Enterococcus faecalis (n=94), ccrABEnt genes were not found. Thirty-eight sequenced ccrABEnt genes from five different enterococcal species showed 94–100 % nucleotide sequence identity and linkage PCRs showed heterogeneity in the ccrABEnt flanking chromosomal genes. Expression analysis of ccrABEnt genes from the E. faecium DO strain showed constitutive expression as a bicistronic mRNA. The ccrABEnt mRNA levels were lower during log phase than stationary phase in relation to total mRNA. Multilocus sequence typing was performed on 39 isolates. ccrABEnt genes were detected in both hospital-related (10/29, 34 %) and non-hospital (4/10, 40 %) strains of E. faecium. Various sequence types were represented by both ccrABEnt positive and negative isolates, suggesting acquisition or loss of ccrABEnt in E. faecium. In summary, ccrABEnt genes, potentially involved in genome plasticity, are expressed in E. faecium and are widely distributed in the E. faecium and E. casseliflavus species groups. PMID:20817645

In vivo (1)H MRS and (31)P MRSI of the response to cyclocreatine in transgenic mouse liver expressing creatine kinase.

PubMed

Cui, Min-Hui; Jayalakshmi, Kamaiah; Liu, Laibin; Guha, Chandan; Branch, Craig A

2015-12-01

Hepatocyte transplantation has been explored as a therapeutic alternative to liver transplantation, but a means to monitor the success of the procedure is lacking. Published findings support the use of in vivo (31)P MRSI of creatine kinase (CK)-expressing hepatocytes to monitor proliferation of implanted hepatocytes. Phosphocreatine tissue level depends upon creatine (Cr) input to the CK enzyme reaction, but Cr measurement by (1)H MRS suffers from low signal-to-noise ratio (SNR). We examine the possibility of using the Cr analog cyclocreatine (CCr, a substrate for CK), which is quickly phosphorylated to phosphocyclocreatine (PCCr), as a higher SNR alternative to Cr. (1)H MRS and (31)P MRSI were employed to measure the effect of incremental supplementation of CCr upon PCCr, γ-ATP, pH and Pi /ATP in the liver of transgenic mice expressing the BB isoform of CK (CKBB) in hepatocytes. Water supplementation with 0.1% CCr led to a peak total PCCr level of 17.15 ± 1.07 mmol/kg wet weight by 6 weeks, while adding 1.0% CCr led to a stable PCCr liver level of 18.12 ± 3.91 mmol/kg by the fourth day of feeding. PCCr was positively correlated with CCr, and ATP concentration and pH declined with increasing PCCr. Feeding with 1% CCr in water induced an apparent saturated level of PCCr, suggesting that CCr quantization may not be necessary for quantifying expression of CK in mice. These findings support the possibility of using (31)P MRS to noninvasively monitor hepatocyte transplant success with CK-expressing hepatocytes. Copyright © 2015 John Wiley & Sons, Ltd.

Relevance of CCL3/CCR5 axis in oral carcinogenesis.

PubMed

da Silva, Janine Mayra; Moreira Dos Santos, Tálita Pollyanna; Sobral, Lays Martin; Queiroz-Junior, Celso Martins; Rachid, Milene Alvarenga; Proudfoot, Amanda E I; Garlet, Gustavo Pompermaier; Batista, Aline Carvalho; Teixeira, Mauro Martins; Leopoldino, Andréia Machado; Russo, Remo Castro; Silva, Tarcília Aparecida

2017-08-01

The chemokine CCL3 is a chemotactic cytokine crucial for inflammatory cell recruitment in homeostatic and pathological conditions. CCL3 might stimulate cancer progression by promoting leukocyte accumulation, angiogenesis and tumour growth. The expression of CCL3 and its receptors CCR1 and CCR5 was demonstrated in oral squamous cell carcinoma (OSCC), but their role was not defined. Here, the functions of CCL3 were assessed using a model of chemically induced tongue carcinogenesis with 4-nitroquinoline-1-oxide (4NQO). Lineages of OSCC were used to analyse the effects of CCL3 in vitro . The 4NQO-induced lesions exhibited increased expression of CCL3, CCR1 and CCR5. CCL3 -/- and CCR5 -/- mice presented reduced incidence of tongue tumours compared to wild-type (WT) and CCR1 -/- mice. Consistently, attenuated cytomorphological atypia and reduced cell proliferation were observed in lesions of CCL3 -/- and CCR5 -/- mice. OSCC from CCL3 -/- mice exhibited lower infiltration of eosinophils and reduced expression of Egf, Fgf1, Tgf-β1, Vegfa, Vegfb, Itga-4, Vtn, Mmp-1a, Mmp-2 and Mmp-9 than WT mice. In vitro , CCL3 induced invasion and production of CCL5, IL-6, MMP -2, -8, -9. Blockage of CCL3 in vitro using α-CCL3 or Evasin-1 (a CCL3-binding protein) impaired tumour cell invasion. In conclusion, CCL3/CCR5 axis has pro-tumourigenic effects in oral carcinogenesis. The induction of inflammatory and angiogenic pathways and eosinophils recruitment appear to be the underlying mechanism explaining these effects. These data reveal potential protective effects of CCL3 blockade in oral cancer.

Molecular cloning and expression analysis of the canine chemokine receptor CCR9.

PubMed

Maeda, Shingo; Ohno, Koichi; Tsukamoto, Atsushi; Nakashima, Ko; Fukushima, Kenjiro; Goto-Koshino, Yuko; Fujino, Yasuhito; Tsujimoto, Hajime

2012-01-15

The chemokine receptor CCR9, which interacts with the thymus-expressed chemokine TECK/CCL25, contributes to the localization of lymphocytes to the small intestine, and is implicated in the development of human inflammatory bowel disease (IBD); however, their role in canine IBD is unknown. The objective of this study was to isolate cDNA encoding CCR9 and to investigate CCR9 expression in normal canine tissues and lymphoid cell lines. The complete open reading frame contained 1104 bp, encoding 367 amino acids, with 85% and 81% identity to human and mouse homologs, respectively. CCR9 mRNA was detected in all tissues investigated with the highest expression level in the small intestine. CCR9 mRNA was also expressed in GL-1, a canine B cell leukemia cell line, but not in CLBL-1, a canine B cell lymphoma cell line. Immunoblot and flow cytometry analyses of these cell lines using an anti-human CCR9 monoclonal antibody revealed that CCR9 protein expression was detected only in GL-1, indicating the cross-reactivity of the antibody. Using the antibody, flow cytometry showed that the proportions of CCR9(+) cells were small (mean, 4.88%; SD, 2.15%) in the normal canine PBMCs. This study will be useful in understanding canine intestinal immunity and the immunopathogenesis of canine IBD. Copyright © 2011 Elsevier B.V. All rights reserved.

Generation and genetic engineering of human induced pluripotent stem cells using designed zinc finger nucleases.

PubMed

Ramalingam, Sivaprakash; London, Viktoriya; Kandavelou, Karthikeyan; Cebotaru, Liudmila; Guggino, William; Civin, Curt; Chandrasegaran, Srinivasan

2013-02-15

Zinc finger nucleases (ZFNs) have become powerful tools to deliver a targeted double-strand break at a pre-determined chromosomal locus in order to insert an exogenous transgene by homology-directed repair. ZFN-mediated gene targeting was used to generate both single-allele chemokine (C-C motif) receptor 5 (CCR5)-modified human induced pluripotent stem cells (hiPSCs) and biallele CCR5-modified hiPSCs from human lung fibroblasts (IMR90 cells) and human primary cord blood mononuclear cells (CBMNCs) by site-specific insertion of stem cell transcription factor genes flanked by LoxP sites into the endogenous CCR5 locus. The Oct4 and Sox2 reprogramming factors, in combination with valproic acid, induced reprogramming of human lung fibroblasts to form CCR5-modified hiPSCs, while 5 factors, Oct4/Sox2/Klf4/Lin28/Nanog, induced reprogramming of CBMNCs. Subsequent Cre recombinase treatment of the CCR5-modified IMR90 hiPSCs resulted in the removal of the Oct4 and Sox2 transgenes. Further genetic engineering of the single-allele CCR5-modified IMR90 hiPSCs was achieved by site-specific addition of the large CFTR transcription unit to the remaining CCR5 wild-type allele, using CCR5-specific ZFNs and a donor construct containing tdTomato and CFTR transgenes flanked by CCR5 homology arms. CFTR was expressed efficiently from the endogenous CCR5 locus of the CCR5-modified tdTomato/CFTR hiPSCs. These results suggest that it might be feasible to use ZFN-evoked strategies to (1) generate precisely targeted genetically well-defined patient-specific hiPSCs, and (2) then to reshape their function by targeted addition and expression of therapeutic genes from the CCR5 chromosomal locus for autologous cell-based transgene-correction therapy to treat various recessive monogenic human diseases in the future.

Loss of CXCR6 coreceptor usage characterizes pathogenic lentiviruses

PubMed Central

Wetzel, Katherine S.; Yi, Yanjie; Bauer, Anya M.; Bibollet-Ruche, Frederic; Hahn, Beatrice H.; Paiardini, Mirko; Silvestri, Guido; Peeters, Martine; Collman, Ronald G.

2018-01-01

Pandemic HIV-1 originated from the cross-species transmission of SIVcpz, which infects chimpanzees, while SIVcpz itself emerged following the cross-species transmission and recombination of monkey SIVs, with env contributed by the SIVgsn/mus/mon lineage that infects greater spot-nosed, mustached and mona monkeys. SIVcpz and HIV-1 are pathogenic in their respective hosts, while the phenotype of their SIVgsn/mus/mon ancestors is unknown. However, two well-studied SIV infected natural hosts, sooty mangabeys (SMs) and African green monkeys (AGMs), typically remain healthy despite high viral loads; these species express low levels of the canonical coreceptor CCR5, and recent work shows that CXCR6 is a major coreceptor for SIV in these hosts. It is not known what coreceptors were used by the precursors of SIVcpz, whether coreceptor use changed during emergence of the SIVcpz/HIV-1 lineage, and what T cell subsets express CXCR6 in natural hosts. Using species-matched coreceptors and CD4, we show here that SIVcpz uses only CCR5 for entry and, like HIV-1, cannot use CXCR6. In contrast, SIVmus efficiently uses both CXCR6 and CCR5. Coreceptor selectivity was determined by Env, with CXCR6 use abrogated by Pro326 in the V3 crown, which is absent in monkey SIVs but highly conserved in SIVcpz/HIV-1. To characterize which cells express CXCR6, we generated a novel antibody that recognizes CXCR6 of multiple primate species. Testing lymphocytes from SM, the best-studied natural host, we found that CXCR6 is restricted to CD4+ effector memory cells, and is expressed by a sub-population distinct from those expressing CCR5. Thus, efficient CXCR6 use, previously identified in SM and AGM infection, also characterizes a member of the SIV lineage that gave rise to SIVcpz/HIV-1. Loss of CXCR6 usage by SIVcpz may have altered its cell tropism, shifting virus from CXCR6-expressing cells that may support replication without disrupting immune function or homeostasis, towards CCR5-expressing cells with pathogenic consequences. PMID:29659623

Loss of CXCR6 coreceptor usage characterizes pathogenic lentiviruses.

PubMed

Wetzel, Katherine S; Yi, Yanjie; Yadav, Anjana; Bauer, Anya M; Bello, Ezekiel A; Romero, Dino C; Bibollet-Ruche, Frederic; Hahn, Beatrice H; Paiardini, Mirko; Silvestri, Guido; Peeters, Martine; Collman, Ronald G

2018-04-01

Pandemic HIV-1 originated from the cross-species transmission of SIVcpz, which infects chimpanzees, while SIVcpz itself emerged following the cross-species transmission and recombination of monkey SIVs, with env contributed by the SIVgsn/mus/mon lineage that infects greater spot-nosed, mustached and mona monkeys. SIVcpz and HIV-1 are pathogenic in their respective hosts, while the phenotype of their SIVgsn/mus/mon ancestors is unknown. However, two well-studied SIV infected natural hosts, sooty mangabeys (SMs) and African green monkeys (AGMs), typically remain healthy despite high viral loads; these species express low levels of the canonical coreceptor CCR5, and recent work shows that CXCR6 is a major coreceptor for SIV in these hosts. It is not known what coreceptors were used by the precursors of SIVcpz, whether coreceptor use changed during emergence of the SIVcpz/HIV-1 lineage, and what T cell subsets express CXCR6 in natural hosts. Using species-matched coreceptors and CD4, we show here that SIVcpz uses only CCR5 for entry and, like HIV-1, cannot use CXCR6. In contrast, SIVmus efficiently uses both CXCR6 and CCR5. Coreceptor selectivity was determined by Env, with CXCR6 use abrogated by Pro326 in the V3 crown, which is absent in monkey SIVs but highly conserved in SIVcpz/HIV-1. To characterize which cells express CXCR6, we generated a novel antibody that recognizes CXCR6 of multiple primate species. Testing lymphocytes from SM, the best-studied natural host, we found that CXCR6 is restricted to CD4+ effector memory cells, and is expressed by a sub-population distinct from those expressing CCR5. Thus, efficient CXCR6 use, previously identified in SM and AGM infection, also characterizes a member of the SIV lineage that gave rise to SIVcpz/HIV-1. Loss of CXCR6 usage by SIVcpz may have altered its cell tropism, shifting virus from CXCR6-expressing cells that may support replication without disrupting immune function or homeostasis, towards CCR5-expressing cells with pathogenic consequences.

Downregulation of mouse CCR3 by lentiviral shRNA inhibits proliferation and induces apoptosis of mouse eosinophils.

PubMed

Zhu, Xin-Hua; Liao, Bing; Xu, Yi; Liu, Ke; Huang, Yun; Huang, Quan-Long; Liu, Yue-Hui

2017-02-01

RNA interference has been considered as an effective gene silencing method in basic and preclinical investigations. The aims of the present study were to construct a lentiviral vector expressing a short hairpin RNA (shRNA) targeting the murine CC chemokine receptor 3 (mCCR3), and to investigate its effects on the proliferation and apoptosis of mouse eosinophils. A recombinant lentiviral vector expressing four fragments of mouse CCR3 shRNA (pLVX‑mCCR3‑1+2+3+4‑shRNA) was constructed using subcloning techniques. This novel lentivirus was then packaged into 293T cells by co‑transduction with plasmids, including Baculo p35, pCMV R8.2 and VSV. The interference effects of the vector were verified using polymerase chain reaction (PCR) and western blot analyses. The effects of the interference on the proliferation and apoptosis of mouse eosinophils were investigated using 3‑(4,5‑dimethylthiazol‑2‑yl)‑5‑(3‑carboxymethoxyphenyl)‑2‑(4‑sulfophenyl)‑2H‑tetrazolium and terminal deoxynucleotidyl transferase dUTP nick end labeling methods, respectively. The results of the PCR and western blot analyses confirmed that the novel recombinant vector, pLVX‑mCCR3‑1+2+3+4‑shRNA, had high efficiency in inhibiting the mRNA and protein expression levels of mCCR3 in mouse eosinophils. The downregulation of mCCR3 significantly inhibited proliferation of the eosinophils. Furthermore, the present study found that the downregulation of mCCR3 significantly promoted apoptosis of the eosinophils. Therefore, the downregulation of mCCR3 led to the inhibition of proliferation and induction of apoptosis in mouse eosinophils. The predominant characteristics of allergic rhinitis are eosinophil infiltration and release of inflammatory mediators, which appear in a variety of clinical manifestations. The results of the present study indicate that mCCR3 silencing may serve as a putative approach for the treatment of allergic rhinitis.

Podocyte-specific chemokine (C-C motif) receptor 2 overexpression mediates diabetic renal injury in mice

PubMed Central

You, Hanning; Gao, Ting; Raup-Konsavage, Wesley M.; Cooper, Timothy K.; Bronson, Sarah K.; Reeves, W. Brian; Awad, Alaa S.

2016-01-01

Inflammation is a central pathophysiologic mechanism that contributes to diabetes mellitus and diabetic nephropathy. Recently, we showed that macrophages directly contribute to diabetic renal injury, and that pharmacological blockade or genetic deficiency of chemokine (C-C motif) receptor 2 (CCR2) confers kidney protection in diabetic nephropathy. However, the direct role of CCR2 in kidney-derived cells such as podocytes in diabetic nephropathy remains unclear. To study this, we developed a transgenic mouse model expressing CCR2 specifically in podocytes (Tg(NPHS2-Ccr2)) on a nephropathy prone (DBA/2J) and CCR2 deficient (Ccr2−/−) background with heterozygous Ccr2+/− littermate controls. Diabetes was induced by streptozotocin. As expected, absence of CCR2 conferred kidney protection after nine weeks of diabetes. In contrast, transgenic CCR2 over expression in the podocytes of Ccr2−/− mice resulted in significantly increased albuminuria, blood urea nitrogen, histopathologic changes, kidney fibronectin and type-1 collagen expression, podocyte loss, and glomerular apoptosis after nine weeks of streptozotocin-induced diabetes. Interestingly, there was no concurrent increase in kidney macrophage recruitment or inflammatory cytokine levels in the mice. These findings support a direct role for CCR2 expression in podocytes to mediate diabetic renal injury, independent of monocyte/macrophage recruitment. Thus, targeting the CCR2 signaling cascade in podocytes could be a novel therapeutic approach for treatment of diabetic nephropathy. PMID:27914709

Genetic Polymorphism at CCL5 Is Associated With Protection in Chagas’ Heart Disease: Antagonistic Participation of CCR1+ and CCR5+ Cells in Chronic Chagasic Cardiomyopathy

PubMed Central

Batista, Angelica Martins; Alvarado-Arnez, Lucia Elena; Alves, Silvia Marinho; Melo, Gloria; Pereira, Isabela Resende; Ruivo, Leonardo Alexandre de Souza; da Silva, Andrea Alice; Gibaldi, Daniel; da Silva, Thayse do E. S. Protásio; de Lorena, Virginia Maria Barros; de Melo, Adriene Siqueira; de Araújo Soares, Ana Karine; Barros, Michelle da Silva; Costa, Vláudia Maria Assis; Cardoso, Cynthia C.; Pacheco, Antonio G.; Carrazzone, Cristina; Oliveira, Wilson; Moraes, Milton Ozório; Lannes-Vieira, Joseli

2018-01-01

Chronic cardiomyopathy is the main clinical manifestation of Chagas disease (CD), a disease caused by Trypanosoma cruzi infection. A hallmark of chronic chagasic cardiomyopathy (CCC) is a fibrogenic inflammation mainly composed of CD8+ and CD4+ T cells and macrophages. CC-chemokine ligands and receptors have been proposed to drive cell migration toward the heart tissue of CD patients. Single nucleotide polymorphisms (SNPs) in CC-chemokine ligand and receptor genes may determine protein expression. Herein, we evaluated the association of SNPs in the CC-chemokines CCL2 (rs1024611) and CCL5 (rs2107538, rs2280788) and the CCL5/RANTES receptors CCR1 (rs3181077, rs1491961, rs3136672) and CCR5 (rs1799987) with risk and progression toward CCC. We performed a cross-sectional association study of 406 seropositive patients from endemic areas for CD in the State of Pernambuco, Northeast Brazil. The patients were classified as non-cardiopathic (A, n = 110) or cardiopathic (mild, B1, n = 163; severe, C, n = 133). Serum levels of CCL5 and CCL2/MCP-1 were elevated in CD patients but were neither associated with risk/severity of CCC nor with SNP genotypes. After logistic regression analysis with adjustment for the covariates gender and ethnicity, CCL5 −403 (rs2107538) CT heterozygotes (OR = 0.5, P-value = 0.04) and T carriers (OR = 0.5, P-value = 0.01) were associated with protection against CCC. To gain insight into the participation of the CCL5–CCR5/CCR1 axis in CCC, mice were infected with the Colombian T. cruzi strain. Increased CCL5 concentrations were detected in cardiac tissue. In spleen, frequencies of CCR1+ CD8+ T cells and CD14+ macrophages were decreased, while frequencies of CCR5+ cells were increased. Importantly, CCR1+CD14+ macrophages were mainly IL-10+, while CCR5+ cells were mostly TNF+. CCR5-deficient infected mice presented reduced TNF concentrations and injury in heart tissue. Selective blockade of CCR1 (Met-RANTES therapy) in infected Ccr5−/− mice supported a protective role for CCR1 in CCC. Furthermore, parasite antigen stimulation of CD patient blood cells increased the frequency of CCR1+CD8+ T cells and CCL5 production. Collectively, our data support that a genetic variant of CCL5 and CCR1+ cells confer protection against Chagas heart disease, identifying the CCL5-CCR1 axis as a target for immunostimulation. PMID:29696014

Genetic Polymorphism at CCL5 Is Associated With Protection in Chagas' Heart Disease: Antagonistic Participation of CCR1+ and CCR5+ Cells in Chronic Chagasic Cardiomyopathy.

PubMed

Batista, Angelica Martins; Alvarado-Arnez, Lucia Elena; Alves, Silvia Marinho; Melo, Gloria; Pereira, Isabela Resende; Ruivo, Leonardo Alexandre de Souza; da Silva, Andrea Alice; Gibaldi, Daniel; da Silva, Thayse do E S Protásio; de Lorena, Virginia Maria Barros; de Melo, Adriene Siqueira; de Araújo Soares, Ana Karine; Barros, Michelle da Silva; Costa, Vláudia Maria Assis; Cardoso, Cynthia C; Pacheco, Antonio G; Carrazzone, Cristina; Oliveira, Wilson; Moraes, Milton Ozório; Lannes-Vieira, Joseli

2018-01-01

Chronic cardiomyopathy is the main clinical manifestation of Chagas disease (CD), a disease caused by Trypanosoma cruzi infection. A hallmark of chronic chagasic cardiomyopathy (CCC) is a fibrogenic inflammation mainly composed of CD8 + and CD4 + T cells and macrophages. CC-chemokine ligands and receptors have been proposed to drive cell migration toward the heart tissue of CD patients. Single nucleotide polymorphisms (SNPs) in CC-chemokine ligand and receptor genes may determine protein expression. Herein, we evaluated the association of SNPs in the CC-chemokines CCL2 (rs1024611) and CCL5 (rs2107538, rs2280788) and the CCL5/RANTES receptors CCR1 (rs3181077, rs1491961, rs3136672) and CCR5 (rs1799987) with risk and progression toward CCC. We performed a cross-sectional association study of 406 seropositive patients from endemic areas for CD in the State of Pernambuco, Northeast Brazil. The patients were classified as non-cardiopathic (A, n  = 110) or cardiopathic (mild, B1, n  = 163; severe, C, n  = 133). Serum levels of CCL5 and CCL2/MCP-1 were elevated in CD patients but were neither associated with risk/severity of CCC nor with SNP genotypes. After logistic regression analysis with adjustment for the covariates gender and ethnicity, CCL5 -403 (rs2107538) CT heterozygotes (OR = 0.5, P -value = 0.04) and T carriers (OR = 0.5, P -value = 0.01) were associated with protection against CCC. To gain insight into the participation of the CCL5-CCR5/CCR1 axis in CCC, mice were infected with the Colombian T. cruzi strain. Increased CCL5 concentrations were detected in cardiac tissue. In spleen, frequencies of CCR1 + CD8 + T cells and CD14 + macrophages were decreased, while frequencies of CCR5 + cells were increased. Importantly, CCR1 + CD14 + macrophages were mainly IL-10 + , while CCR5 + cells were mostly TNF + . CCR5-deficient infected mice presented reduced TNF concentrations and injury in heart tissue. Selective blockade of CCR1 (Met-RANTES therapy) in infected Ccr5 -/- mice supported a protective role for CCR1 in CCC. Furthermore, parasite antigen stimulation of CD patient blood cells increased the frequency of CCR1 + CD8 + T cells and CCL5 production. Collectively, our data support that a genetic variant of CCL5 and CCR1 + cells confer protection against Chagas heart disease, identifying the CCL5-CCR1 axis as a target for immunostimulation.

Elucidating a Key Anti-HIV-1 and Cancer-Associated Axis: The Structure of CCL5 (Rantes) in Complex with CCR5

NASA Astrophysics Data System (ADS)

Tamamis, Phanourios; Floudas, Christodoulos A.

2014-06-01

CCL5 (RANTES) is an inflammatory chemokine which binds to chemokine receptor CCR5 and induces signaling. The CCL5:CCR5 associated chemotactic signaling is of critical biological importance and is a potential HIV-1 therapeutic axis. Several studies provided growing evidence for the expression of CCL5 and CCR5 in non-hematological malignancies. Therefore, the delineation of the CCL5:CCR5 complex structure can pave the way for novel CCR5-targeted drugs. We employed a computational protocol which is primarily based on free energy calculations and molecular dynamics simulations, and report, what is to our knowledge, the first computationally derived CCL5:CCR5 complex structure which is in excellent agreement with experimental findings and clarifies the functional role of CCL5 and CCR5 residues which are associated with binding and signaling. A wealth of polar and non-polar interactions contributes to the tight CCL5:CCR5 binding. The structure of an HIV-1 gp120 V3 loop in complex with CCR5 has recently been derived through a similar computational protocol. A comparison between the CCL5 : CCR5 and the HIV-1 gp120 V3 loop : CCR5 complex structures depicts that both the chemokine and the virus primarily interact with the same CCR5 residues. The present work provides insights into the blocking mechanism of HIV-1 by CCL5.

Plasmacytoid Dendritic Cell Infection and Sensing Capacity during Pathogenic and Nonpathogenic Simian Immunodeficiency Virus Infection

PubMed Central

Jochems, Simon P.; Jacquelin, Beatrice; Chauveau, Lise; Huot, Nicolas; Petitjean, Gaël; Lepelley, Alice; Liovat, Anne-Sophie; Ploquin, Mickaël J.; Cartwright, Emily K.; Bosinger, Steven E.; Silvestri, Guido; Barré-Sinoussi, Françoise; Lebon, Pierre; Schwartz, Olivier

2015-01-01

ABSTRACT Human immunodeficiency virus (HIV) in humans and simian immunodeficiency virus (SIV) in macaques (MAC) lead to chronic inflammation and AIDS. Natural hosts, such as African green monkeys (AGM) and sooty mangabeys (SM), are protected against SIV-induced chronic inflammation and AIDS. Here, we report that AGM plasmacytoid dendritic cells (pDC) express extremely low levels of CD4, unlike MAC and human pDC. Despite this, AGM pDC efficiently sensed SIVagm, but not heterologous HIV/SIV isolates, indicating a virus-host adaptation. Moreover, both AGM and SM pDC were found to be, in contrast to MAC pDC, predominantly negative for CCR5. Despite such limited CD4 and CCR5 expression, lymphoid tissue pDC were infected to a degree similar to that seen with CD4+ T cells in both MAC and AGM. Altogether, our finding of efficient pDC infection by SIV in vivo identifies pDC as a potential viral reservoir in lymphoid tissues. We discovered low expression of CD4 on AGM pDC, which did not preclude efficient sensing of host-adapted viruses. Therefore, pDC infection and efficient sensing are not prerequisites for chronic inflammation. The high level of pDC infection by SIVagm suggests that if CCR5 paucity on immune cells is important for nonpathogenesis of natural hosts, it is possibly not due to its role as a coreceptor. IMPORTANCE The ability of certain key immune cell subsets to resist infection might contribute to the asymptomatic nature of simian immunodeficiency virus (SIV) infection in its natural hosts, such as African green monkeys (AGM) and sooty mangabeys (SM). This relative resistance to infection has been correlated with reduced expression of CD4 and/or CCR5. We show that plasmacytoid dendritic cells (pDC) of natural hosts display reduced CD4 and/or CCR5 expression, unlike macaque pDC. Surprisingly, this did not protect AGM pDC, as infection levels were similar to those found in MAC pDC. Furthermore, we show that AGM pDC did not consistently produce type I interferon (IFN-I) upon heterologous SIVmac/HIV type 1 (HIV-1) encounter, while they sensed autologous SIVagm isolates. Pseudotyping SIVmac/HIV-1 overcame this deficiency, suggesting that reduced uptake of heterologous viral strains underlays this lack of sensing. The distinct IFN-I responses depending on host species and HIV/SIV isolates reveal the host/virus species specificity of pDC sensing. PMID:25903334

Expression and function of CCL2/CCR2 in rat micturition reflexes and somatic sensitivity with urinary bladder inflammation

PubMed Central

Arms, Lauren; Girard, Beatrice M.; Malley, Susan E.

2013-01-01

Chemokines are proinflammatory mediators of the immune response, and there is growing evidence for chemokine/receptor signaling involvement in pronociception. Bladder pain syndrome (BPS)/interstitial cystitis (IC) is a chronic pain syndrome characterized by pain, pressure, or discomfort perceived to be bladder-related with at least one urinary symptom. We have explored the expression and functional roles of CCL2 (monocyte chemoattractant protein-1) and its high-affinity receptor, CCR2, in micturition reflex function and somatic sensitivity in rats with urinary bladder inflammation induced by cyclophosphamide (CYP) treatment of varying duration (4 h, 48 h, chronic). Real-time quantitative RT-PCR, ELISAs, and immunohistochemistry demonstrated significant (P ≤ 0.01) increases in CCL2 and CCR2 expression in the urothelium and in Fast Blue-labeled bladder afferent neurons in lumbosacral dorsal root ganglia with CYP-induced cystitis. Intravesical infusion of RS504393 (5 μM), a specific CCR2 antagonist, reduced voiding frequency and increased bladder capacity and void volume in rats with CYP-induced cystitis (4 h), as determined with open outlet, conscious cystometry. In addition, CCR2 blockade, at the level of the urinary bladder, reduced referred somatic sensitivity of the hindpaw and pelvic region in rats with CYP treatment, as determined with von Frey filament testing. We provide evidence of functional roles for CCL2/CCR2 signaling at the level of the urinary bladder in reducing voiding frequency and somatic sensitivity following CYP-induced cystitis (4 h). These studies suggest that chemokines/receptors may be novel targets with therapeutic potential in the context of urinary bladder inflammation. PMID:23594826

Chemokine Receptor CCR5 Antagonist Maraviroc: Medicinal Chemistry and Clinical Applications

PubMed Central

Xu, Guoyan G.; Guo, Jia; Wu, Yuntao

2015-01-01

The human immunodeficiency virus (HIV) causes acquired immumodeficiency syndrome (AIDS), one of the worst global pandemic. The virus infects human CD4 T cells and macrophages, and causes CD4 depletion. HIV enters target cells through the binding of the viral envelope glycoprotein to CD4 and the chemokine coreceptor, CXCR4 or CCR5. In particular, the CCR5-utilizing viruses predominate in the blood during the disease course. CCR5 is expressed on the surface of various immune cells including macrophages, monocytes, microglia, dendric cells, and active memory CD4 T cells. In the human population, the CCR5 genomic mutation, CCR5Δ32, is associated with relative resistance to HIV. These findings paved the way for the discovery and development of CCR5 inhibitors to block HIV transmission and replication. Maraviroc, discovered as a CCR5 antagonist, is the only CCR5 inhibitor that has been approved by both US FDA and the European Medicines Agency (EMA) for treating HIV/AIDS patients. In this review, we summarize the medicinal chemistry and clinical studies of Maraviroc. PMID:25159165

Concordance of CCR5 genotypes that influence cell-mediated immunity and HIV-1 disease progression rates.

PubMed

Catano, Gabriel; Chykarenko, Zoya A; Mangano, Andrea; Anaya, J-M; He, Weijing; Smith, Alison; Bologna, Rosa; Sen, Luisa; Clark, Robert A; Lloyd, Andrew; Shostakovich-Koretskaya, Ludmila; Ahuja, Sunil K

2011-01-15

We used cutaneous delayed-type hypersensitivity responses, a powerful in vivo measure of cell-mediated immunity, to evaluate the relationships among cell-mediated immunity, AIDS, and polymorphisms in CCR5, the HIV-1 coreceptor. There was high concordance between CCR5 polymorphisms and haplotype pairs that influenced delayed-type hypersensitivity responses in healthy persons and HIV disease progression. In the cohorts examined, CCR5 genotypes containing -2459G/G (HHA/HHA, HHA/HHC, HHC/HHC) or -2459A/A (HHE/HHE) associated with salutary or detrimental delayed-type hypersensitivity and AIDS phenotypes, respectively. Accordingly, the CCR5-Δ32 allele, when paired with non-Δ32-bearing haplotypes that correlate with low (HHA, HHC) versus high (HHE) CCR5 transcriptional activity, associates with disease retardation or acceleration, respectively. Thus, the associations of CCR5-Δ32 heterozygosity partly reflect the effect of the non-▵32 haplotype in a background of CCR5 haploinsufficiency. The correlations of increased delayed-type hypersensitivity with -2459G/G-containing CCR5 genotypes, reduced CCR5 expression, decreased viral replication, and disease retardation suggest that CCR5 may influence HIV infection and AIDS, at least in part, through effects on cell-mediated immunity.

The chemokine receptor CXCR3 and its splice variant are expressed in human airway epithelial cells.

PubMed

Kelsen, Steven G; Aksoy, Mark O; Yang, Yi; Shahabuddin, Syed; Litvin, Judith; Safadi, Fayez; Rogers, Thomas J

2004-09-01

Activation of the chemokine receptor CXCR3 by its cognate ligands induces several differentiated cellular responses important to the growth and migration of a variety of hematopoietic and structural cells. In the human respiratory tract, human airway epithelial cells (HAEC) release the CXCR3 ligands Mig/CXCL9, IP-10/CXCL10, and I-TAC/CXCL11. Simultaneous expression of CXCR3 by HAEC would have important implications for the processes of airway inflammation and repair. Accordingly, in the present study we sought to determine whether HAEC also express the classic CXCR3 chemokine receptor CXCR3-A and its splice variant CXCR3-B and hence may respond in autocrine fashion to its ligands. We found that cultured HAEC (16-HBE and tracheocytes) constitutively expressed CXCR3 mRNA and protein. CXCR3 mRNA levels assessed by expression array were approximately 35% of beta-actin expression. In contrast, CCR3, CCR4, CCR5, CCR8, and CX3CR1 were <5% beta-actin. Both CXCR3-A and -B were expressed. Furthermore, tracheocytes freshly harvested by bronchoscopy stained positively for CXCR3 by immunofluorescence microscopy, and 68% of cytokeratin-positive tracheocytes (i.e., the epithelial cell population) were positive for CXCR3 by flow cytometry. In 16-HBE cells, CXCR3 receptor density was approximately 78,000 receptors/cell when assessed by competitive displacement of 125I-labeled IP-10/CXCL10. Finally, CXCR3 ligands induced chemotactic responses and actin reorganization in 16-HBE cells. These findings indicate constitutive expression by HAEC of a functional CXC chemokine receptor, CXCR3. Our data suggest the possibility that autocrine activation of CXCR3 expressed by HAEC may contribute to airway inflammation and remodeling in obstructive lung disease by regulating HAEC migration.

Role of CCR5 and its ligands in the control of vascular inflammation and leukocyte recruitment required for acute excitotoxic seizure induction and neural damage

PubMed Central

Louboutin, Jean-Pierre; Chekmasova, Alena; Marusich, Elena; Agrawal, Lokesh; Strayer, David S.

2011-01-01

Chemokines may play a role in leukocyte migration across the blood-brain barrier (BBB) during neuroinflammation and other neuropathological processes, such as epilepsy. We investigated the role of the chemokine receptor CCR5 in seizures. We used a rat model based on intraperitoneal kainic acid (KA) administration. Four months before KA injection, adult rats were given femoral intramarrow inoculations of SV (RNAiR5-RevM10.AU1), which carries an interfering RNA (RNAi) against CCR5, plus a marker epitope (AU1), or its monofunctional RNAi-carrying homologue, SV(RNAiR5). This treatment lowered expression of CCR5 in circulating cells. In control rats, seizures induced elevated expression of CCR5 ligands MIP-1α and RANTES in the microvasculature, increased BBB leakage and CCR5+ cells, as well as neuronal loss, inflammation, and gliosis in the hippocampi. Animals given either the bifunctional or the monofunctional vector were largely protected from KA-induced seizures, neuroinflammation, BBB damage, and neuron loss. Brain CCR5 mRNA was reduced. Rats receiving RNAiR5-bearing vectors showed far greater repair responses: increased neuronal proliferation, and decreased production of MIP-1α and RANTES. Controls received unrelated SV(BUGT) vectors. Decrease in CCR5 in circulating cells strongly protected from excitotoxin-induced seizures, BBB leakage, CNS injury, and inflammation, and facilitated neurogenic repair.—Louboutin, J.-P., Chekmasova, A., Marusich, E., Agrawal, L., Strayer, D. S. Role of CCR5 and its ligands in the control of vascular inflammation and leukocyte recruitment required for acute excitotoxic seizure induction and neural damage. PMID:20940264

Dynamics of CCR5 Expression by CD4+ T Cells in Lymphoid Tissues during Simian Immunodeficiency Virus Infection

PubMed Central

Veazey, Ronald S.; Mansfield, Keith G.; Tham, Irene C.; Carville, Angela C.; Shvetz, Daniel E.; Forand, Amy E.; Lackner, Andrew A.

2000-01-01

Early viral replication and profound CD4+ T-cell depletion occur preferentially in intestinal tissues of macaques infected with simian immunodeficiency virus (SIV). Here we show that a much higher percentage of CD4+ T cells in the intestine express CCR5 compared with those found in the peripheral blood, spleen, or lymph nodes. In addition, the selectivity and extent of the CD4+ T-cell loss in SIV infection may depend upon these cells coexpressing CCR5 and having a “memory” phenotype (CD45RA−). Following intravenous infection with SIVmac251, memory CD4+ CCR5+ T cells were selectively eliminated within 14 days in all major lymphoid tissues (intestine, spleen, and lymph nodes). However, the effect on CD4+ T-cell numbers was most profound in the intestine, where cells of this phenotype predominate. The CD4+ T cells that remain after 14 days of infection lacked CCR5 and/or were naive (CD45RA+). Furthermore, when animals in the terminal stages of SIV infection (with AIDS) were examined, virtually no CCR5-expressing CD4+ T cells were found in lymphoid tissues, and all of the remaining CD4+ T cells were naive and coexpressed CXCR4. These findings suggest that chemokine receptor usage determines which cells are targeted for SIV infection and elimination in vivo. PMID:11069995

Involvement of both the V2 and V3 Regions of the CCR5-Tropic Human Immunodeficiency Virus Type 1 Envelope in Reduced Sensitivity to Macrophage Inflammatory Protein 1α

PubMed Central

Maeda, Yosuke; Foda, Mohamed; Matsushita, Shuzo; Harada, Shinji

2000-01-01

To determine whether C-C chemokines play an important role in the phenotype switch of human immunodeficiency virus (HIV) from CCR5 to CXCR4 usage during the course of an infection in vivo, macrophage inflammatory protein (MIP)-1α-resistant variants were isolated from CCR5-tropic (R5) HIV-1 in vitro. The selected variants displayed reduced sensitivities to MIP-1α (fourfold) through CCR5-expressing CD4-HeLa/long terminal repeat–β-galactosidase (MAGI/CCR5) cells. The variants were also resistant to other natural ligands for CCR5, namely, MIP-1β (>4-fold) and RANTES (regulated upon activation, normal T-cell expressed and secreted) (6-fold). The env sequence analyses revealed that the variants had amino acid substitutions in V2 (valine 166 to methionine) and V3 (serine 303 to glycine), although the same V3 substitution appeared in virus passaged without MIP-1α. A single-round replication assay using a luciferase reporter HIV-1 strain pseudotyped with mutant envelopes confirmed that mutations in both V2 and V3 were necessary to confer the reduced sensitivity to MIP-1α, MIP-1β, and RANTES. However, the double mutant did not switch its chemokine receptor usage from CCR5 to CXCR4, indicating the altered recognition of CCR5 by this mutant. These results indicated that V2 combined with the V3 region of the CCR5-tropic HIV-1 envelope modulates the sensitivity of HIV-1 to C-C chemokines without altering the ability to use chemokine receptors. PMID:10644351

Involvement of both the V2 and V3 regions of the CCR5-tropic human immunodeficiency virus type 1 envelope in reduced sensitivity to macrophage inflammatory protein 1alpha.

PubMed

Maeda, Y; Foda, M; Matsushita, S; Harada, S

2000-02-01

To determine whether C-C chemokines play an important role in the phenotype switch of human immunodeficiency virus (HIV) from CCR5 to CXCR4 usage during the course of an infection in vivo, macrophage inflammatory protein (MIP)-1alpha-resistant variants were isolated from CCR5-tropic (R5) HIV-1 in vitro. The selected variants displayed reduced sensitivities to MIP-1alpha (fourfold) through CCR5-expressing CD4-HeLa/long terminal repeat-beta-galactosidase (MAGI/CCR5) cells. The variants were also resistant to other natural ligands for CCR5, namely, MIP-1beta (>4-fold) and RANTES (regulated upon activation, normal T-cell expressed and secreted) (6-fold). The env sequence analyses revealed that the variants had amino acid substitutions in V2 (valine 166 to methionine) and V3 (serine 303 to glycine), although the same V3 substitution appeared in virus passaged without MIP-1alpha. A single-round replication assay using a luciferase reporter HIV-1 strain pseudotyped with mutant envelopes confirmed that mutations in both V2 and V3 were necessary to confer the reduced sensitivity to MIP-1alpha, MIP-1beta, and RANTES. However, the double mutant did not switch its chemokine receptor usage from CCR5 to CXCR4, indicating the altered recognition of CCR5 by this mutant. These results indicated that V2 combined with the V3 region of the CCR5-tropic HIV-1 envelope modulates the sensitivity of HIV-1 to C-C chemokines without altering the ability to use chemokine receptors.

Changes in gene expression induced by histamine, fexofenadine and osthole: Expression of histamine H1 receptor, COX-2, NF-κB, CCR1, chemokine CCL5/RANTES and interleukin-1β in PBMC allergic and non-allergic patients.

PubMed

Kordulewska, Natalia Karolina; Kostyra, Elżbieta; Cieślińska, Anna; Matysiewicz, Michał; Fiedorowicz, Ewa; Sienkiewicz-Szłapka, Edyta

2017-03-01

Fexofenadine (FXF) is a third-generation antihistamine drug and osthole is assumed as a natural antihistamine alternative. This paper compares results of histamine, FXF and osthole impact on HRH-1, COX-2, NF-κB-p50, CCR1 mRNA expression. We also measured mRNA expression of IL-1β and CCL5/RANTES in incubated peripheral blood mononuclear cells (PBMC) to compared how histamine, FXF and osthole had influence on expression level and interacts on product secretion. The purpose was to investigate expression pattern in asthma PBMC. The cultures were treated 72h with FXF and osthole. We measured mRNA expression of histamine HRH-1, COX-2, NF-κB-p50, CCR1, IL-1β and CCL5/RANTES with Real-Time PCR (RT-PCR). The present study suggest that osthole may be a potential inhibitor of histamine H 1 receptor activity. We also demonstrated that cells cultured with histamine increase COX-2 mRNA expression and osthole reduce it. Allergy remains one of the most common chronic diseases in Europe and it is rapidly approaching epidemic proportions; with current predictions estimating that the number of allergy-afflicted will equal the healthy population by 2020. It is therefore paramount to find new pharmaceuticals which successfully combat allergic disease. Copyright © 2016 Elsevier GmbH. All rights reserved.

Efficient modification of CCR5 in primary human hematopoietic cells using a megaTAL nuclease and AAV donor template.

PubMed

Sather, Blythe D; Romano Ibarra, Guillermo S; Sommer, Karen; Curinga, Gabrielle; Hale, Malika; Khan, Iram F; Singh, Swati; Song, Yumei; Gwiazda, Kamila; Sahni, Jaya; Jarjour, Jordan; Astrakhan, Alexander; Wagner, Thor A; Scharenberg, Andrew M; Rawlings, David J

2015-09-30

Genetic mutations or engineered nucleases that disrupt the HIV co-receptor CCR5 block HIV infection of CD4(+) T cells. These findings have motivated the engineering of CCR5-specific nucleases for application as HIV therapies. The efficacy of this approach relies on efficient biallelic disruption of CCR5, and the ability to efficiently target sequences that confer HIV resistance to the CCR5 locus has the potential to further improve clinical outcomes. We used RNA-based nuclease expression paired with adeno-associated virus (AAV)-mediated delivery of a CCR5-targeting donor template to achieve highly efficient targeted recombination in primary human T cells. This method consistently achieved 8 to 60% rates of homology-directed recombination into the CCR5 locus in T cells, with over 80% of cells modified with an MND-GFP expression cassette exhibiting biallelic modification. MND-GFP-modified T cells maintained a diverse repertoire and engrafted in immune-deficient mice as efficiently as unmodified cells. Using this method, we integrated sequences coding chimeric antigen receptors (CARs) into the CCR5 locus, and the resulting targeted CAR T cells exhibited antitumor or anti-HIV activity. Alternatively, we introduced the C46 HIV fusion inhibitor, generating T cell populations with high rates of biallelic CCR5 disruption paired with potential protection from HIV with CXCR4 co-receptor tropism. Finally, this protocol was applied to adult human mobilized CD34(+) cells, resulting in 15 to 20% homologous gene targeting. Our results demonstrate that high-efficiency targeted integration is feasible in primary human hematopoietic cells and highlight the potential of gene editing to engineer T cell products with myriad functional properties. Copyright © 2015, American Association for the Advancement of Science.

Eotaxin-1 promotes prostate cancer cell invasion via activation of the CCR3-ERK pathway and upregulation of MMP-3 expression.

PubMed

Zhu, Feng; Liu, Pei; Li, Jun; Zhang, Yan

2014-05-01

Chemokines have been reported to play crucial roles in tumor progression. Eotaxin-1 (CCL11), a member of the CC chemokine family, is elevated in many types of human cancer. Here, to reveal the molecular mechanisms of eotaxin-1 in prostate cancer cell invasion, the expression of eotaxin-1 receptors [CC chemokine receptor (CCR)2, CCR3 and CCR5] were silenced by small interfering RNA (siRNA). The ERK pathway was inhibited by the specific MEK inhibitor U0126. The role of eotaxin-1 and the CCR3-ERK pathway in prostate cancer cell invasion was assessed by invasion and migration assays. MMP-3 expression was detected by real-time PCR and ELISA assay. The results demonstrated that eotaxin-1 promoted the invasion and migration of DU-145 cells, and increased ERK1/2 activation and MMP-3 expression. Knockdown of CCR3 inhibited the invasion and migration of prostate cancer cells, and attenuated the eotaxin-1-induced ERK1/2 activation and MMP-3 expression. Furthermore, inactivation of the ERK pathway suppressed the eotaxin‑1-promoted invasion and migration, and decreased MMP-3 expression in the prostate cancer cells. Together, the present study suggests that eotaxin-1 increases MMP-3 expression via the CCR3-ERK pathway, thereby promoting prostate cancer cell invasion and migration. Thus, therapies that block eotaxin-1 and CCR3 may be effective interventions for prostate cancer.

CCR5+ CD8 T-cell levels and monocyte activation precede the onset of acute coronary syndrome in HIV-infected patients on antiretroviral therapy.

PubMed

Tarancon-Diez, Laura; De Pablo-Bernal, Rebeca S; Álvarez-Rios, Ana I; Rosado-Sánchez, Isaac; Dominguez-Molina, Beatriz; Genebat, Miguel; Pacheco, Yolanda M; Jiménez, José Luis; Muñoz-Fernández, M Ángeles; Ruiz-Mateos, Ezequiel; Leal, Manuel

2017-06-02

Acute coronary syndrome (ACS) is nowadays one of the leading causes of morbid-mortality in HIV-infected population, but innate and adaptive immune mechanisms preceding this event are unknown. In this work we comprehensively and longitudinally observed, by multiparametric flow cytometry and following a case-control design, increased CCR5 + CD8 + T-cells levels and monocytes expressing activation and adhesion markers in HIV-infected patients who are going to suffer ACS. In addition, we found direct associations between activated CD8 + T-cells and myeloid cells that were only statistically significant in the group of patients with ACS and in the follow up time point just before the ACS. Our data highlight the important role of CCR5 in the onset of ACS and suggest this receptor as a marker of cardiovascular risk and potential therapeutic target to prevent the development of such non-AIDS-related event in HIV-infected patients.

Effector stage CC chemokine receptor-1 selective antagonism reduces multiple sclerosis-like rat disease.

PubMed

Eltayeb, Sana; Sunnemark, Dan; Berg, Anna-Lena; Nordvall, Gunnar; Malmberg, Asa; Lassmann, Hans; Wallström, Erik; Olsson, Tomas; Ericsson-Dahlstrand, Anders

2003-09-01

We have studied the role of the chemokine receptor CCR1 during the effector stage of myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis in DA rats. In situ hybridization histochemistry revealed local production of the CCR1 ligands CCL3 (MIP-1 alpha) and CCL5 (RANTES), as well as large numbers of CCR1 and CCR5 expressing cells within inflammatory brain lesions. A low-molecular weight CCR1 selective antagonist potently abrogated both clinical and histopathological disease signs during a 5-day treatment period, without signs of peripheral immune compromise. Thus, we demonstrate therapeutic targeting of CCR1-dependent leukocyte recruitment to the central nervous system in a multiple sclerosis (MS)-like rat model.

CCR2 deficiency leads to increased eosinophils, alternative macrophage activation, and type 2 cytokine expression in adipose tissue.

PubMed

Bolus, W Reid; Gutierrez, Dario A; Kennedy, Arion J; Anderson-Baucum, Emily K; Hasty, Alyssa H

2015-10-01

Adipose tissue (AT) inflammation during obesity is mediated by immune cells and closely correlates with systemic insulin resistance. In lean AT, eosinophils are present in low but significant numbers and capable of promoting alternative macrophage activation in an IL-4/IL-13-dependent manner. In WT mice, obesity causes the proportion of AT eosinophils to decline, concomitant with inflammation and classical activation of AT macrophages. In this study, we show that CCR2 deficiency leads to increased eosinophil accumulation in AT. Furthermore, in contrast to WT mice, the increase in eosinophils in CCR2(-/-) AT is sustained and even amplified during obesity. Interestingly, a significant portion of eosinophils is found in CLSs in AT of obese CCR2(-/-) mice, which is the first time eosinophils have been shown to localize to these inflammatory hot spots. CCR2(-/-) bone marrow precursors displayed increased expression of various key eosinophil genes during in vitro differentiation to eosinophils, suggesting a potentially altered eosinophil phenotype in the absence of CCR2. In addition, the proportion of eosinophils in AT positively correlated with local expression of Il5, a potent eosinophil stimulator. The increase in eosinophils in CCR2(-/-) mice was detected in all white fat pads analyzed and in the peritoneal cavity but not in bone marrow, blood, spleen, or liver. In AT of CCR2(-/-) mice, an increased eosinophil number positively correlated with M2-like macrophages, expression of the Treg marker Foxp3, and type 2 cytokines, Il4, Il5, and Il13. This is the first study to link CCR2 function with regulation of AT eosinophil accumulation. © Society for Leukocyte Biology.

CCR2 deficiency leads to increased eosinophils, alternative macrophage activation, and type 2 cytokine expression in adipose tissue

PubMed Central

Bolus, W. Reid; Gutierrez, Dario A.; Kennedy, Arion J.; Anderson-Baucum, Emily K.; Hasty, Alyssa H.

2015-01-01

Adipose tissue (AT) inflammation during obesity is mediated by immune cells and closely correlates with systemic insulin resistance. In lean AT, eosinophils are present in low but significant numbers and capable of promoting alternative macrophage activation in an IL-4/IL-13-dependent manner. In WT mice, obesity causes the proportion of AT eosinophils to decline, concomitant with inflammation and classical activation of AT macrophages. In this study, we show that CCR2 deficiency leads to increased eosinophil accumulation in AT. Furthermore, in contrast to WT mice, the increase in eosinophils in CCR2−/− AT is sustained and even amplified during obesity. Interestingly, a significant portion of eosinophils is found in CLSs in AT of obese CCR2−/− mice, which is the first time eosinophils have been shown to localize to these inflammatory hot spots. CCR2−/− bone marrow precursors displayed increased expression of various key eosinophil genes during in vitro differentiation to eosinophils, suggesting a potentially altered eosinophil phenotype in the absence of CCR2. In addition, the proportion of eosinophils in AT positively correlated with local expression of Il5, a potent eosinophil stimulator. The increase in eosinophils in CCR2−/− mice was detected in all white fat pads analyzed and in the peritoneal cavity but not in bone marrow, blood, spleen, or liver. In AT of CCR2−/− mice, an increased eosinophil number positively correlated with M2-like macrophages, expression of the Treg marker Foxp3, and type 2 cytokines, Il4, Il5, and Il13. This is the first study to link CCR2 function with regulation of AT eosinophil accumulation. PMID:25934927

Substance P (SP) enhances CCL5-induced chemotaxis and intracellular signaling in human monocytes, which express the truncated neurokinin-1 receptor (NK1R)

PubMed Central

Chernova, Irene; Lai, Jian-Ping; Li, Haiying; Schwartz, Lynnae; Tuluc, Florin; Korchak, Helen M.; Douglas, Steven D.; Kilpatrick, Laurie E.

2009-01-01

Substance P (SP) is a potent modulator of monocyte/macrophage function. The SP-preferring receptor neurokinin-1 receptor (NK1R) has two forms: a full-length NK1R (NK1R-F) isoform and a truncated NK1R (NK1R-T) isoform, which lacks the terminal cytoplasmic 96-aa residues. The distribution of these receptor isoforms in human monocytes is not known. We previously identified an interaction among SP, NK1R, and HIV viral strains that use the chemokine receptor CCR5 as a coreceptor, suggesting crosstalk between NK1R and CCR5. The purpose of this study was to determine which form(s) of NK1R are expressed in human peripheral blood monocytes and to determine whether SP affects proinflammatory cellular responses mediated through the CCR5 receptor. Human peripheral blood monocytes were found to express NK1R-T but not NK1R-F. SP interactions with NK1R-T did not mobilize calcium (Ca2+), but SP mobilized Ca2+ when the NK1R-F was transfected into monocytes. However, the NK1R-T was functional in monocytes, as SP enhanced the CCR5 ligand CCL5-elicited Ca2+ mobilization, a response inhibited by the NK1R antagonist aprepitant. SP interactions with the NK1R-T also enhanced CCL5-mediated chemotaxis, which was ERK1/2-dependent. NK1R-T selectively activated ERK2 but increased ERK1 and ERK2 activation by CCL5. Activation of NK1R-T elicited serine phosphorylation of CCR5, indicating that crosstalk between CCL5 and SP may occur at the level of the receptor. Thus, NK1R-T is functional in human monocytes and activates select signaling pathways, and the NK1R-T-mediated enhancement of CCL5 responses does not require the NK1R terminal cytoplasmic domain. PMID:18835883

Dengue virus requires the CC-chemokine receptor CCR5 for replication and infection development.

PubMed

Marques, Rafael E; Guabiraba, Rodrigo; Del Sarto, Juliana L; Rocha, Rebeca F; Queiroz, Ana Luiza; Cisalpino, Daniel; Marques, Pedro E; Pacca, Carolina C; Fagundes, Caio T; Menezes, Gustavo B; Nogueira, Maurício L; Souza, Danielle G; Teixeira, Mauro M

2015-08-01

Dengue is a mosquito-borne disease that affects millions of people worldwide yearly. Currently, there is no vaccine or specific treatment available. Further investigation on dengue pathogenesis is required to better understand the disease and to identify potential therapeutic targets. The chemokine system has been implicated in dengue pathogenesis, although the specific role of chemokines and their receptors remains elusive. Here we describe the role of the CC-chemokine receptor CCR5 in Dengue virus (DENV-2) infection. In vitro experiments showed that CCR5 is a host factor required for DENV-2 replication in human and mouse macrophages. DENV-2 infection induces the expression of CCR5 ligands. Incubation with an antagonist prevents CCR5 activation and reduces DENV-2 positive-stranded (+) RNA inside macrophages. Using an immunocompetent mouse model of DENV-2 infection we found that CCR5(-/-) mice were resistant to lethal infection, presenting at least 100-fold reduction of viral load in target organs and significant reduction in disease severity. This phenotype was reproduced in wild-type mice treated with CCR5-blocking compounds. Therefore, CCR5 is a host factor required for DENV-2 replication and disease development. Targeting CCR5 might represent a therapeutic strategy for dengue fever. These data bring new insights on the association between viral infections and the chemokine receptor CCR5. © 2015 John Wiley & Sons Ltd.

TNF/TNFR1 signaling up-regulates CCR5 expression by CD8+ T lymphocytes and promotes heart tissue damage during Trypanosoma cruzi infection: beneficial effects of TNF-alpha blockade.

PubMed

Kroll-Palhares, Karina; Silvério, Jaline Coutinho; Silva, Andrea Alice da; Michailowsky, Vladimir; Marino, Ana Paula; Silva, Neide Maria; Carvalho, Cristiano Marcelo Espinola; Pinto, Luzia Maria de Oliveira; Gazzinelli, Ricardo Tostes; Lannes-Vieira, Joseli

2008-06-01

In Chagas disease, understanding how the immune response controls parasite growth but also leads to heart damage may provide insight into the design of new therapeutic strategies. Tumor necrosis factor-alpha (TNF-alpha) is important for resistance to acute Trypanosoma cruzi infection; however, in patients suffering from chronic T. cruzi infection, plasma TNF-alpha levels correlate with cardiomyopathy. Recent data suggest that CD8-enriched chagasic myocarditis formation involves CCR1/CCR5-mediated cell migration. Herein, the contribution of TNF-alpha, especially signaling through the receptor TNFR1/p55, to the pathophysiology of T. cruzi infection was evaluated with a focus on the development of myocarditis and heart dysfunction. Colombian strain-infected C57BL/6 mice had increased frequencies of TNFR1/p55+ and TNF-alpha+ splenocytes. Although TNFR1-/- mice exhibited reduced myocarditis in the absence of parasite burden, they succumbed to acute infection. Similar to C57BL/6 mice, Benznidazole-treated TNFR1-/- mice survived acute infection. In TNFR1-/- mice, reduced CD8-enriched myocarditis was associated with defective activation of CD44+CD62Llow/- and CCR5+ CD8+ lymphocytes. Also, anti-TNF-alpha treatment reduced the frequency of CD8+CCR5+ circulating cells and myocarditis, though parasite load was unaltered in infected C3H/HeJ mice. TNFR1-/- and anti-TNF-alpha-treated infected mice showed regular expression of connexin-43 and reduced fibronectin deposition, respectively. Furthermore, anti-TNF-alpha treatment resulted in lower levels of CK-MB, a cardiomyocyte lesion marker. Our results suggest that TNF/TNFR1 signaling promotes CD8-enriched myocarditis formation and heart tissue damage, implicating the TNF/TNFR1 signaling pathway as a potential therapeutic target for control of T. cruzi-elicited cardiomyopathy.

CCL5/CCR5 axis induces vascular endothelial growth factor-mediated tumor angiogenesis in human osteosarcoma microenvironment.

PubMed

Wang, Shih-Wei; Liu, Shih-Chia; Sun, Hui-Lung; Huang, Te-Yang; Chan, Chia-Han; Yang, Chen-Yu; Yeh, Hung-I; Huang, Yuan-Li; Chou, Wen-Yi; Lin, Yu-Min; Tang, Chih-Hsin

2015-01-01

Chemokines modulate angiogenesis and metastasis that dictate cancer development in tumor microenvironment. Osteosarcoma is the most frequent bone tumor and is characterized by a high metastatic potential. Chemokine CCL5 (previously called RANTES) has been reported to facilitate tumor progression and metastasis. However, the crosstalk between chemokine CCL5 and vascular endothelial growth factor (VEGF) as well as tumor angiogenesis in human osteosarcoma microenvironment has not been well explored. In this study, we found that CCL5 increased VEGF expression and production in human osteosarcoma cells. The conditioned medium (CM) from CCL5-treated osteosarcoma cells significantly induced tube formation and migration of human endothelial progenitor cells. Pretreatment of cells with CCR5 antibody or transfection with CCR5 specific siRNA blocked CCL5-induced VEGF expression and angiogenesis. CCL5/CCR5 axis demonstrably activated protein kinase Cδ (PKCδ), c-Src and hypoxia-inducible factor-1 alpha (HIF-1α) signaling cascades to induce VEGF-dependent angiogenesis. Furthermore, knockdown of CCL5 suppressed VEGF expression and attenuated osteosarcoma CM-induced angiogenesis in vitro and in vivo. CCL5 knockdown dramatically abolished tumor growth and angiogenesis in the osteosarcoma xenograft animal model. Importantly, we demonstrated that the expression of CCL5 and VEGF were correlated with tumor stage according the immunohistochemistry analysis of human osteosarcoma tissues. Taken together, our findings provide evidence that CCL5/CCR5 axis promotes VEGF-dependent tumor angiogenesis in human osteosarcoma microenvironment through PKCδ/c-Src/HIF-1α signaling pathway. CCL5 may represent a potential therapeutic target against human osteosarcoma. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Loss of a single N-linked glycan allows CD4-independent human immunodeficiency virus type 1 infection by altering the position of the gp120 V1/V2 variable loops.

PubMed

Kolchinsky, P; Kiprilov, E; Bartley, P; Rubinstein, R; Sodroski, J

2001-04-01

The gp120 envelope glycoprotein of primary human immunodeficiency virus type 1 (HIV-1) promotes virus entry by sequentially binding CD4 and the CCR5 chemokine receptor on the target cell. Previously, we adapted a primary HIV-1 isolate, ADA, to replicate in CD4-negative canine cells expressing human CCR5. The gp120 changes responsible for CD4-independent replication were limited to the V2 loop-V1/V2 stem. Here we show that elimination of a single glycosylation site at asparagine 197 in the V1/V2 stem is sufficient for CD4-independent gp120 binding to CCR5 and for HIV-1 entry into CD4-negative cells expressing CCR5. Deletion of the V1/V2 loops also allowed CD4-independent viral entry and gp120 binding to CCR5. The binding of the wild-type ADA gp120 to CCR5 was less dependent upon CD4 at 4 degrees C than at 37 degrees C. In the absence of the V1/V2 loops, neither removal of the N-linked carbohydrate at asparagine 197 nor lowering of the temperature increased the CD4-independent phenotypes. A CCR5-binding conformation of gp120, achieved by CD4 interaction or by modification of temperature, glycosylation, or variable loops, was preferentially recognized by the monoclonal antibody 48d. These results suggest that the CCR5-binding region of gp120 is occluded by the V1/V2 variable loops, the position of which can be modulated by temperature, CD4 binding, or an N-linked glycan in the V1/V2 stem.

CXC chemokine ligand 4 (CXCL4) down-regulates CC chemokine receptor expression on human monocytes.

PubMed

Schwartzkopff, Franziska; Petersen, Frank; Grimm, Tobias Alexander; Brandt, Ernst

2012-02-01

During acute inflammation, monocytes are essential in abolishing invading micro-organisms and encouraging wound healing. Recruitment by CC chemokines is an important step in targeting monocytes to the inflamed tissue. However, cell surface expression of the corresponding chemokine receptors is subject to regulation by various endogenous stimuli which so far have not been comprehensively identified. We report that the platelet-derived CXC chemokine ligand 4 (CXCL4), a known activator of human monocytes, induces down-regulation of CC chemokine receptors (CCR) 1, -2, and -5, resulting in drastic impairment of monocyte chemotactic migration towards cognate CC chemokine ligands (CCL) for these receptors. Interestingly, CXCL4-mediated down-regulation of CCR1, CCR2 and CCR5 was strongly dependent on the chemokine's ability to stimulate autocrine/paracrine release of TNF-α. In turn, TNF-α induced the secretion CCL3 and CCL4, two chemokines selective for CCR1 and CCR5, while the secretion of CCR2-ligand CCL2 was TNF-α-independent. Culture supernatants of CXCL4-stimulated monocytes as well as chemokine-enriched preparations thereof reproduced CXCL4-induced CCR down-regulation. In conclusion, CXCL4 may act as a selective regulator of monocyte migration by stimulating the release of autocrine, receptor-desensitizing chemokine ligands. Our results stress a co-ordinating role for CXCL4 in the cross-talk between platelets and monocytes during early inflammation.

Tumor cell apoptosis induces tumor-specific immunity in a CC chemokine receptor 1- and 5-dependent manner in mice.

PubMed

Iida, Noriho; Nakamoto, Yasunari; Baba, Tomohisa; Kakinoki, Kaheita; Li, Ying-Yi; Wu, Yu; Matsushima, Kouji; Kaneko, Shuichi; Mukaida, Naofumi

2008-10-01

The first step in the generation of tumor immunity is the migration of dendritic cells (DCs) to the apoptotic tumor, which is presumed to be mediated by various chemokines. To clarify the roles of chemokines, we induced apoptosis using suicide gene therapy and investigated the immune responses following tumor apoptosis. We injected mice with a murine hepatoma cell line, BNL 1ME A.7R.1 (BNL), transfected with HSV-thymidine kinase (tk) gene and then treated the animals with ganciclovir (GCV). GCV treatment induced massive tumor cell apoptosis accompanied with intratumoral DC infiltration. Tumor-infiltrating DCs expressed chemokine receptors CCR1 and CCR5, and T cells and macrophages expressed CCL3, a ligand for CCR1 and CCR5. Moreover, tumor apoptosis increased the numbers of DCs migrating into the draining lymph nodes and eventually generated a specific cytotoxic cell population against BNL cells. Although GCV completely eradicated HSV-tk-transfected BNL cells in CCR1-, CCR5-, or CCL3-deficient mice, intratumoral and intranodal DC infiltration and the subsequent cytotoxicity generation were attenuated in these mice. When parental cells were injected again after complete eradication of primary tumors by GCV treatment, the wild-type mice completely rejected the rechallenged cells, but the deficient mice exhibited impairment in rejection. Thus, we provide definitive evidence indicating that CCR1 and CCR5 and their ligand CCL3 play a crucial role in the regulation of intratumoral DC accumulation and the subsequent establishment of tumor immunity following induction of tumor apoptosis by suicide genes.

The chemokines CCR1 and CCRL2 have a role in colorectal cancer liver metastasis.

PubMed

Akram, Israa G; Georges, Rania; Hielscher, Thomas; Adwan, Hassan; Berger, Martin R

2016-02-01

C-C chemokine receptor type 1 (CCR1) and chemokine C-C motif receptor-like 2 (CCRL2) have not yet been sufficiently investigated for their role in colorectal cancer (CRC). Here, we investigated their expression in rat and human CRC samples, their modulation of expression in a rat liver metastasis model, as well as the effects on cellular properties resulting from their knockdown. One rat and five human colorectal cancer cell lines were used. CC531 rat colorectal cells were injected via the portal vein into rats and re-isolated from rat livers after defined periods. Following mRNA isolation, the gene expression was investigated by microarray. In addition, all cell lines were screened for mRNA expression of CCR1 and CCRL2 by reverse transcription polymerase chain reaction (RT-PCR). Cell lines with detectable expression were used for knockdown experiments; and the respective influence was determined on the cells' proliferation, scratch closure, and colony formation. Finally, specimens from the primaries of 50 patients with CRC were monitored by quantitative RT-PCR for CCR1 and CCRL2 expression levels. The microarray studies showed peak increases of CCR1 and CCRL2 in the early phase of liver colonization. Knockdown was sufficient at mRNA but only moderate at protein levels and resulted in modest but significant inhibition of proliferation (p < 0.05), scratch closure, and colony formation (p < 0.05). All human CRC samples were positive for CCR1 and CCRL2 and showed a significant pairwise correlation (p < 0.0004), but there was no correlation with tumor stage or age of patients. In summary, the data point to an important role of CCR1 and CCRL2 under conditions of organ colonization and both chemokine receptors qualify as targets of treatment during early colorectal cancer liver metastasis.

A Cell Line-Based Neutralization Assay for Primary Human Immunodeficiency Virus Type 1 Isolates That Use either the CCR5 or the CXCR4 Coreceptor

PubMed Central

Trkola, Alexandra; Matthews, Jamie; Gordon, Cynthia; Ketas, Tom; Moore, John P.

1999-01-01

We describe here a cell line-based assay for the evaluation of human immunodeficiency virus type 1 (HIV-1) neutralization. The assay is based on CEM.NKR cells, transfected to express the HIV-1 coreceptor CCR5 to supplement the endogenous expression of CD4 and the CXCR4 coreceptor. The resulting CEM.NKR-CCR5 cells efficiently replicate primary HIV-1 isolates of both R5 and X4 phenotypes. A comparison of the CEM.NKR-CCR5 cells with mitogen-activated peripheral blood mononuclear cells (PBMC) in neutralization assays with sera from HIV-1-infected individuals or specific anti-HIV-1 monoclonal antibodies shows that the sensitivity of HIV-1 neutralization is similar in the two cell types. The CEM.NKR-CCR5 cell assay, however, is more convenient to perform and eliminates the donor-to-donor variation in HIV-1 replication efficiency, which is one of the principal drawbacks of the PBMC-based neutralization assay. We suggest that this new assay is suitable for the general measurement of HIV-1 neutralization by antibodies. PMID:10516002

High CD4(+) T-cell surface CXCR4 density as a risk factor for R5 to X4 switch in the course of HIV-1 infection.

PubMed

Fiser, Anne-Laure; Vincent, Thierry; Brieu, Natalie; Lin, Yea-Lih; Portalès, Pierre; Mettling, Clément; Reynes, Jacques; Corbeau, Pierre

2010-12-15

For unclear reasons, about 50% of HIV-infected subjects harbour CXCR4-using (X4) viral strains in addition of CCR5-using (R5) viral strains at late stages of the disease. One hypothesis is that a low CD4(+) T-cell surface CCR5 density could facilitate the emergence of X4 strains. Alternatively, one could argue that a high CD4(+) T-cell surface CXCR4 density that is observed in individuals presenting with X4 strains, could favour R5 to X4 switch. Here, we tested both hypotheses. In vivo, we observed by quantitative flow cytometry no difference in CD4(+) T-cell surface CCR5 densities between patients with or without X4 strains. In the course of an in vitro R5 infection, the delay of emergence of X4 mutants was similar between cells expressing 2 distinct cell surface CCR5 densities, but shorter (12 ± 0 days and 21 ± 0 days, respectively, P = 0.01) in cells expressing a high surface CXCR4 density as compared with cells with a low surface CXCR4 density. These data argue for a role of CXCR4 density, but not of CCR5 density, in the emergence of X4 strains. They are reassuring concerning the risk of inducing an R5 to X4 switch using CCR5 antagonists to treat HIV infection.

Th1/Th17/Th22 immune response and their association with joint pain, imagenological bone loss, RANKL expression and osteoclast activity in temporomandibular joint osteoarthritis: A preliminary report.

PubMed

Monasterio, G; Castillo, F; Rojas, L; Cafferata, E A; Alvarez, C; Carvajal, P; Núñez, C; Flores, G; Díaz, W; Vernal, R

2018-05-15

It is well accepted that the presence of cytokines belonging to the Th1/Th17/Th22 axis of immuno-inflammatory response in the joint environment, such as IL-1β, IL-17 and IL-22, respectively, are associated with pathogenesis of several synovial joint degenerative disorders. During temporomandibular joint osteoarthritis (TMJ-OA), IL-1β and IL-17 have been implicated in the inflammation and resorption of sub-chondral bone; however, the role of Th22 response in the TMJ-OA pathophysiology has not been established. This study aimed to compare the expression of Th1/Th17/Th22-type cytokines, chemokines and chemokine receptors in synovial fluid samples obtained from TMJ-OA or disk displacement with reduction (DDWR) patients. In addition, it aimed to associate these levels with joint pain, imagenological signs of bone degeneration, RANKL production, osteoclastogenesis and osteoclast-induced bone resorption. Higher levels of IL-1β, IL-17 and IL-22 were expressed in TMJ-OA compared with DDWR subjects, and these increased levels significantly correlated with RANKL expression, joint pain and articular bone degeneration. Higher levels of CCR5, CCR6 and CCR7, as well as their respective ligands CCL5 and CCL20, responsible for recruitment of IL-1β, IL-17 and IL-22-producing cells, were over-expressed in TMJ-OA compared with DDWR subjects. Osteoclastogenesis and osteoclast-induced bone resorption were significantly greater in presence of synovial fluid from TMJ-OA compared with DDWR subjects. These data demonstrate that cytokines, CCLs and CCRs associated with the Th1/Th17/Th22 axis of immuno-inflammatory response are involved in TMJ-OA pathogenesis. These findings suggest that IL-22 is involved in the RANKL expression in TMJ-OA, which in turn induces differentiation of osteoclasts and subsequent resorption of sub-chondral bone. © 2018 John Wiley & Sons Ltd.

HIV turns plasmacytoid dendritic cells (pDC) into TRAIL-expressing killer pDC and down-regulates HIV coreceptors by Toll-like receptor 7-induced IFN-alpha.

PubMed

Hardy, Andrew W; Graham, David R; Shearer, Gene M; Herbeuval, Jean-Philippe

2007-10-30

Plasmacytoid dendritic cells (pDC) are key players in viral immunity and produce IFN-alpha after HIV-1 exposure, which in turn regulates TNF-related apoptosis-inducing ligand (TRAIL) expression by CD4(+) T cells. We show here that infectious and noninfectious HIV-1 virions induce activation of pDC into TRAIL-expressing IFN-producing killer pDC (IKpDC). IKpDC expressed high levels of activation markers (HLA-DR, CD80, CD83, and CD86) and the migration marker CCR7. Surprisingly, CXCR4 and CCR5 were down-regulated on IKpDC. We also show that HIV-1-induced IKpDC depended on Toll-like receptor 7 (TLR7) activation. HIV-1 or TLR7 agonistexposed IKpDC induced apoptosis of the CD4(+) T cell line SupT1 via the TRAIL pathway. Furthermore, IFN-alpha produced after HIV-induced TLR7 stimulation was responsible for TRAIL expression and the down-regulation of both CXCR4 and CCR5 by IKpDC. In contrast, activation and migration markers were not regulated by IFN-alpha. Finally, IFN-alpha increased the survival of IKpDC. We characterized a subset of pDC with a killer activity that is activated by endosomal-associated viral RNA and not by infection.

Antigen-driven C–C Chemokine-mediated HIV-1 Suppression by CD4+ T Cells from Exposed Uninfected Individuals Expressing the Wild-type CCR-5 Allele

PubMed Central

Furci, Lucinda; Scarlatti, Gabriella; Burastero, Samuele; Tambussi, Giuseppe; Colognesi, Claudia; Quillent, Caroline; Longhi, Renato; Loverro, Patrizia; Borgonovo, Barbara; Gaffi, Davide; Carrow, Emily; Malnati, Mauro; Lusso, Paolo; Siccardi, Antonio G.; Lazzarin, Adriano; Beretta, Alberto

1997-01-01

Despite repeated exposure to HIV-1, certain individuals remain persistently uninfected. Such exposed uninfected (EU) people show evidence of HIV-1–specific T cell immunity and, in rare cases, selective resistance to infection by macrophage-tropic strains of HIV-1. The latter has been associated with a 32–base pair deletion in the C–C chemokine receptor gene CCR-5, the major coreceptor of macrophage-tropic strains of HIV-1. We have undertaken an analysis of the HIV-specific T cell responses in 12 EU individuals who were either homozygous for the wild-type CCR-5 allele or heterozygous for the deletion allele (CCR-5Δ32). We have found evidence of an oligoclonal T cell response mediated by helper T cells specific for a conserved region of the HIV-1 envelope. These cells produce very high levels of C–C chemokines when stimulated by the specific antigen and suppress selectively the replication of macrophage-tropic, but not T cell–tropic, strains of HIV-1. These chemokine-producing helper cells may be part of a protective immune response that could be potentially exploited for vaccine development. PMID:9236198

Bioinformatic analysis of neurotropic HIV envelope sequences identifies polymorphisms in the gp120 bridging sheet that increase macrophage-tropism through enhanced interactions with CCR5

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mefford, Megan E., E-mail: megan_mefford@hms.harvard.edu; Kunstman, Kevin, E-mail: kunstman@northwestern.edu; Wolinsky, Steven M., E-mail: s-wolinsky@northwestern.edu

Macrophages express low levels of the CD4 receptor compared to T-cells. Macrophage-tropic HIV strains replicating in brain of untreated patients with HIV-associated dementia (HAD) express Envs that are adapted to overcome this restriction through mechanisms that are poorly understood. Here, bioinformatic analysis of env sequence datasets together with functional studies identified polymorphisms in the β3 strand of the HIV gp120 bridging sheet that increase M-tropism. D197, which results in loss of an N-glycan located near the HIV Env trimer apex, was detected in brain in some HAD patients, while position 200 was estimated to be under positive selection. D197 andmore » T/V200 increased fusion and infection of cells expressing low CD4 by enhancing gp120 binding to CCR5. These results identify polymorphisms in the HIV gp120 bridging sheet that overcome the restriction to macrophage infection imposed by low CD4 through enhanced gp120–CCR5 interactions, thereby promoting infection of brain and other macrophage-rich tissues. - Highlights: • We analyze HIV Env sequences and identify amino acids in beta 3 of the gp120 bridging sheet that enhance macrophage tropism. • These amino acids at positions 197 and 200 are present in brain of some patients with HIV-associated dementia. • D197 results in loss of a glycan near the HIV Env trimer apex, which may increase exposure of V3. • These variants may promote infection of macrophages in the brain by enhancing gp120–CCR5 interactions.« less

Synovial chemokine expression and relationship with knee symptoms in patients with meniscal tears

PubMed Central

Nair, Anjali; Gan, Justin; Bush-Joseph, Charles; Verma, Nikhil; Tetreault, Matthew W.; Saha, Kanta; Margulis, Arkady; Fogg, Louis; Scanzello, Carla R.

2015-01-01

Objective In patients with knee OA, synovitis is associated with knee pain and symptoms. We previously identified synovial mRNA expression of a set of chemokines (CCL19, IL-8, CCL5, XCL-1, CCR7) associated with synovitis in patients with meniscal tears but without radiographic OA. CCL19 and CCR7 were also associated with knee symptoms. This study sought to validate expression of these chemokines and association with knee symptoms in more typical patients presenting for meniscal arthroscopy, many who have pre-existing OA. Design Synovial biopsies and fluid (SF) were collected from patients undergoing meniscal arthroscopy. Synovial mRNA expression was measured using quantitative RT-PCR. The Knee Injury and Osteoarthritis Outcome Score (KOOS) was administered preoperatively. Regression analyses determined if associations between chemokine mRNA levels and KOOS scores were independent of other factors including radiographic OA. CCL19 in SF was measured by ELISA, and compared to patients with advanced knee OA and asymptomatic organ donors. Results 90% of patients had intra-operative evidence of early cartilage degeneration. CCL19, IL-8, CCL5, XCL1, CCR7 transcripts were detected in all patients. Synovial CCL19 mRNA levels independently correlated with KOOS Activities of Daily Living scores (95% CI [-8.071, -0.331], p= 0.036), indicating higher expression was associated with more knee-related dysfunction. SF CCL19 was detected in 7 of 10 patients, compared to 4 of 10 asymptomatic donors. Conclusion In typical patients presenting for meniscal arthroscopy, synovial CCL19 mRNA expression was associated with knee-related difficulty with activities of daily living, independent of other factors including presence of radiographic knee OA. PMID:25724256

Chemokine and Chemokine Receptor Profiles in Metastatic Salivary Adenoid Cystic Carcinoma.

PubMed

Mays, Ashley C; Feng, Xin; Browne, James D; Sullivan, Christopher A

2016-08-01

To characterize the chemokine pattern in metastatic salivary adenoid cystic carcinoma (SACC). Real-time polymerase chain reaction (RT-PCR) was used to compare chemokine and chemokine receptor gene expression in two SACC cell lines: SACC-83 and SACC-LM (lung metastasis). Chemokines and receptor genes were then screened and their expression pattern characterized in human tissue samples of non-recurrent SACC and recurrent SACC with perineural invasion. Expression of chemokine receptors C5AR1, CCR1, CCR3, CCR6, CCR7, CCR9, CCR10, CXCR4, CXCR6, CXCR7, CCRL1 and CCRL2 were higher in SACC-83 compared to SACC-LM. CCRL1, CCBP2, CMKLR1, XCR1 and CXCR2 and 6 chemokine genes (CCL13, CCL27, CXCL14, CMTM1, CMTM2, CKLF) were more highly expressed in tissues of patients without tumor recurrence/perineural invasion compared to those with tumor recurrence. CCRL1 (receptor), CCL27, CMTM1, CMTM2, and CKLF (chemokine) genes were more highly expressed in SACC-83 and human tissues of patients without tumor recurrence/perineural invasion. CCRL1, CCL27, CMTM1, CMTM2 and CKLF may play important roles in the development of tumor metastases in SACC. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Vaccination with live attenuated simian immunodeficiency virus causes dynamic changes in intestinal CD4+CCR5+ T cells

PubMed Central

2011-01-01

Background Vaccination with live attenuated SIV can protect against detectable infection with wild-type virus. We have investigated whether target cell depletion contributes to the protection observed. Following vaccination with live attenuated SIV the frequency of intestinal CD4+CCR5+ T cells, an early target of wild-type SIV infection and destruction, was determined at days 3, 7, 10, 21 and 125 post inoculation. Results In naive controls, modest frequencies of intestinal CD4+CCR5+ T cells were predominantly found within the LPL TTrM-1 and IEL TTrM-2 subsets. At day 3, LPL and IEL CD4+CCR5+ TEM cells were dramatically increased whilst less differentiated subsets were greatly reduced, consistent with activation-induced maturation. CCR5 expression remained high at day 7, although there was a shift in subset balance from CD4+CCR5+ TEM to less differentiated TTrM-2 cells. This increase in intestinal CD4+CCR5+ T cells preceded the peak of SIV RNA plasma loads measured at day 10. Greater than 65.9% depletion of intestinal CD4+CCR5+ T cells followed at day 10, but overall CD4+ T cell homeostasis was maintained by increased CD4+CCR5- T cells. At days 21 and 125, high numbers of intestinal CD4+CCR5- naive TN cells were detected concurrent with greatly increased CD4+CCR5+ LPL TTrM-2 and IEL TEM cells at day 125, yet SIV RNA plasma loads remained low. Conclusions This increase in intestinal CD4+CCR5+ T cells, following vaccination with live attenuated SIV, does not correlate with target cell depletion as a mechanism of protection. Instead, increased intestinal CD4+CCR5+ T cells may correlate with or contribute to the protection conferred by vaccination with live attenuated SIV. PMID:21291552

HIV Type 1 from a Patient with Baseline Resistance to CCR5 Antagonists Uses Drug-Bound Receptor for Entry

PubMed Central

Tilton, John C.; Amrine-Madsen, Heather; Miamidian, John L.; Kitrinos, Kathryn M.; Pfaff, Jennifer; Demarest, James F.; Ray, Neelanjana; Jeffrey, Jerry L.; Labranche, Celia C.

2010-01-01

Abstract CCR5 antagonists are a new class of antiretroviral drugs that block viral entry by disrupting interactions between the viral envelope (Env) glycoprotein and coreceptor. During the CCR100136 (EPIC) Phase IIb study of the CCR5 antagonist aplaviroc (APL) in treatment-naive individuals, a patient was identified who harbored virus strains that exhibited partial resistance to APL at the time of virologic failure. Retrospectively, it was found that APL resistance was present at baseline as well. To investigate the mechanism of APL resistance in this patient, we cloned HIV-1 env genes from plasma obtained at baseline and after virologic failure. Approximately 85% of cloned Envs were functional, and all exhibited partial resistance to APL. All Envs were R5-tropic, were partially resistant to other CCR5 antagonists including maraviroc on cells with high CCR5 expression, but remained sensitive to the fusion inhibitor enfuvirtide. Competition studies with natural CCR5 ligands revealed that the mechanism of drug resistance entailed the use of the drug-bound conformation of CCR5 by the Env proteins obtained from this individual. The degree of drug resistance varied between Env clones, and also varied depending on the cell line used or the donor from whom the primary T cells were obtained. Thus, both virus and host factors contribute to CCR5 antagonist resistance. This study shows that R5 HIV-1 strains resistant to CCR5 inhibitors can arise in patients, confirming a mechanism of resistance previously characterized in vitro. In addition, some patients can harbor CCR5 antagonist-resistant viruses prior to treatment, which may have implications for the clinical use of this new class of antiretrovirals. PMID:20055594

Enhanced Chemokine Receptor Expression on Leukocytes of Patients with Alzheimer's Disease.

PubMed

Goldeck, David; Larbi, Anis; Pellicanó, Mariavaleria; Alam, Iftikhar; Zerr, Inga; Schmidt, Christian; Fulop, Tamas; Pawelec, Graham

2013-01-01

Although primarily a neurological complaint, systemic inflammation is present in Alzheimer's Disease, with higher than normal levels of proinflammatory cytokines and chemokines in the periphery as well as the brain. A gradient of these factors may enhance recruitment of activated immune cells into the brain via chemotaxis. Here, we investigated the phenotypes of circulating immune cells in AD patients with multi-colour flow cytometry to determine whether their expression of chemokine receptors is consistent with this hypothesis. In this study, we confirmed our previously reported data on the shift of early- to late-differentiated CD4+ T-cells in AD patients. The percentage of cells expressing CD25, a marker of acute T-cell activation, was higher in patients than in age-matched controls, and percentages of CCR6+ cells were elevated. This chemokine receptor is primarily expressed on pro-inflammatory memory cells and Th17 cells. The proportion of cells expressing CCR4 (expressed on Th2 cells) and CCR5 (Th1 cells and dendritic cells) was also greater in patients, and was more pronounced on CD4+ than CD8+ T-cells. These findings allow a more detailed insight into the systemic immune status of patients with Alzheimer's disease and suggest possible novel targets for immune therapy.

A study on the association of autoantibodies, chemokine, and its receptor with disease activity in systemic lupus erythematosus in North Indian population.

PubMed

Bidyalaxmi Devi, Leishangthem; Bhatnagar, Archana; Wanchu, Ajay; Sharma, Aman

2013-11-01

Systemic lupus erythematosus (SLE) is a chronic and complex autoimmune disease characterized by the production of autoantibodies against a spectrum of nuclear antigens. RANTES and its receptor CCR5 have been associated with the pathogenesis of SLE. The objective of this study is to analyze autoantibodies (DNA/RNA), allelic distribution of RANTES and the association of levels of RANTES and its receptor CCR5 in SLE patients in North Indian region. The RANTES-403 and RANTES-28 polymorphism in the promoter region of RANTES gene was studied in 80 patients and 80 healthy controls. The levels of chemokine RANTES, its receptor CCR5, anti-dsDNA, and anti-SSA antibodies levels were determined. Disease activity was assessed with the systemic lupus erythematosus disease activity index (SLEDAI) score. All the parameters were studied for statistical analysis by using t test (graph pad prism) and correlation by SPSS data. PCR-RFLP performed showed 28C/C and the 403G/G genotypes in both patients and controls, but no other genotypes such as 28C/G, 28G/G and 403A/G, 403A/A were found. Patients had higher levels of RANTES (1840.48 ± 739.42 vs. 835.44 ± 70.48 pg/ml; P < 0.0001) and its receptor CCR5 expression (26.49 ± 0.16 vs. 24.72 ± 3.02 %; P < 0.05) compared to controls. The levels of autoantibodies anti-dsDNA and anti-SSA were also higher in patients than controls. The patients showing elevated anti-dsDNA had negative correlation with SLEDAI score (P < 0.05) while borderline patients were not found to be correlated. In case of anti-Ro/anti-SSA antibody levels, the borderline patients showed a moderately significant negative correlation as compared to controls than patients with elevated autoantibody (P < 0.01). The levels of RANTES and CCR5 were also higher in case of patients than controls. But there was no significant correlation of RANTES and CCR5 with disease activity. We were unable to find an association of RANTES polymorphism with SLE in North Indian population in our sample. No significant difference in allele distribution of RANTES-28 and RANTES-403 in the sample of 160 individuals was detected. Of the two autoantibodies studied, anti-Ro/anti-SSA levels in borderline lupus patients appeared as an important parameter for monitoring/diagnosis of lupus patients.

Effect of C-C Chemokine Receptor 2 (CCR2) Knockout on Type-2 (Schistosomal Antigen-Elicited) Pulmonary Granuloma Formation

PubMed Central

Warmington, Kelly S.; Boring, Landin; Ruth, Jeffrey H.; Sonstein, Joanne; Hogaboam, Cory M.; Curtis, Jeffrey L.; Kunkel, Steven L.; Charo, Israel R.; Chensue, Stephen W.

1999-01-01

Monocyte chemotactic protein (MCP)-1 is postulated to play a role in cellular recruitment during inflammatory reactions. C-C chemokine receptor 2 (CCR2) is considered the major G-protein coupled receptor for MCP-1/JE. We reported that mice with knockout of the CCR2 gene display partially impaired type-1 granuloma formation. The present study similarly examined the effect of CCR2 deficiency on synchronously developing type-2 (Th2) cytokine-mediated lung granulomas elicited by embolization of beads coated with Ags of Schistosoma mansoni eggs. Systemically, blood monocytes were reduced by about half throughout the 8-day study period. At the local level, granuloma size and macrophage content were impaired during the early growth phase (days 1 to 2). By day 4, granuloma sizes were similar to controls. In granulomatous lungs, CCR2 knockout increased mRNA for CCR2 agonists, MCP-1, MCP-3, and MCP-5, but reduced IL-4 and IFNγ mRNA. The latter was possibly related to decreased CD4+ T cell recruitment. Regionally, draining lymph nodes showed panlymphoid hyperplasia with impaired production of IFNγ, IL-2, and IL-4, but not IL-5, IL-10, or IL-13. Analysis of procollagen gene expression indicated transient impairment of procollagen III transcripts on day 4 of granuloma formation. These findings indicate that agonists of CCR2 contribute to multiple facets of type-2 hypersensitivity granulomatous inflammation. PMID:10329593

Intestinal recruiting and activation profiles in peripheral blood mononuclear cells in response to pathogen-associated molecular patterns stimulation in patients with IBS.

PubMed

Rodríguez-Fandiño, O; Hernández-Ruíz, J; López-Vidal, Y; Charúa, L; Bandeh-Moghaddam, H; Minzoni, A; Guzmán, C; Schmulson, M

2013-11-01

Immune activation, increased Toll-like Receptors (TLR) expression, and gut epithelial diffusion of bacterial molecules have been reported in irritable bowel syndrome (IBS). Thus, we sought to relate these factors by analyzing gut homing (integrin α4β7), intestinal recruiting (CCR5) and activation (CD28) phenotypes, and the cytokines and chemokines concentration in peripheral blood T-lymphocytes stimulated with TLR-ligands. Twenty-one IBS-Rome II (1 PI-IBS) patients and 19 controls were studied. Isolated peripheral blood mononuclear cells were cultured with and without Escherichia coli lipopolysaccharide (LPS), Staphylococcus aureus peptidoglycan (PGN), and unmethylated cytosine-phosphate-guanine motifs (CpG). Phenotypes were investigated by flow cytometry and supernatant cytokines and chemokines were also measured. After LPS, CCR5 expression in CD4⁺ α4β7⁺ cells remained unchanged in IBS, but decreased in controls (p = 0.002), to lower levels than in IBS (Mean fluorescence intensity [MFI]: 1590 ± 126.9 vs 2417 ± 88.4, p < 0.001). There were less CD8(+) α4β7⁺ CCR5⁺ cells (85.7 ± 1.5 vs 90.8 ± 0.9%, p = 0.006) after LPS and CD3⁺ α4β7⁺ CCR5⁺ (40.0 ± 1.7 vs 51.2 ± 4.3%, p = 0.006) after PGN in controls. Also, after LPS, CD28 decreased in CD4⁺ α4β7⁺ CCR5⁺ in IBS (MFI: 2337 ± 47.2 vs 1779 ± 179.2, p < 0.001), but not in controls. Cytokines and chemokines were similar, except for lower IL8/CXCL8 in the unstimulated condition in IBS (4.18, 95% CI: 3.94-4.42 vs 3.77, 3.59-3.95; p = 0.006). Pathogen-associated molecular patterns stimulation of peripheral blood T cells expressing gut homing marker in IBS compared with controls resulted in an unsuccessful down-regulation of the co-expression of intestinal recruiting/residence phenotype and a state of activation. These findings support an interaction between an innate immune predisposition and microbial triggers, which may unleash or exacerbate IBS. © 2013 John Wiley & Sons Ltd.

Multi-functional regulation of 4E-BP gene expression by the Ccr4-Not complex.

PubMed

Okada, Hirokazu; Schittenhelm, Ralf B; Straessle, Anna; Hafen, Ernst

2015-01-01

The mechanistic target of rapamycin (mTOR) signaling pathway is highly conserved from yeast to humans. It senses various environmental cues to regulate cellular growth and homeostasis. Deregulation of the pathway has been implicated in many pathological conditions including cancer. Phosphorylation cascades through the pathway have been extensively studied but not much is known about the regulation of gene expression of the pathway components. Here, we report that the mRNA level of eukaryotic translation initiation factor (eIF) subunit 4E-binding protein (4E-BP) gene, one of the key mTOR signaling components, is regulated by the highly conserved Ccr4-Not complex. RNAi knockdown of Not1, a putative scaffold protein of this protein complex, increases the mRNA level of 4E-BP in Drosophila Kc cells. Examination of the gene expression mechanism using reporter swap constructs reveals that Not1 depletion increases reporter mRNAs with the 3'UTR of 4E-BP gene, but decreases the ones with the 4E-BP promoter region, suggesting that Ccr4-Not complex regulates both degradation and transcription of 4E-BP mRNA. These results indicate that the Ccr4-Not complex controls expression of a single gene at multiple levels and adjusts the magnitude of the total effect. Thus, our study reveals a novel regulatory mechanism of a key component of the mTOR signaling pathway at the level of gene expression.

MKEY, a Peptide Inhibitor of CXCL4-CCL5 Heterodimer Formation, Protects Against Stroke in Mice.

PubMed

Fan, Yifang; Xiong, Xiaoxing; Zhang, Yongming; Yan, Dongmei; Jian, Zhihong; Xu, Baohui; Zhao, Heng

2016-09-15

MKEY, a synthetic cyclic peptide inhibitor of CXCL4-CCL5 heterodimer formation, has been shown to protect against atherosclerosis and aortic aneurysm formation by mediating inflammation, but whether it modulates neuroinflammation and brain injury has not been studied. We therefore studied the role of MKEY in stroke-induced brain injury in mice. MKEY was injected into mice after stroke with 60 minutes of middle cerebral artery occlusion. Infarct volume and neurological deficit scores were measured. Protein levels of CCL5 and its receptor CCR5 were detected by Western blot and fluorescence-activated cell sorting (FACS), respectively. Numbers of microglia-derived macrophages (MiMΦs) and monocyte-derived MΦs (MoMΦs) in the brain, and their subsets, based on the surface markers CD45, CD11b, CCR2, CX3CR1, and Ly6C, were analyzed by FACS. MΦs and neutrophil infiltration in the ischemic brain were stained with CD68 and myeloperoxidase (MPO), respectively, and assessed by immunofluorescent confocal microscopy. The results showed that expressions of CCL5 and its receptor CCR5, were increased in the ischemic brain after stroke. MKEY injection significantly reduced infarct sizes and improved neurological deficit scores measured 72 hours after stroke. In addition, MKEY injection inhibited the number of MoMΦs, but not MiMΦs, in the ischemic brain. Furthermore, MKEY inhibited protein expression levels of Ly6C,CCR2, and CX3CR1 on MoMΦs. Lastly, the confocal study also suggests that the number of CD68-positive MΦs and MPO-positive neutrophils was inhibited by MKEY injection. MKEY injection protects against stroke-induced brain injury, probably by inhibiting MoMΦ-mediated neuroinflammation. © 2016 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.

Toll-Like Receptor 2 Ligation Enhances HIV-1 Replication in Activated CCR6+ CD4+ T Cells by Increasing Virus Entry and Establishing a More Permissive Environment to Infection.

PubMed

Bolduc, Jean-François; Ouellet, Michel; Hany, Laurent; Tremblay, Michel J

2017-02-15

In this study, we investigated the effect of Toll-like receptor 2 (TLR2) ligation on the permissiveness of activated CD4 + T cells to HIV-1 infection by focusing our experiments on the relative susceptibility of cell subsets based on their expression of CCR6. Purified primary human CD4 + T cells were first subjected to a CD3/CD28 costimulation before treatment with the TLR2 agonist Pam3CSK4. Finally, cells were inoculated with R5-tropic HIV-1 particles that permit us to study the effect of TLR2 triggering on virus production at both population and single-cell levels. We report here that HIV-1 replication is augmented in CD3/CD28-costimulated CCR6 + CD4 + T cells upon engagement of the cell surface TLR2. Additional studies indicate that a higher virus entry and polymerization of the cortical actin are seen in this cell subset following TLR2 stimulation. A TLR2-mediated increase in the level of phosphorylated NF-κB p65 subunit was also detected in CD3/CD28-costimulated CCR6 + CD4 + T cells. We propose that, upon antigenic presentation, an engagement of TLR2 acts specifically on CCR6 + CD4 + T cells by promoting virus entry in an intracellular milieu more favorable for productive HIV-1 infection. Following primary infection, HIV-1 induces an immunological and structural disruption of the gut mucosa, leading to bacterial translocation and release of microbial components in the bloodstream. These pathogen-derived constituents include several agonists of Toll-like receptors that may affect gut-homing CD4 + T cells, such as those expressing the chemokine receptor CCR6, which are highly permissive to HIV-1 infection. We demonstrate that TLR2 ligation in CD3/CD28-costimulated CCR6 + CD4 + T cells leads to enhanced virus production. Our results highlight the potential impact of bacterial translocation on the overall permissiveness of CCR6 + CD4 + T cells to productive HIV-1 infection. Copyright © 2017 American Society for Microbiology.

Role of LAP+CD4+ T cells in the tumor microenvironment of colorectal cancer.

PubMed

Zhong, Wu; Jiang, Zhi-Yuan; Zhang, Lei; Huang, Jia-Hao; Wang, Shi-Jun; Liao, Cun; Cai, Bin; Chen, Li-Sheng; Zhang, Sen; Guo, Yun; Cao, Yun-Fei; Gao, Feng

2017-01-21

To investigate the abundance and potential functions of LAP + CD4 + T cells in colorectal cancer (CRC). Proportions of LAP + CD4 + T cells were examined in peripheral blood and tumor/paratumor tissues of CRC patients and healthy controls using flow cytometry. Expression of phenotypic markers such as forkhead box (Fox)p3, cytotoxic T-lymphocyte-associated protein (CTLA)-4, chemokine CC receptor (CCR)4 and CCR5 was measured using flow cytometry. LAP - CD4 + and LAP + CD4 + T cells were isolated using a magnetic cell-sorting system and cell purity was analyzed by flow cytometry. Real-time quantitative polymerase chain reaction was used to measure expression of cytokines interleukin (IL)-10 and transforming growth factor (TGF)-β. The proportion of LAP + CD4 + T cells was significantly higher in peripheral blood from patients (9.44% ± 3.18%) than healthy controls (1.49% ± 1.00%, P < 0.001). Among patients, the proportion of LAP + CD4 + T cells was significantly higher in tumor tissues (11.76% ± 3.74%) compared with paratumor tissues (3.87% ± 1.64%, P < 0.001). We also observed positive correlations between the proportion of LAP + CD4 + T cells and TNM stage ( P < 0.001), distant metastasis ( P < 0.001) and serum level of carcinoembryonic antigen ( P < 0.05). Magnetic-activated cell sorting gave an overall enrichment of LAP + CD4 + T cells (95.02% ± 2.87%), which was similar for LAP - CD4 + T cells (94.75% ± 2.76%). In contrast to LAP - CD4 + T cells, LAP + CD4 + T cells showed lower Foxp3 expression but significantly higher levels of CTLA-4, CCR4 and CCR5 ( P < 0.01). LAP + CD4 + T cells expressed significantly larger amounts of IL-10 and TGF-β but lower levels of IL-2, IL-4, IL-17 and interferon-γ, compared with LAP - CD4 + T cells. LAP + CD4 + T cells accumulated in the tumor microenvironment of CRC patients and were involved in immune evasion mediated by IL-10 and TGF-β.

Flexible use of CCR5 in the absence of CXCR4 use explains the immune deficiency in HIV-1 infected children.

PubMed

Cavarelli, Mariangela; Karlsson, Ingrid; Ripamonti, Chiara; Plebani, Anna; Fenyo, Eva Maria; Scarlatti, Gabriella

2010-10-23

CCR5-using HIV-1 (R5 viruses) are usually isolated during acute infection from both adults and children. We have recently demonstrated that R5 viruses with a flexible use of CCR5 (called R5broad) can be detected in children close to birth and are predictive of a fast immunological failure. The aim of the present work was to investigate viral phenotype variation during disease progression in HIV-1 infected children, six slow and eight fast progressors. A total of 74 viral isolates obtained sequentially from 14 HIV-1 infected children were tested for their ability to infect U87.CD4 cells expressing a set of six different CCR5/CXCR4 chimeric receptors or wild-type coreceptors. The sensitivity of 35 R5 viruses to inhibition with the CC-chemokine RANTES (regulated upon activation, normal T-cell expressed and secreted) was evaluated in a peripheral blood mononuclear cells based assay. Viral evolution to R5broad or to R5X4 phenotype occurred with one exception, in all children, although at a different time point according to rate of disease progression. Immune deficiency in the children was significantly associated with the appearance of R5broad phenotype or R5X4 viruses. Analysis of the sensitivity to inhibition by RANTES revealed a significant correlation between the R5broad phenotype and an augmented resistance to this CC-chemokine. We demonstrate that the viral evolution to a more flexible CCR5-use is sufficient to explain the immunological failure in the absence of CXCR4 usage. These results warrant detailed analysis of the R5 phenotype in forthcoming clinical studies introducing CCR5 inhibitors for the treatment of pediatric HIV-1 infection.

The Lupane-type Triterpene 30-Oxo-calenduladiol Is a CCR5 Antagonist with Anti-HIV-1 and Anti-chemotactic Activities*

PubMed Central

Barroso-González, Jonathan; El Jaber-Vazdekis, Nabil; García-Expósito, Laura; Machado, José-David; Zárate, Rafael; Ravelo, Ángel G.; Estévez-Braun, Ana; Valenzuela-Fernández, Agustín

2009-01-01

The existence of drug-resistant human immunodeficiency virus (HIV) viruses in patients receiving antiretroviral treatment urgently requires the characterization and development of new antiretroviral drugs designed to inhibit resistant viruses and to complement the existing antiretroviral strategies against AIDS. We assayed several natural or semi-synthetic lupane-type pentacyclic triterpenes in their ability to inhibit HIV-1 infection in permissive cells. We observed that the 30-oxo-calenduladiol triterpene, compound 1, specifically impaired R5-tropic HIV-1 envelope-mediated viral infection and cell fusion in permissive cells, without affecting X4-tropic virus. This lupane derivative competed for the binding of a specific anti-CCR5 monoclonal antibody or the natural CCL5 chemokine to the CCR5 viral coreceptor with high affinity. 30-Oxo-calenduladiol seems not to interact with the CD4 antigen, the main HIV receptor, or the CXCR4 viral coreceptor. Our results suggest that compound 1 is a specific CCR5 antagonist, because it binds to the CCR5 receptor without triggering cell signaling or receptor internalization, and inhibits RANTES (regulated on activation normal T cell expressed and secreted)-mediated CCR5 internalization, intracellular calcium mobilization, and cell chemotaxis. Furthermore, compound 1 appeared not to interact with β-chemokine receptors CCR1, CCR2b, CCR3, or CCR4. Thereby, the 30-oxo-calenduladiol-associated anti-HIV-1 activity against R5-tropic virus appears to rely on the selective occupancy of the CCR5 receptor to inhibit CCR5-mediated HIV-1 infection. Therefore, it is plausible that the chemical structure of 30-oxo-calenduladiol or other related dihydroxylated lupane-type triterpenes could represent a good model to develop more potent anti-HIV-1 molecules to inhibit viral infection by interfering with early fusion and entry steps in the HIV life cycle. PMID:19386595

Glial cell activation, recruitment, and survival of B-lineage cells following MCMV brain infection.

PubMed

Lokensgard, James R; Mutnal, Manohar B; Prasad, Sujata; Sheng, Wen; Hu, Shuxian

2016-05-20

Chemokines produced by reactive glia drive migration of immune cells and previous studies from our laboratory have demonstrated that CD19(+) B cells infiltrate the brain. In this study, in vivo and in vitro experiments investigated the role of reactive glial cells in recruitment and survival of B-lineage cells in response to (murine cytomegalovirus) MCMV infection. Flow cytometric analysis was used to assess chemokine receptor expression on brain-infiltrating B cells. Real-time RT-PCR and ELISA were used to measure chemokine levels. Dual-immunohistochemical staining was used to co-localize chemokine production by reactive glia. Primary glial cell cultures and migration assays were used to examine chemokine-mediated recruitment. Astrocyte: B cell co-cultures were used to investigate survival and proliferation. The chemokine receptors CXCR3, CXCR5, CCR5, and CCR7 were detected on CD19(+) cells isolated from the brain during MCMV infection. In particular, CXCR3 was found to be elevated on an increasing number of cells over the time course of infection, and it was the primary chemokine receptor expressed at 60 days post infection Quite different expression kinetics were observed for CXCR5, CCR5, and CCR7, which were elevated on the highest number of cells early during infection and decreased by 14, 30, and 60 days post infection Correspondingly, elevated levels of CXCL9, CXCL10, and CXCL13, as well as CCL5, were found within the brains of infected animals, and only low levels of CCL3 and CCL19 were detected. Differential expression of CXCL9/CXCL10 and CXCL13 between microglia and astrocytes was apparent, and B cells moved towards supernatants from MCMV-infected microglia, but not astrocytes. Pretreatment with neutralizing Abs to CXCL9 and CXCL10 inhibited this migration. In contrast, neutralizing Abs to the ligand of CXCR5 (i.e., CXCL13) did not significantly block chemotaxis. Proliferation of brain-infiltrating B cells was detected at 7 days post infection and persisted through the latest time tested (60 days post infection). Finally, astrocytes produce BAFF (B cell activating factor of the TNF family) and promote proliferation of B cells via cell-to-cell contact. CXCR3 is the primary chemokine receptor on CD19(+) B cells persisting within the brain, and migration to microglial cell supernatants is mediated through this receptor. Correspondingly, microglial cells produce CXCL9 and CXCL10, but not CXCL13. Reactive astrocytes promote B cell proliferation.

Human liver-resident CD56(bright)/CD16(neg) NK cells are retained within hepatic sinusoids via the engagement of CCR5 and CXCR6 pathways.

PubMed

Hudspeth, Kelly; Donadon, Matteo; Cimino, Matteo; Pontarini, Elena; Tentorio, Paolo; Preti, Max; Hong, Michelle; Bertoletti, Antonio; Bicciato, Silvio; Invernizzi, Pietro; Lugli, Enrico; Torzilli, Guido; Gershwin, M Eric; Mavilio, Domenico

2016-01-01

The liver-specific natural killer (NK) cell population is critical for local innate immune responses, but the mechanisms that lead to their selective homing and the definition of their functionally relevance remain enigmatic. We took advantage of the availability of healthy human liver to rigorously define the mechanisms regulating the homing of NK cells to liver and the repertoire of receptors that distinguish liver-resident NK (lr-NK) cells from circulating counterparts. Nearly 50% of the entire liver NK cell population is composed of functionally relevant CD56(bright) lr-NK cells that localize within hepatic sinusoids. CD56(bright) lr-NK cells express CD69, CCR5 and CXCR6 and this unique repertoire of chemokine receptors is functionally critical as it determines selective migration in response to the chemotactic stimuli exerted by CCL3, CCL5 and CXCL16. Here, we also show that hepatic sinusoids express CCL3(pos) Kupffer cells, CXCL16(pos) endothelial cells and CCL5(pos) T and NK lymphocytes. The selective presence of these chemokines in sinusoidal spaces creates a unique tissue niche for lr-CD56(bright) NK cells that constitutively express CCR5 and CXCR6. CD56(bright) lr-NK cells co-exist with CD56(dim) conventional NK (c-NK) cells that are, interestingly, transcriptionally and phenotypically similar to their peripheral circulating counterparts. Indeed, CD56(dim) c-NK cells lack expression of CD69, CCR5, and CXCR6 but express selectins, integrins and CX3CR1. Our findings disclosing the phenotypic and functional differences between lr-Nk cells and c-NK cells are critical to distinguish liver-specific innate immune responses. Hence, any therapeutic attempts at modifying the large population of CD56(bright) lr-NK cells will require modification of hepatic CCR5 and CXCR6. Copyright © 2015 Elsevier Ltd. All rights reserved.

Human liver-resident CD56bright/CD16neg NK cells are retained within hepatic sinusoids via the engagement of CCR5 and CXCR6 pathways

PubMed Central

Hudspeth, Kelly; Donadon, Matteo; Cimino, Matteo; Pontarini, Elena; Tentorio, Paolo; Preti, Max; Hong, Michelle; Bertoletti, Antonio; Bicciato, Silvio; Invernizzi, Pietro; Lugli, Enrico; Torzilli, Guido; Gershwin, M. Eric; Mavilio, Domenico

2015-01-01

Rationale The liver-specific natural killer (NK) cell population is critical for local innate immune responses, but the mechanisms that lead to their selective homing and the definition of their functionally relevance remain enigmatic. Objectives We took advantage of the availability of healthy human liver to rigorously define the mechanisms regulating the homing of NK cells to liver and the repertoire of receptors that distinguish liver-resident NK (lr-NK) cells from circulating counterparts. Findings Nearly 50% of the entire liver NK cell population is composed of functionally relevant CD56bright lr-NK cells that localize within hepatic sinusoids. Further, CD56bright lr-NK cells express CD69, CCR5 and CXCR6 and this unique repertoire of chemokine receptors is functionally critical as it determines selective migration in response to the chemotactic stimuli exerted by CCL3, CCL5 and CXCL16. In addition, hepatic sinusoids express CCL3pos Kupffer cells, CXCL16pos endothelial cells and CCL5pos T and NK lymphocytes. The selective presence of these chemokines in sinusoidal spaces creates a tissue niche for lr-CD56bright NK cells that constitutively express CCR5 and CXCR6. CD56bright lr-NK cells co-exist with CD56dim conventional NK (c-NK) cells that are, interestingly, transcriptionally and phenotypically similar to their peripheral circulating counterparts. Indeed, CD56dim c-NK cells lack expression of CD69, CCR5, and CXCR6 but express selectins, integrins and CX3CR1. Conclusion Our findings disclosing the phenotypic and functional differences between lr-Nk cells and c-NK cells are critical to distinguish liver-specific innate immune responses. Hence, any therapeutic attempts at modifying the large population of CD56bright lr-NK cells will require modification of hepatic CCR5 and CXCR6. PMID:26330348

Both Cerebral and Hematopoietic Deficiencies in CCR2 Result in Uncontrolled Herpes Simplex Virus Infection of the Central Nervous System in Mice.

PubMed

Menasria, Rafik; Canivet, Coraline; Piret, Jocelyne; Gosselin, Jean; Boivin, Guy

2016-01-01

CCR2 is a chemokine receptor expressed on the surface of blood leukocytes, particularly «Ly6Chi» inflammatory monocytes and microglia. Signaling through this receptor is thought to influence the immune activity of microglia as well as monocytes egress from the bone marrow (BM) and their trafficking into the central nervous system (CNS) in several neurological diseases. During experimental herpes simplex virus 1 (HSV-1) encephalitis (HSE), CCR2 deficiency has been reported to exacerbate the outcome of the disease. However, the precise contribution of CCR2 expressed in cells of the CNS or peripheral monocytes in the protection against HSE remains unclear. To dissect the differential role of CCR2 during HSE, chimeric mice with receptor deficiency in the brain or blood cells were generated by transplanting wild-type (WT) C57BL/6 or CCR2-/- BM-derived cells in CCR2-/- (WT→CCR2-/-) and WT (CCR2-/-→WT) mice, respectively. Our results indicate that following intranasal infection with 1.2x106 plaque forming units of HSV-1, CCR2 deficiency in hematopoietic cells and, to a lesser extent, in CNS exacerbates the outcome of HSE. Mortality rates of CCR2-/- (71.4%) and CCR2-/-→WT (57.1%) mice were significantly higher than that of WT (15.3%; P<0.01 and P<0.05, respectively) but the difference did not reach statistical significance for WT→CCR2-/- animals (42.8%; P = 0.16). Both peripheral and CNS deficiencies in CCR2 resulted in increased infectious viral titers and wider dissemination of HSV antigens in the brain as well as an overproduction of inflammatory cytokines and chemokines including IL-1β, IL-6, CCL2, CCL3 and CCL5. Furthermore, CCR2 deficiency in the hematopoietic system altered monocytes egress from the BM and their recruitment to the CNS, which may contribute to the failure in HSV-1 containment. Collectively, these data suggest that CCR2 expressed on cells of CNS and especially on peripheral monocytes is important for the control of HSV-1 replication and inflammatory environment during experimental HSE.

Both Cerebral and Hematopoietic Deficiencies in CCR2 Result in Uncontrolled Herpes Simplex Virus Infection of the Central Nervous System in Mice

PubMed Central

Menasria, Rafik; Canivet, Coraline; Piret, Jocelyne; Gosselin, Jean; Boivin, Guy

2016-01-01

CCR2 is a chemokine receptor expressed on the surface of blood leukocytes, particularly «Ly6Chi» inflammatory monocytes and microglia. Signaling through this receptor is thought to influence the immune activity of microglia as well as monocytes egress from the bone marrow (BM) and their trafficking into the central nervous system (CNS) in several neurological diseases. During experimental herpes simplex virus 1 (HSV-1) encephalitis (HSE), CCR2 deficiency has been reported to exacerbate the outcome of the disease. However, the precise contribution of CCR2 expressed in cells of the CNS or peripheral monocytes in the protection against HSE remains unclear. To dissect the differential role of CCR2 during HSE, chimeric mice with receptor deficiency in the brain or blood cells were generated by transplanting wild-type (WT) C57BL/6 or CCR2-/- BM-derived cells in CCR2-/- (WT→CCR2-/-) and WT (CCR2-/-→WT) mice, respectively. Our results indicate that following intranasal infection with 1.2x106 plaque forming units of HSV-1, CCR2 deficiency in hematopoietic cells and, to a lesser extent, in CNS exacerbates the outcome of HSE. Mortality rates of CCR2-/- (71.4%) and CCR2-/-→WT (57.1%) mice were significantly higher than that of WT (15.3%; P<0.01 and P<0.05, respectively) but the difference did not reach statistical significance for WT→CCR2-/- animals (42.8%; P = 0.16). Both peripheral and CNS deficiencies in CCR2 resulted in increased infectious viral titers and wider dissemination of HSV antigens in the brain as well as an overproduction of inflammatory cytokines and chemokines including IL-1β, IL-6, CCL2, CCL3 and CCL5. Furthermore, CCR2 deficiency in the hematopoietic system altered monocytes egress from the BM and their recruitment to the CNS, which may contribute to the failure in HSV-1 containment. Collectively, these data suggest that CCR2 expressed on cells of CNS and especially on peripheral monocytes is important for the control of HSV-1 replication and inflammatory environment during experimental HSE. PMID:27930721

Upregulation of CC Chemokine Receptor 7 (CCR7) Enables Migration of Xenogeneic Human Adipose-Derived Mesenchymal Stem Cells to Rat Secondary Lymphoid Organs.

PubMed

Ma, Tian; Luan, Shao-Liang; Huang, Hong; Sun, Xing-Kun; Yang, Yan-Mei; Zhang, Hui; Han, Wei-Dong; Li, Hong; Han, Yan

2016-12-30

BACKGROUND CC chemokine receptor 7 (CCR7) expression is vital for cell migration to secondary lymphoid organs (SLOs). Our previous work showed that inducing CCR7 expression enabled syngeneic mesenchymal stem cells (MSCs) to migrate into SLOs, resulting in enhanced immunosuppressive performance in mice. Given that human adipose-derived stem cells (hASCs) are widely used in clinical therapy, we further investigated whether upregulation of CCR7 enables xenogeneic hASCs to migrate to rat SLOs. MATERIAL AND METHODS hASCs rarely express CCR7; therefore, hASCs were transfected with lentivirus encoding rat CCR7 (rCCR7) plus green fluorescence protein (GFP) or GFP alone. CCR7 mRNA and cell surface expression of rCCR7-hASCs and GFP-hASCs were examined by reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometry (FCM), respectively. The phenotype, differentiation, and proliferation capacity of each cell type was also determined. To examine migration, rCCR7-hASCs and GFP-hASCs were injected intravenously into Lewis rats, and the proportion of GFP-positive cells in the spleen and lymph nodes was determined with FCM. RESULTS mRNA and cell surface protein expression of CCR7 was essentially undetectable in hASCs and GFP-ASCs; however, CCR7 was highly expressed in rCCR7-ASCs. rCCR7-hASCs, GFP-hASCs, and hASCs shared a similar immunophenotype, and maintained the ability of multilineage differentiation and proliferation. In addition, the average proportion of GFP-positive cells was significantly higher following transplantation of rCCR7-hASCs compared with GFP-hASCs (p<0.01). CONCLUSIONS These results suggest that upregulation of rat CCR7 expression does not change the phenotype, differentiation, or proliferation capacity of hASCs, but does enable efficient migration of hASCs to rat SLOs.

FaPOD27 functions in the metabolism of polyphenols in strawberry fruit (Fragaria sp.)

PubMed Central

Yeh, Su-Ying; Huang, Fong-Chin; Hoffmann, Thomas; Mayershofer, Mechthild; Schwab, Wilfried

2014-01-01

The strawberry (Fragaria × ananassa) is one of the most preferred fresh fruit worldwide, accumulates numerous flavonoids but has limited shelf life due to excessive tissue softening caused by cell wall degradation. Since lignin is one of the polymers that strengthen plant cell walls and might contribute to some extent to fruit firmness monolignol biosynthesis was studied in strawberry fruit. Cinnamoyl-CoA reductase (CCR), cinnamyl alcohol dehydrogenase (CAD), and a peroxidase (POD27) gene were strongly expressed in red, ripe fruit whereas a second POD gene was primarily expressed in green, immature fruit. Moreover, FaPOD27 transcripts were strongly and constitutively induced in fruits exposed to Agrobacterium infection. Gene expression levels and enzymatic activities of FaCCR and FaCAD were efficiently suppressed through RNAi in FaCCR- and FaCAD-silenced strawberries. Besides, significantly elevated FaPOD transcript levels were detected after agroinfiltration of pBI-FaPOD constructs in fruits. At the same time, levels of G-monomers were considerably reduced in FaCCR-silenced fruits whereas the proportion of both G- and S-monomers decisively decreased in FaCAD-silenced and pBI-FaPOD fruits. Development, firmness, and lignin level of the treated fruits were similar to pBI-intron control fruits, presumably attributed to increased expression levels of FaPOD27 upon agroinfiltration. Additionally, enhanced firmness, accompanied with elevated lignin levels, was revealed in chalcone synthase-deficient fruits (CHS−), independent of down- or up-regulation of individual and combined FaCCR. FaCAD, and FaPOD genes by agroinfiltration, when compared to CHS−/pBI-intron control fruits. These approaches provide further insight into the genetic control of flavonoid and lignin synthesis in strawberries. The results suggest that FaPOD27 is a key gene for lignin biosynthesis in strawberry fruit and thus to improving the firmness of strawberries. PMID:25346738

CXCR6 and CCR5 localize T lymphocyte subsets in nasopharyngeal carcinoma.

PubMed

Parsonage, Greg; Machado, Lee Richard; Hui, Jan Wai-Ying; McLarnon, Andrew; Schmaler, Tilo; Balasothy, Meenarani; To, Ka-Fai; Vlantis, Alexander C; van Hasselt, Charles A; Lo, Kwok-Wai; Wong, Wai-Lap; Hui, Edwin Pun; Chan, Anthony Tak Cheung; Lee, Steven P

2012-03-01

The substantial T lymphocyte infiltrate found in cases of nasopharyngeal carcinoma (NPC) has been implicated in the promotion of both tumor growth and immune escape. Conversely, because malignant NPC cells harbor the Epstein-Barr virus, this tumor is a candidate for virus-specific T cell-based therapies. Preventing the accumulation of tumor-promoting T cells or enhancing the recruitment of tumor-specific cytotoxic T cells offers therapeutic potential. However, the mechanisms involved in T cell recruitment to this tumor are poorly understood. Comparing memory T cell subsets that have naturally infiltrated NPC tissue with their counterparts from matched blood revealed enrichment of CD8(+), CD4(+), and regulatory T cells expressing the chemokine receptor CXCR6 in tumor tissue. CD8(+) and (nonregulatory) CD4(+) T cells also were more frequently CCR5(+) in tumor than in blood. Ex vivo studies demonstrated that both receptors were functional. CXCL16 and CCL4, unique chemokine ligands for CXCR6 and CCR5, respectively, were expressed by the malignant cells in tumor tissue from the majority of NPC cases, as was another CCR5 ligand, CCL5. The strongest expression of CXCL16 was found on tumor-infiltrating cells. CCL4 was detected on the tumor vasculature in a majority of cases. These findings suggest that CXCR6 and CCR5 play important roles in T cell recruitment and/or retention in NPC and have implications for the pathogenesis and treatment of this tumor. Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Glutamine supplementation attenuates expressions of adhesion molecules and chemokine receptors on T cells in a murine model of acute colitis.

PubMed

Hou, Yu-Chen; Wu, Jin-Ming; Wang, Ming-Yang; Wu, Ming-Hsun; Chen, Kuen-Yuan; Yeh, Sung-Ling; Lin, Ming-Tsan

2014-01-01

Migration of T cells into the colon plays a major role in the pathogenesis in inflammatory bowel disease. This study investigated the effects of glutamine (Gln) supplementation on chemokine receptors and adhesion molecules expressed by T cells in mice with dextran sulfate sodium- (DSS-) induced colitis. C57BL/6 mice were fed either a standard diet or a Gln diet replacing 25% of the total nitrogen. After being fed the diets for 5 days, half of the mice from both groups were given 1.5% DSS in drinking water to induce colitis. Mice were killed after 5 days of DSS exposure. DSS colitis resulted in higher expression levels of P-selectin glycoprotein ligand- (PSGL-) 1, leukocyte function-associated antigen- (LFA-) 1, and C-C chemokine receptor type 9 (CCR9) by T helper (Th) and cytotoxic T (Tc) cells, and mRNA levels of endothelial adhesion molecules in colons were upregulated. Gln supplementation decreased expressions of PSGL-1, LFA-1, and CCR9 by Th cells. Colonic gene expressions of endothelial adhesion molecules were also lower in Gln-colitis mice. Histological finding showed that colon infiltrating Th cells were less in the DSS group with Gln administration. Gln supplementation may ameliorate the inflammation of colitis possibly via suppression of T cell migration.

Chemokines and their receptors in whiplash injury: elevated RANTES and CCR-5.

PubMed

Kivioja, J; Rinaldi, L; Ozenci, V; Kouwenhoven, M; Kostulas, N; Lindgren, U; Link, H

2001-07-01

The human sufferings and socioeconomic burden due to whip-lash-associated disorders (WAD) are obvious but the pathogenesis of WAD is obscure. The possible involvement of the immune system during the disease process in WAD is not known. Effector molecules including chemokines and their receptors could play a role in WAD. In a prospective study using flow cytometry, we examined percentages of blood mononuclear cells (MNC) expressing the chemokines RANTES, MCP-1, MIP-1alpha, MIP-1beta, and IL-8, the chemokine receptor CCR-5, the T cell activation marker CD25, and the T cell chemoattractant IL-16 in patients with WAD and, for reference, in healthy controls. Higher percentages of RANTES-expressing blood MNC and T cells were observed in patients with WAD examined within 3 days compared to 14 days after the whiplash injury and, likewise, compared with healthy controls. The patients with WAD examined within 3 days after the accident also had higher percentages of CCR-5-expressing blood MNC, T cells, and CD45RO+ T cells compared to healthy controls. In contrast, there were no differences for any of these variables between patients with WAD examined 14 days after injury and healthy controls. In conclusion, WAD is associated with a systemic but transient dysregulation in percentages of RANTES and CCR-5 expressing MNC and T cells.

The role of ZAP70 kinase in acute lymphoblastic leukemia infiltration into the central nervous system.

PubMed

Alsadeq, Ameera; Fedders, Henning; Vokuhl, Christian; Belau, Nele M; Zimmermann, Martin; Wirbelauer, Tim; Spielberg, Steffi; Vossen-Gajcy, Michaela; Cario, Gunnar; Schrappe, Martin; Schewe, Denis M

2017-02-01

Central nervous system infiltration and relapse are poorly understood in childhood acute lymphoblastic leukemia. We examined the role of zeta-chain-associated protein kinase 70 in preclinical models of central nervous system leukemia and performed correlative studies in patients. Zeta-chain-associated protein kinase 70 expression in acute lymphoblastic leukemia cells was modulated using short hairpin ribonucleic acid-mediated knockdown or ectopic expression. We show that zeta-chain-associated protein kinase 70 regulates CCR7/CXCR4 via activation of extracellular signal-regulated kinases. High expression of zeta-chain-associated protein kinase 70 in acute lymphoblastic leukemia cells resulted in a higher proportion of central nervous system leukemia in xenografts as compared to zeta-chain-associated protein kinase 70 low expressing counterparts. High zeta-chain-associated protein kinase 70 also enhanced the migration potential towards CCL19/CXCL12 gradients in vitro CCR7 blockade almost abrogated homing of acute lymphoblastic leukemia cells to the central nervous system in xenografts. In 130 B-cell precursor acute lymphoblastic leukemia and 117 T-cell acute lymphoblastic leukemia patients, zeta-chain-associated protein kinase 70 and CCR7/CXCR4 expression levels were significantly correlated. Zeta-chain-associated protein kinase 70 expression correlated with central nervous system disease in B-cell precursor acute lymphoblastic leukemia, and CCR7/CXCR4 correlated with central nervous system involvement in T-cell acute lymphoblastic leukemia patients. In multivariate analysis, zeta-chain-associated protein kinase 70 expression levels in the upper third and fourth quartiles were associated with central nervous system involvement in B-cell precursor acute lymphoblastic leukemia (odds ratio=7.48, 95% confidence interval, 2.06-27.17; odds ratio=6.86, 95% confidence interval, 1.86-25.26, respectively). CCR7 expression in the upper fourth quartile correlated with central nervous system positivity in T-cell acute lymphoblastic leukemia (odds ratio=11.00, 95% confidence interval, 2.00-60.62). We propose zeta-chain-associated protein kinase 70, CCR7 and CXCR4 as markers of central nervous system infiltration in acute lymphoblastic leukemia warranting prospective investigation. Copyright© Ferrata Storti Foundation.

Absence of Association between CCR5 rs333 Polymorphism and Childhood Acute Lymphoblastic Leukemia

PubMed Central

de Oliveira, Carlos Eduardo Coral; Perim, Aparecida de Lourdes; Ozawa, Patricia Midori Murobushi; Freire Vitiello, Glauco Akelinghton; Losi Guembarovski, Roberta; Watanabe, Maria Angelica Ehara

2014-01-01

Acute lymphoblastic leukemia (ALL) is a malignant disorder that originates from one single hematopoietic precursor committed to B- or T-cell lineage. Ordinarily, these cells express CCR5 chemokine receptor, which directs the immune response to a cellular pattern and is involved in cancer pathobiology. The genetic rs333 polymorphism of CCR5 (Δ32), results in a diminished receptor expression, thus leading to impaired cell trafficking. The objective of the present study was to investigate the effect of CCR5 chemokine receptor rs333 polymorphism in the pathogenesis of ALL. The genotype distribution was studied in 79 patients and compared with 80 control subjects, in a childhood population of Southern Brazil. Genotyping was performed using DNA samples amplified by polymerase chain reaction with sequence-specific primers (PCR-SSP). The homozygous (Δ32/Δ32) deletion was not observed in any subject involved in the study. Heterozygous genotype was not associated with ALL risk (OR 0.7%; 95% CI 0.21–2.32; P > 0.05), nor recurrence status of ALL (OR 0.86; 95% CI 0.13–5.48; P > 0.05). This work demonstrated, for the first time, no significant differences in the frequency of the CCR5/Δ32 genotype between ALL and control groups, indicating no effect of this genetic variant on the ALL susceptibility and recurrence risk. PMID:24822066

Recombinant Human T-Cell Leukemia Virus Types 1 and 2 Tax Proteins Induce High Levels of CC-Chemokines and Downregulate CCR5 in Human Peripheral Blood Mononuclear Cells

PubMed Central

Barrios, Christy S.; Abuerreish, Muna; Lairmore, Michael D.; Castillo, Laura; Giam, Chou-Zen

2011-01-01

Abstract Human T-cell leukemia viruses types 1 (HTLV-1) and 2 (HTLV-2) produce key transcriptional regulatory gene products, known as Tax1 and Tax2, respectively. Tax1 and Tax2 transactivate multiple host genes involved in cellular immune responses within the cellular microenvironment, including induction of genes encoding expression of CC-chemokines. It is speculated that HTLV Tax proteins may act as immune modulators. In this study, recombinant Tax1 and Tax2 proteins were tested for their effects on the viability of cultured peripheral blood mononuclear cells (PBMCs), and their ability to induce expression of CC-chemokines and to downregulate the level of CCR5 expression in PBMCs. PBMCs obtained from uninfected donors were cultured in a range of Tax1 and Tax2 concentrations (10–100 pM), and supernatant fluids were harvested at multiple time points for quantitative determinations of MIP-1α/CCL3, MIP-1β/CCL4, and RANTES/CCL5. Treatment of PBMCs with Tax1 and Tax2 proteins (100 pM) resulted in a significant increase in viability over a 7-d period compared to controls (p<0.01). Both Tax1 and Tax2 induced high levels of all three CC-chemokines over the dosing range compared to mock-treated controls (p<0.05). The gated population of lymphocytes treated with Tax2, as well as lymphocytes from HTLV-2-infected donors, showed a significantly lower percentage of CCR5-positive cells compared to those of uninfected donors and from mock-treated lymphocytes, respectively (p<0.05). These results suggest that Tax1 and Tax2 could promote innate immunity in the extracellular environment during HTLV-1 and HTLV-2 infections via CC-chemokine ligands and receptors. PMID:22111594

The puzzling role of CXCR4 in human immunodeficiency virus infection.

PubMed

Vicenzi, Elisa; Liò, Pietro; Poli, Guido

2013-01-01

The human immunodeficiency virus type-1 (HIV-1) is the etiological agent of the acquired immunodeficiency syndrome (AIDS), a disease highly lethal in the absence of combination antiretroviral therapy. HIV infects CD4(+) cells of the immune system (T cells, monocyte-macrophages and dendritic cells) via interaction with a universal primary receptor, the CD4 molecule, followed by a mandatory interaction with a second receptor (co-receptor) belonging to the chemokine receptor family. Apart from some rare cases, two chemokine receptors have been evolutionarily selected to accomplish this need for HIV-1: CCR5 and CXCR4. Yet, usage of these two receptors appears to be neither casual nor simply explained by their levels of cell surface expression. While CCR5 use is the universal rule at the start of every infection regardless of the transmission route (blood-related, sexual or mother to child), CXCR4 utilization emerges later in disease coinciding with the immunological deficient phase of infection. Moreover, in most instances CXCR4 use as viral entry co-receptor is associated with maintenance of CCR5 use. Since antiviral agents preventing CCR5 utilization by the virus are already in use, while others targeting either CCR5 or CXCR4 (or both) are under investigation, understanding the biological correlates of this "asymmetrical" utilization of HIV entry co-receptors bears relevance for the clinical choice of which therapeutics should be administered to infected individuals. We will here summarize the basic knowledge and the hypotheses underlying the puzzling and yet unequivocal role of CXCR4 in HIV-1 infection.

Involvement of chemokine receptors in breast cancer metastasis

NASA Astrophysics Data System (ADS)

Müller, Anja; Homey, Bernhard; Soto, Hortensia; Ge, Nianfeng; Catron, Daniel; Buchanan, Matthew E.; McClanahan, Terri; Murphy, Erin; Yuan, Wei; Wagner, Stephan N.; Barrera, Jose Luis; Mohar, Alejandro; Verástegui, Emma; Zlotnik, Albert

2001-03-01

Breast cancer is characterized by a distinct metastatic pattern involving the regional lymph nodes, bone marrow, lung and liver. Tumour cell migration and metastasis share many similarities with leukocyte trafficking, which is critically regulated by chemokines and their receptors. Here we report that the chemokine receptors CXCR4 and CCR7 are highly expressed in human breast cancer cells, malignant breast tumours and metastases. Their respective ligands CXCL12/SDF-1α and CCL21/6Ckine exhibit peak levels of expression in organs representing the first destinations of breast cancer metastasis. In breast cancer cells, signalling through CXCR4 or CCR7 mediates actin polymerization and pseudopodia formation, and subsequently induces chemotactic and invasive responses. In vivo, neutralizing the interactions of CXCL12/CXCR4 significantly impairs metastasis of breast cancer cells to regional lymph nodes and lung. Malignant melanoma, which has a similar metastatic pattern as breast cancer but also a high incidence of skin metastases, shows high expression levels of CCR10 in addition to CXCR4 and CCR7. Our findings indicate that chemokines and their receptors have a critical role in determining the metastatic destination of tumour cells.

Contribution of enzymatic properties, cell permeability, and enzyme expression to microbiological activities of beta-lactams in three Bacteroides fragilis isolates that harbor a metallo-beta-lactamase gene.

PubMed

Rasmussen, B A; Yang, Y; Jacobus, N; Bush, K

1994-09-01

The metallo-beta-lactamase gene, ccrA, has been cloned from three clinical isolates of Bacteroides fragilis, TAL3636, QMCN3, and QMCN4. Although all three isolates harbored a gene encoding a potent beta-lactamase, the MICs of benzylpenicillin, piperacillin, cefotaxime, ceftazidime, imipenem, and biapenem for the three isolates varied from 4- to > 128-fold. QMCN4 was the most susceptible of the three isolates, followed by QMCN3. TAL3636 was resistant to all of the beta-lactams. Previous DNA sequence analysis of the three ccrA genes revealed that the enzymes differed at 5 amino acid residues (B. A. Rasmussen, Y. Gluzman, and F. P. Tally, Mol. Microbiol. 5:1211-1219, 1991). Biochemical characterization of the three enzymes revealed only small differences in kcat and Km values for the majority of beta-lactams tested. Thus, the 5 amino acid substitutions affected the hydrolyzing activity of the enzymes only modestly. Crypticity differences between the three isolates showed that QMCN4 was the least permeable of the isolates to cephaloridine, followed by TAL3636, and that QMCN3 was highly permeable to cephaloridine. Therefore, neither catalytic activity nor permeability was a major contributor to the dramatic differences in the MICs. Instead, microbiological susceptibility was closely related to the level of metallo-beta-lactamase present in each isolate. Both biochemical and physical studies indicated that TAL3636 produced 5- to 10-fold and 50- to 100-fold more metallo-beta-lactamase than QMCN3 and QMCN4, respectively. Therefore, the level of CcrA enzyme production is the dominant contributing factor to high-level resistance among strains harboring a ccrA gene.

Particulate Air pollution mediated effects on insulin resistance in mice are independent of CCR2.

PubMed

Liu, Cuiqing; Xu, Xiaohua; Bai, Yuntao; Zhong, Jixin; Wang, Aixia; Sun, Lixian; Kong, Liya; Ying, Zhekang; Sun, Qinghua; Rajagopalan, Sanjay

2017-03-03

Chronic exposure to fine ambient particulate matter (PM 2.5 ) induces insulin resistance. CC-chemokine receptor 2 (CCR2) appears to be essential in diet-induced insulin resistance implicating an important role for systemic cellular inflammation in the process. We have previously suggested that CCR2 is important in PM 2.5 exposure-mediated inflammation leading to insulin resistance under high fat diet situation. The present study assessed the importance of CCR2 in PM 2.5 exposure-induced insulin resistance in the context of normal diet. C57BL/6 and CCR2 -/- mice were subjected to exposure to concentrated ambient PM 2.5 or filtered air for 6 months. In C57BL/6 mice, concentrated ambient PM 2.5 exposure induced whole-body insulin resistance, macrophage infiltration into the adipose tissue, and upregulation of phosphoenolpyruvate carboxykinase (PEPCK) in the liver. While CCR2 deficiency reduced adipose macrophage content in the PM 2.5 -exposed animals, it did not improve systemic insulin resistance. This lack of improvement in insulin resistance was paralleled by increased hepatic expression of genes in PEPCK and inflammation. CCR2 deletion failed to attenuate PM 2.5 exposure-induced insulin resistance in mice fed on normal diet. The present study indicates that PM 2.5 may dysregulate glucose metabolism directly without exerting proinflammatory effects.

Overexpression of hsa-miR-125a-5p enhances proliferation, migration and invasion of head and neck squamous cell carcinoma cell lines by upregulating C-C chemokine receptor type 7.

PubMed

Jin, Shan; Liu, Min-Da; Wu, Hong; Pang, Pai; Wang, Song; Li, Zhen-Ning; Sun, Chang-Fu; Liu, Fa-Yu

2018-06-01

Head and neck squamous cell carcinoma (HNSCC) is usually diagnosed accompanied by lymph node metastasis. C-C chemokine receptor type 7 (CCR7) is associated with the invasion and metastasis of tumors in HNSCC through various signaling pathways. The role of hsa-miR-125a-5p in HNSCC remains unclear. The present study was performed to investigate the association between hsa-miR-125a-5p and CCR7 in HNSCC. Reverse transcription-quantitative polymerase chain reaction was applied to analyze the expression of hsa-miR-125a-5p in clinical samples. Cell Counting Kit-8, Transwell and wound healing assays were used to detect cell proliferation, invasion, and metastasis, respectively, following overexpression of hsa-miR-125a-5p. Changes in protein expression of CCR7 were observed using western blotting. In the survival analysis, Student's t-tests and log rank tests were performed to analyze the association between the expression of hsa-miR-125a-5p, and HNSCC according to the Cancer Genome Atlas database. The expression of hsa-miR-125a-5p was identified to be significantly lower in cancer tissue compared with the corresponding adjacent normal tissues in clinical samples (P=0.038). The results of western blotting indicated that there was a positive regulatory association between hsa-miR-125a-5p and CCR7. Furthermore, overexpression of hsa-miR-125a-5p significantly enhanced the ability of cell proliferation, migration and invasion in HNSCC, with upregulation of CCR7. The results of survival analysis revealed that patients in the low expression group of hsa-miR-125a-5p tended to have longer survival times compared with the high expression group (P=0.045). Altogether, the data raised the possibility that hsa-miR-125a-5p has a significant role in promoting cancer in HNSCC, which may provide a basis for the treatment of HNSCC in molecular targeted therapy. Further studies are required to ascertain the role of hsa-miR-125a-5p in other HNSCC cell lines and in vivo .

The RORγt-CCR6-CCL20 axis augments Th17 cells invasion into the synovia of rheumatoid arthritis patients.

PubMed

Kaneko, Shunta; Kondo, Yuya; Yokosawa, Masahiro; Furuyama, Kotona; Segawa, Seiji; Tsuboi, Hiroto; Kanamori, Akihiro; Matsumoto, Isao; Yamazaki, Masashi; Sumida, Takayuki

2018-01-22

To clarify the pathogenic role of transcription factor expression of CD4 + T helper (Th) cell subsets in the development of rheumatoid arthritis (RA). We collected CD4 + T cells from peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) by magnetic cell sorting. The proportion of Th cell subsets were classified from cell surface markers (CD45RA, CXCR5, CXCR3, CCR6) and the expression of their transcription factors (T-bet, GATA3, RORγt) were analyzed by flow cytometry before and at 24 weeks after anti-rheumatic treatment. Chemotaxis assays quantified migratory ability. The expression of CCR6 and RORγt in Th17 cells from PBMC of RA patients was significantly higher than in healthy control volunteers and osteoarthritis patients. The proportion of Th17 cells in SFMCs of RA patients was significantly higher than that in PBMCs. Chemotaxis assays revealed that the migration index of Th17 cells towards CCL20 was remarkably enhanced in RA patients. The expression of CCR6 and RORγt in Th17 cells at 24 weeks post-therapeutic intervention was significantly decreased compared to before treatment. The high expression of RORγt might facilitate the migration of Th17 cells to inflamed joints via the enhanced expression of CCR6 and contribute to the pathology of RA.

Expression of chemokine receptor CCR7 is a negative prognostic factor for patients with gastric cancer: a meta-analysis.

PubMed

Du, Peizhun; Liu, Yongchao; Ren, Hong; Zhao, Jing; Zhang, Xiaodan; Patel, Rajan; Hu, Chenen; Gan, Jun; Huang, Guangjian

2017-03-01

The prognostic significance of CC chemokine receptor type 7 (CCR7) for survival of patients with gastric cancer remains controversial. To investigate the impacts of CCR7 on clinicopathological findings and survival outcome in gastric cancer, we performed a meta-analysis. A comprehensive search in PubMed, Embase, the Cochrane Library, and the CNKI database (1966 to November 2015) was undertaken for relevant studies. The relative risk and hazard ratios with their 95 % confidence intervals were used as measures to investigate the correlation between CCR7 expression and clinicopathological findings and overall survival rate. Sensitivity analysis was conducted to assess the stability of outcomes. Fifteen eligible studies comprising 1697 participants were included in our analysis. The pooled relative risks indicated CCR7 expression was significantly associated with deeper tumor invasion [0.61, 95 % confidence interval (CI) 0.45-0.84, p = 0.003], advanced stage (0.47, 95 % CI 0.32-0.69, p < 0.001), vascular invasion (2.12, 95 % CI 1.20-3.73, p = 0.009), lymph node metastasis (2.00, 95 % CI 1.48-2.70, p < 0.001), and lymphatic invasion (1.98, 95 % CI 1.43-2.72, p < 0.001) but not with age, tumor size, and histological type. The pooling of hazard ratios showed a significant relationship between positive CCR7 expression and worse 5-year overall survival rate (0.46, 95 % CI 0.31-0.70, p < 0.001). Our meta-analysis indicated high CCR7 expression is likely to be a negative clinicopathological prognostic factor for patients with gastric cancer and to predict a worse long-term survival outcome.

Beyond the known functions of the CCR4-NOT complex in gene expression regulatory mechanisms: New structural insights to unravel CCR4-NOT mRNA processing machinery.

PubMed

Ukleja, Marta; Valpuesta, José María; Dziembowski, Andrzej; Cuellar, Jorge

2016-10-01

Large protein assemblies are usually the effectors of major cellular processes. The intricate cell homeostasis network is divided into numerous interconnected pathways, each controlled by a set of protein machines. One of these master regulators is the CCR4-NOT complex, which ultimately controls protein expression levels. This multisubunit complex assembles around a scaffold platform, which enables a wide variety of well-studied functions from mRNA synthesis to transcript decay, as well as other tasks still being identified. Solving the structure of the entire CCR4-NOT complex will help to define the distribution of its functions. The recently published three-dimensional reconstruction of the complex, in combination with the known crystal structures of some of the components, has begun to address this. Methodological improvements in structural biology, especially in cryoelectron microscopy, encourage further structural and protein-protein interaction studies, which will advance our comprehension of the gene expression machinery. © 2016 WILEY Periodicals, Inc.

A role for MCP-1/CCR2 in interstitial lung disease in children

PubMed Central

Hartl, Dominik; Griese, Matthias; Nicolai, Thomas; Zissel, Gernot; Prell, Christine; Reinhardt, Dietrich; Schendel, Dolores J; Krauss-Etschmann, Susanne

2005-01-01

Background Interstitial lung diseases (ILD) are chronic inflammatory disorders leading to pulmonary fibrosis. Monocyte chemotactic protein 1 (MCP-1) promotes collagen synthesis and deletion of the MCP-1 receptor CCR2 protects from pulmonary fibrosis in ILD mouse models. We hypothesized that pulmonary MCP-1 and CCR2+ T cells accumulate in pediatric ILD and are related to disease severity. Methods Bronchoalveolar lavage fluid was obtained from 25 children with ILD and 10 healthy children. Levels of pulmonary MCP-1 and Th1/Th2-associated cytokines were quantified at the protein and the mRNA levels. Pulmonary CCR2+, CCR4+, CCR3+, CCR5+ and CXCR3+ T cells were quantified by flow-cytometry. Results CCR2+ T cells and MCP-1 levels were significantly elevated in children with ILD and correlated with forced vital capacity, total lung capacity and ILD disease severity scores. Children with lung fibrosis had significantly higher MCP-1 levels and CCR2+ T cells in bronchoalveolar lavage fluid compared to non-fibrotic children. Conclusion The results indicate that pulmonary CCR2+ T cells and MCP-1 contribute to the pathogenesis of pediatric ILD and might provide a novel target for therapeutic strategies. PMID:16095529

The expression of chemokine receptors CCR6, CXCR2 and CXCR4 is not organ-specific for distant metastasis in colorectal cancer: a comparative study.

PubMed

Hu, Dongzhi; Du, Changzheng; Xue, Weicheng; Dou, Fangyuan; Yao, Yunfeng; Gu, Jin

2013-08-01

The liver and lung are the organs most commonly affected by metastasis in colorectal cancer (CRC), and the interaction of chemokines and chemokine receptors (CKRs) plays an important role in the metastatic process. The aim of this study was to investigate the organ specificity of CKRs in CRC distant metastasis. Surgical specimens of primary tumours from 46 patients with metachronous distant metastases were retrieved retrospectively (20 lung metastases; 26 liver metastases). As a control, the records of 29 patients without distant metastases were randomly retrieved from our database, and their specimens were reassessed. The expression rates of CKRs, including CCR6, CXCR2, and CXCR4, were determined by immunohistochemistry, and were compared among the groups. The expression rates of CCR6 and CXCR2 were both significantly higher in the metastasis group than in the non-metastasis group (P < 0.05), but there was no statistical difference between the lung metastasis and liver metastasis subgroups. The expression of CXCR4 was not significantly different between the metastasis and non-metastasis groups. Multivariable analysis suggested that preoperative serum carcinoembryonic antigen level, CCR6 and CXCR2 were independent factors associated with distant metastasis. The expression of CCR6 and CXCR2 in CRC could predict metachronous distant metastasis, but they have no organ specificity for metastasis. © 2013 John Wiley & Sons Ltd.

Dual R3R5 tropism characterizes cerebrospinal fluid HIV-1 isolates from individuals with high cerebrospinal fluid viral load.

PubMed

Karlsson, Ulf; Antonsson, Liselotte; Ljungberg, Bengt; Medstrand, Patrik; Esbjörnsson, Joakim; Jansson, Marianne; Gisslen, Magnus

2012-09-10

To study the use of major and alternative coreceptors by HIV-1 isolates obtained from paired plasma and cerebrospinal fluid (CSF) samples. Paired plasma and CSF isolates from HIV-1-infected individuals with varying clinical, virologic, and immunologic parameters were assessed for the ability to infect indicator cells expressing a panel of coreceptors with documented expression in the central nervous system (CNS). HIV-1 isolates obtained from plasma and CSF in 28 individuals with varying viral load, CD4 T-cell counts, and with or without AIDS-defining disease were analyzed for the ability to infect NP2.CD4 cells stably expressing a panel of HIV coreceptors (CCR5, CXCR4, CCR3, CXCR6, GPR1, APJ, ChemR23, RDC-1 or BLT1). All isolates from both plasma and CSF utilized CCR5 and/or CXCR4. However, the ability to use both CCR3 and CCR5 (R3R5) was more pronounced in CSF isolates and correlated with high CSF viral load and low CD4 T-cell count. Notably, four out of five CSF isolates of subtype C origin exhibited CXCR6 use, which coincided with high CSF viral load despite preserved CD4 T-cell counts. The use of other alternative coreceptors was less pronounced. Dual-tropic R3R5 HIV-1 isolates in CSF coincide with high CSF viral load and low CD4 T-cell counts. Frequent CXCR6 use by CSF-derived subtype C isolates indicates that subtype-specific differences in coreceptor use may exist that will not be acknowledged when assessing plasma virus isolates. The findings may also bare relevance for HIV-1 replication within the CNS, and consequently, for the neuropathogenesis of AIDS.

Hypoxia modulates CCR7 expression in head and neck cancers.

PubMed

Basheer, Haneen A; Pakanavicius, Edvinas; Cooper, Patricia A; Shnyder, Steven D; Martin, Lisette; Hunter, Keith D; Vinader, Victoria; Afarinkia, Kamyar

2018-05-01

The chemokine receptor CCR7 is expressed on lymphocytes and dendritic cells and is responsible for trafficking of these cells in and out of secondary lymphoid organs. It has recently been shown that CCR7 expression is elevated in a number of cancers, including head and neck cancers, and that its expression correlates to lymph node (LN) metastasis. However, little is known about the factors that can induce CCR7 expression in head and neck cancers. We compared the protein expression and functional responses of CCR7 under normoxia and hypoxia in head and neck cancer cell lines OSC-19, FaDu, SCC-4, A-253 and Detroit-562 cultured as monolayers, spheroids, and grown in vivo as xenografts in balb/c mice. In addition, we analysed the correlation between hypoxia marker HIF-1α and CCR7 expression in a tissue microarray comprising 80 clinical samples with various stages and grades of malignant tumour and normal tissue. Under hypoxia, the expression of CCR7 is elevated in both in vitro and in vivo models. Furthermore, in malignant tissue, a correlation is observed between hypoxia marker HIF-1α and CCR7 across all clinical stages. This correlation is also strong in early histological grade of tumours. Hypoxia plays a role in the regulation of the expression of CCR7 and it may contribute to the development of a metastatic phenotype in head and neck cancers through this axis. Copyright © 2018. Published by Elsevier Ltd.

Rational CCL5 mutagenesis integration in a lactobacilli platform generates extremely potent HIV-1 blockers.

PubMed

Secchi, Massimiliano; Grampa, Valentina; Vangelista, Luca

2018-01-30

Efforts to improve existing anti-HIV-1 therapies or develop preventatives have identified CCR5 as an important target and CCL5 as an ideal scaffold to sculpt potent HIV-1 entry inhibitors. We created novel human CCL5 variants that exhibit exceptional anti-HIV-1 features using recombinant lactobacilli (exploited for live microbicide development) as a screening platform. Protein design, expression and anti-HIV-1 activity flowed in iterative cycles, with a stepwise integration of successful mutations and refinement of an initial CCL5 mutant battery towards the generation of two ultimate CCL5 derivatives, a CCR5 agonist and a CCR5 antagonist with similar anti-HIV-1 potency. The CCR5 antagonist was tested in human macrophages and against primary R5 HIV-1 strains, exhibiting cross-clade low picomolar IC 50 activity. Moreover, its successful combination with several HIV-1 inhibitors provided the ground for conceiving therapeutic and preventative anti-HIV-1 cocktails. Beyond HIV-1 infection, these CCL5 derivatives may now be tested against several inflammation-related pathologies where the CCL5:CCR5 axis plays a relevant role.

MCP-1 and CCR2 Contribute to Non-Lymphocyte-Mediated Brain Disease Induced by Fr98 Polytropic Retrovirus Infection in Mice: Role for Astrocytes in Retroviral Neuropathogenesis

PubMed Central

Peterson, Karin E.; Errett, John S.; Wei, Tao; Dimcheff, Derek E.; Ransohoff, Richard; Kuziel, William A.; Evans, Leonard; Chesebro, Bruce

2004-01-01

Virus infection of the central nervous system (CNS) often results in chemokine upregulation. Although often associated with lymphocyte recruitment, increased chemokine expression is also associated with non-lymphocyte-mediated CNS disease. In these instances, the effect of chemokine upregulation on neurological disease is unclear. In vitro, several chemokines including monocyte chemotactic protein 1 (MCP-1) protect neurons from apoptosis. Therefore, in vivo, chemokine upregulation may be a protective host response to CNS damage. Alternatively, chemokines may contribute to pathogenesis by stimulating intrinsic brain cells or recruiting macrophages to the brain. To investigate these possibilities, we studied a neurovirulent retrovirus, Fr98, that induces severe non-lymphocyte-mediated neurological disease and causes the upregulation of several chemokines that bind to chemokine receptors CCR2 and CCR5. Knockout mice deficient in CCR2 had reduced susceptibility to Fr98 pathogenesis, with significantly fewer mice developing clinical disease than did wild-type controls. In contrast, no reduction in Fr98-induced disease was observed in CCR5 knockout mice. Thus, signaling through CCR2, but not CCR5, plays an important role in Fr98-mediated pathogenesis. Three ligands for CCR2 (MCP-1, MCP-3, and MCP-5) were upregulated during Fr98 infection of the brain. Antibody-blocking experiments demonstrated that MCP-1 was important for retrovirus-induced neurological disease. In situ hybridization analysis revealed that MCP-1 was expressed by glial fibrillary acidic protein-positive astrocytes. Thus, astrocytes, previously not thought to play an effector role in the disease process were found to contribute to pathogenesis through the production of MCP-1. This study also demonstrates that chemokines can mediate pathogenesis in the CNS in the absence of lymphocytic infiltrate and gives credence to the hypothesis that chemokine upregulation is a mechanism by which retroviruses such as human immunodeficiency virus induce neurological damage. PMID:15163738

A Role for the Chemokine Receptor CCR6 in Mammalian Sperm Motility and Chemotaxis

PubMed Central

Caballero-Campo, Pedro; Buffone, Mariano G.; Benencia, Fabian; Conejo-García, José R.; Rinaudo, Paolo F.; Gerton, George L.

2013-01-01

Although recent evidence indicates that several chemokines and defensins, well-known as inflammatory mediators, are expressed in the male and female reproductive tracts, the location and functional significance of chemokine networks in sperm physiology and sperm reproductive tract interactions are poorly understood. To address this deficiency in our knowledge, we examined the expression and function in sperm of CCR6, a receptor common to several chemoattractant peptides, and screened several reproductive tract fluids for the presence of specific ligands. CCR6 protein is present in mouse and human sperm and mainly localized in the sperm tail with other minor patterns in sperm from mice (neck and acrosomal region) and men (neck and midpiece regions). As expected from the protein immunoblotting and immunofluorescence results, mouse Ccr6 mRNA is expressed in the testis. Furthermore, the Defb29 mRNA encoding the CCR6 ligand, β-defensin DEFB29, is expressed at high levels in the epididymis. As determined by protein chip analysis, several chemokines (including some that act through CCR6, such as CCL20/MIP-3α (formerly Macrophage Inflammatory Protein 3α) and protein hormones were present in human follicular fluid, endometrial secretions, and seminal plasma. In functional chemotaxis assays, capacitated human sperm exhibited a directional movement towards CCL20, and displayed modifications in motility parameters. Our data indicate that chemokine ligand/receptor interactions in the male and female genital tracts promote sperm motility and chemotaxis under non-inflammatory conditions. Therefore, some of the physiological reactions mediated by CCR6 ligands in male reproduction extend beyond a pro-inflammatory response and might find application in clinical reproduction and/or contraception. PMID:23765988

Characterization of Developmental- and Stress-Mediated Expression of Cinnamoyl-CoA Reductase in Kenaf (Hibiscus cannabinus L.)

PubMed Central

Lim, Hyoun-Sub; Park, Sang-Un; Bae, Hyeun-Jong; Natarajan, Savithiry

2014-01-01

Cinnamoyl-CoA reductase (CCR) is an important enzyme for lignin biosynthesis as it catalyzes the first specific committed step in monolignol biosynthesis. We have cloned a full length coding sequence of CCR from kenaf (Hibiscus cannabinus L.), which contains a 1,020-bp open reading frame (ORF), encoding 339 amino acids of 37.37 kDa, with an isoelectric point (pI) of 6.27 (JX524276, HcCCR2). BLAST result found that it has high homology with other plant CCR orthologs. Multiple alignment with other plant CCR sequences showed that it contains two highly conserved motifs: NAD(P) binding domain (VTGAGGFIASWMVKLLLEKGY) at N-terminal and probable catalytic domain (NWYCYGK). According to phylogenetic analysis, it was closely related to CCR sequences of Gossypium hirsutum (ACQ59094) and Populus trichocarpa (CAC07424). HcCCR2 showed ubiquitous expression in various kenaf tissues and the highest expression was detected in mature flower. HcCCR2 was expressed differentially in response to various stresses, and the highest expression was observed by drought and NaCl treatments. PMID:24723816

Cyclooxygenase-2 up-regulates CCR7 expression via AKT-mediated phosphorylation and activation of Sp1 in breast cancer cells.

PubMed

Chuang, Chun-Wei; Pan, Mei-Ren; Hou, Ming-Feng; Hung, Wen-Chun

2013-02-01

Up-regulation of cyclooxygenase-2 (COX-2) is frequently found in human cancers and is significantly associated with tumor metastasis. Our previous results demonstrate that COX-2 and its metabolite prostaglandin E2 (PGE2) stimulate the expression of CCR7 chemokine receptor via EP2/EP4 receptors to promote lymphatic invasion in breast cancer cells. In this study, we address the underlying mechanism of COX-2/PGE2-induced CCR7 expression. We find that COX-2/PGE2 increase CCR7 expression via the AKT signaling pathway in breast cancer cells. Promoter deletion and mutation assays identify the Sp1 site located at the -60/-57 region of CCR7 gene promoter is critical for stimulation. Chromatin immunoprecipitation (ChIP) assay confirms that in vivo binding of Sp1 to human CCR7 promoter is increased by COX-2 and PGE2. Knockdown of Sp1 by shRNA reduces the induction of CCR7 by PGE2. We demonstrate for the first time that AKT may directly phosphorylate Sp1 at S42, T679, and S698. Phosphorylation-mimic Sp1 protein harboring S42D, T679D, and S698D mutation strongly activates CCR7 expression. In contrast, change of these three residues to alanine completely blocks the induction of CCR7 by PGE2. Pathological investigation demonstrates that CCR7 expression is strongly associated with phospho-AKT and Sp1 in 120 breast cancer tissues. Collectively, our results demonstrate that COX-2 up-regulates CCR7 expression via AKT-mediated phosphorylation and activation of Sp1 and this pathway is highly activated in metastatic breast cancer. Copyright © 2012 Wiley Periodicals, Inc.

Repression of Ccr9 transcription in mouse T lymphocyte progenitors by the Notch signaling pathway.

PubMed

Krishnamoorthy, Veena; Carr, Tiffany; de Pooter, Renee F; Emanuelle, Akinola Olumide; Akinola, Emanuelle Olumide; Gounari, Fotini; Kee, Barbara L

2015-04-01

The chemokine receptor CCR9 controls the immigration of multipotent hematopoietic progenitor cells into the thymus to sustain T cell development. Postimmigration, thymocytes downregulate CCR9 and migrate toward the subcapsular zone where they recombine their TCR β-chain and γ-chain gene loci. CCR9 is subsequently upregulated and participates in the localization of thymocytes during their selection for self-tolerant receptor specificities. Although the dynamic regulation of CCR9 is essential for early T cell development, the mechanisms controlling CCR9 expression have not been determined. In this article, we show that key regulators of T cell development, Notch1 and the E protein transcription factors E2A and HEB, coordinately control the expression of Ccr9. E2A and HEB bind at two putative enhancers upstream of Ccr9 and positively regulate CCR9 expression at multiple stages of T cell development. In contrast, the canonical Notch signaling pathway prevents the recruitment of p300 to the putative Ccr9 enhancers, resulting in decreased acetylation of histone H3 and a failure to recruit RNA polymerase II to the Ccr9 promoter. Although Notch signaling modestly modulates the binding of E proteins to one of the two Ccr9 enhancers, we found that Notch signaling represses Ccr9 in T cell lymphoma lines in which Ccr9 transcription is independent of E protein function. Our data support the hypothesis that activation of Notch1 has a dominant-negative effect on Ccr9 transcription and that Notch1 and E proteins control the dynamic expression of Ccr9 during T cell development. Copyright © 2015 by The American Association of Immunologists, Inc.

Effect of female genital schistosomiasis and anti-schistosomal treatment on monocytes, CD4+ T-cells and CCR5 expression in the female genital tract.

PubMed

Kleppa, Elisabeth; Ramsuran, Veron; Zulu, Siphosenkosi; Karlsen, Gunn Hege; Bere, Alfred; Passmore, Jo-Ann S; Ndhlovu, Patricia; Lillebø, Kristine; Holmen, Sigve D; Onsrud, Mathias; Gundersen, Svein Gunnar; Taylor, Myra; Kjetland, Eyrun F; Ndung'u, Thumbi

2014-01-01

Schistosoma haematobium is a waterborne parasite that may cause female genital schistosomiasis (FGS), characterized by genital mucosal lesions. There is clinical and epidemiological evidence for a relationship between FGS and HIV. We investigated the impact of FGS on HIV target cell density and expression of the HIV co-receptor CCR5 in blood and cervical cytobrush samples. Furthermore we evaluated the effect of anti-schistosomal treatment on these cell populations. The study followed a case-control design with post treatment follow-up, nested in an on-going field study on FGS. Blood and cervical cytobrush samples were collected from FGS negative and positive women for flow cytometry analyses. Urine samples were investigated for schistosome ova by microscopy and polymerase chain reaction (PCR). FGS was associated with a higher frequency of CD14+ cells (monocytes) in blood (11.5% in FGS+ vs. 2.2% in FGS-, p = 0.042). Frequencies of CD4+ cells expressing CCR5 were higher in blood samples from FGS+ than from FGS- women (4.7% vs. 1.5%, p = 0.018). The CD14+ cell population decreased significantly in both compartments after anti-schistosomal treatment (p = 0.043). Although the frequency of CD4+ cells did not change after treatment, frequencies of CCR5 expression by CD4+ cells decreased significantly in both compartments (from 3.4% to 0.5% in blood, p = 0.036; and from 42.4% to 5.6% in genital samples, p = 0.025). The results support the hypothesis that FGS may increase the risk of HIV acquisition, not only through damage of the mucosal epithelial barrier, but also by affecting HIV target cell populations, and that anti-schistosomal treatment can modify this.

CCR5 gene polymorphism is a genetic risk factor for radiographic severity of rheumatoid arthritis.

PubMed

Han, S W; Sa, K H; Kim, S I; Lee, S I; Park, Y W; Lee, S S; Yoo, W H; Soe, J S; Nam, E J; Lee, J; Park, J Y; Kang, Y M

2012-11-01

The chemokine receptor [C-C chemokine receptor 5 (CCR5)] is expressed on diverse immune effecter cells and has been implicated in the pathogenesis of rheumatoid arthritis (RA). This study sought to determine whether single-nucleotide polymorphisms (SNPs) in the CCR5 gene and their haplotypes were associated with susceptibility to and severity of RA. Three hundred fifty-seven patients with RA and 383 healthy unrelated controls were recruited. Using a pyrosequencing assay, we examined four polymorphisms -1118 CTAT(ins) (/del) (rs10577983), 303 A>G (rs1799987), 927 C>T (rs1800024), and 4838 G>T (rs1800874) of the CCR5 gene, which were distributed over the promoter region as well as the 5' and 3' untranslated regions. No significant difference in the genotype, allele, and haplotype frequencies of the four selected SNPs was observed between RA patients and controls. CCR5 polymorphisms of -1118 CTAT(del) (P = 0.012; corrected P = 0.048) and 303 A>G (P = 0.012; corrected P = 0.048) showed a significant association with radiographic severity in a recessive model, and, as a result of multivariate logistic regression analysis, were found to be an independent predictor of radiographic severity. When we separated the erosion score from the total Sharp score, the statistical significance of CCR5 polymorphisms showed an increase; -1118 CTAT(ins) (/del) (P = 0.007; corrected P = 0.028) and 303 A>G (P = 0.007; corrected P = 0.028). Neither SNPs nor haplotypes of the CCR5 gene showed a significant association with joint space narrowing score. These results indicate that genetic polymorphisms of CCR5 are an independent risk factor for radiographic severity denoted by modified Sharp score, particularly joint erosion in RA. © 2012 John Wiley & Sons A/S.

Effect of Vaginal Immunization with HIVgp140 and HSP70 on HIV-1 Replication and Innate and T Cell Adaptive Immunity in Women

PubMed Central

Lewis, David J. M.; Wang, Yufei; Huo, Zhiming; Giemza, Raphaela; Babaahmady, Kaboutar; Rahman, Durdana; Shattock, Robin J.; Singh, Mahavir

2014-01-01

ABSTRACT The international effort to prevent HIV-1 infection by vaccination has failed to develop an effective vaccine. The aim of this vaccine trial in women was to administer by the vaginal mucosal route a vaccine consisting of HIV-1 gp140 linked to the chaperone 70-kDa heat shock protein (HSP70). The primary objective was to determine the safety of the vaccine. The secondary objective was to examine HIV-1 infectivity ex vivo and innate and adaptive immunity to HIV-1. Protocol-defined female volunteers were recruited. HIV-1 CN54gp140 linked to HSP70 was administered by the vaginal route. Significant adverse reactions were not detected. HIV-1 was significantly inhibited ex vivo in postimmunization CD4+ T cells compared with preimmunization CD4+ T cells. The innate antiviral restrictive factor APOBEC3G was significantly upregulated, as were CC chemokines which induce downregulation of CCR5 in CD4+ T cells. Indeed, a significant inverse correlation between the proportion of CCR5+ T cells and the concentration of CCL-3 or CCL-5 was found. Importantly, the upregulation of APOBEC3G showed a significant inverse correlation, whereas CCR5 exhibited a trend to correlate with inhibition of HIV-1 infection (r = 0.51). Furthermore, specific CD4+ and CD8+ T cell proliferative responses were significantly increased and CD4+ T cells showed a trend to have an inverse correlation with the viral load (r = −0.60). However, HIVgp140-specific IgG or IgA antibodies were not detected. The results provide proof of concept that an innate mechanism consisting of CC chemokines, APOBEC3G, and adaptive immunity by CD4 and CD8 T cells might be involved in controlling HIV-1 infectivity following vaginal mucosal immunization in women. (This study has been registered at ClinicalTrials.gov under registration no. NCT01285141.) IMPORTANCE Vaginal immunization of women with a vaccine consisting of HIVgp140 linked to the 70-kDa heat shock protein (HSP70) elicited ex vivo significant inhibition of HIV-1 replication in postimmunization CD4+ T cells compared with that in preimmunization peripheral blood mononuclear cells. There were no significant adverse events. The vaccine induced the significant upregulation of CC chemokines and the downmodulation of CCR5 expression in CD4+ T cells, as well as an inverse correlation between them. Furthermore, the level of CCR5 expression was directly correlated with the viral load, consistent with the protective mechanism in which a decrease in CCR5 molecules on CD4+ T cells decreases HIV-1 envelope binding. Expression of the antiviral restriction factor APOBEC3G was inversely correlated with the viral load, suggesting that it may inhibit intracellular HIV-1 replication. Both CD4+ and CD8+ T cells showed HIVgp140- and HSP70-specific proliferation. A strong inverse correlation between the proportion of CC chemokine-modulated CCR5-expressing CD4+ T cells and the stimulation of CD4+ or CD8+ T cell proliferation by HIVgp140 was found, demonstrating a significant interaction between innate and adaptive immunity. This is the first clinical trial of vaginal immunization in women using only HIVgp140 and HSP70 administered by the mucosal route (3 times) in which a dual innate protective mechanism was induced and enhanced by significant adaptive CD4+ and CD8+ T cell proliferative responses. PMID:25008917

Effect of vaginal immunization with HIVgp140 and HSP70 on HIV-1 replication and innate and T cell adaptive immunity in women.

PubMed

Lewis, David J M; Wang, Yufei; Huo, Zhiming; Giemza, Raphaela; Babaahmady, Kaboutar; Rahman, Durdana; Shattock, Robin J; Singh, Mahavir; Lehner, Thomas

2014-10-01

The international effort to prevent HIV-1 infection by vaccination has failed to develop an effective vaccine. The aim of this vaccine trial in women was to administer by the vaginal mucosal route a vaccine consisting of HIV-1 gp140 linked to the chaperone 70-kDa heat shock protein (HSP70). The primary objective was to determine the safety of the vaccine. The secondary objective was to examine HIV-1 infectivity ex vivo and innate and adaptive immunity to HIV-1. Protocol-defined female volunteers were recruited. HIV-1 CN54gp140 linked to HSP70 was administered by the vaginal route. Significant adverse reactions were not detected. HIV-1 was significantly inhibited ex vivo in postimmunization CD4(+) T cells compared with preimmunization CD4(+) T cells. The innate antiviral restrictive factor APOBEC3G was significantly upregulated, as were CC chemokines which induce downregulation of CCR5 in CD4(+) T cells. Indeed, a significant inverse correlation between the proportion of CCR5(+) T cells and the concentration of CCL-3 or CCL-5 was found. Importantly, the upregulation of APOBEC3G showed a significant inverse correlation, whereas CCR5 exhibited a trend to correlate with inhibition of HIV-1 infection (r = 0.51). Furthermore, specific CD4(+) and CD8(+) T cell proliferative responses were significantly increased and CD4(+) T cells showed a trend to have an inverse correlation with the viral load (r = -0.60). However, HIVgp140-specific IgG or IgA antibodies were not detected. The results provide proof of concept that an innate mechanism consisting of CC chemokines, APOBEC3G, and adaptive immunity by CD4 and CD8 T cells might be involved in controlling HIV-1 infectivity following vaginal mucosal immunization in women. (This study has been registered at ClinicalTrials.gov under registration no. NCT01285141.) Importance: Vaginal immunization of women with a vaccine consisting of HIVgp140 linked to the 70-kDa heat shock protein (HSP70) elicited ex vivo significant inhibition of HIV-1 replication in postimmunization CD4(+) T cells compared with that in preimmunization peripheral blood mononuclear cells. There were no significant adverse events. The vaccine induced the significant upregulation of CC chemokines and the downmodulation of CCR5 expression in CD4(+) T cells, as well as an inverse correlation between them. Furthermore, the level of CCR5 expression was directly correlated with the viral load, consistent with the protective mechanism in which a decrease in CCR5 molecules on CD4(+) T cells decreases HIV-1 envelope binding. Expression of the antiviral restriction factor APOBEC3G was inversely correlated with the viral load, suggesting that it may inhibit intracellular HIV-1 replication. Both CD4(+) and CD8(+) T cells showed HIVgp140- and HSP70-specific proliferation. A strong inverse correlation between the proportion of CC chemokine-modulated CCR5-expressing CD4(+) T cells and the stimulation of CD4(+) or CD8(+) T cell proliferation by HIVgp140 was found, demonstrating a significant interaction between innate and adaptive immunity. This is the first clinical trial of vaginal immunization in women using only HIVgp140 and HSP70 administered by the mucosal route (3 times) in which a dual innate protective mechanism was induced and enhanced by significant adaptive CD4(+) and CD8(+) T cell proliferative responses. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Depletion of host CCR7(+) dendritic cells prevented donor T cell tissue tropism in anti-CD3-conditioned recipients.

PubMed

He, Wei; Racine, Jeremy J; Johnston, Heather F; Li, Xiaofan; Li, Nainong; Cassady, Kaniel; Liu, Can; Deng, Ruishu; Martin, Paul; Forman, Stephen; Zeng, Defu

2014-07-01

We reported previously that anti-CD3 mAb treatment before hematopoietic cell transplantation (HCT) prevented graft-versus-host disease (GVHD) and preserved graft-versus-leukemia (GVL) effects in mice. These effects were associated with downregulated donor T cell expression of tissue-specific homing and chemokine receptors, marked reduction of donor T cell migration into GVHD target tissues, and deletion of CD103(+) dendritic cells (DCs) in mesenteric lymph nodes (MLN). MLN CD103(+) DCs and peripheral lymph node (PLN) DCs include CCR7(+) and CCR7(-) subsets, but the role of these DC subsets in regulating donor T cell expression of homing and chemokine receptors remain unclear. Here, we show that recipient CCR7(+), but not CCR7(-), DCs in MLN induced donor T cell expression of gut-specific homing and chemokine receptors in a retinoid acid-dependent manner. CCR7 regulated activated DC migration from tissue to draining lymph node, but it was not required for the ability of DCs to induce donor T cell expression of tissue-specific homing and chemokine receptors. Finally, anti-CD3 treatment depleted CCR7(+) but not CCR7(-) DCs by inducing sequential expansion and apoptosis of CCR7(+) DCs in MLN and PLN. Apoptosis of CCR7(+) DCs was associated with DC upregulation of Fas expression and natural killer cell but not T, B, or dendritic cell upregulation of FasL expression in the lymph nodes. These results suggest that depletion of CCR7(+) host-type DCs, with subsequent inhibition of donor T cell migration into GVHD target tissues, can be an effective approach in prevention of acute GVHD and preservation of GVL effects. Copyright © 2014 American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.

Exaggerated Hepatic Injury Due to Acetaminophen Challenge in Mice Lacking C-C Chemokine Receptor 2

PubMed Central

Hogaboam, Cory M.; Bone-Larson, Cynthia L.; Steinhauser, Matthew L.; Matsukawa, Akihiro; Gosling, Jennifa; Boring, Landin; Charo, Israel F.; Simpson, Kenneth J.; Lukacs, Nicholas W.; Kunkel, Steven L.

2000-01-01

Monocyte chemoattractant protein-1 is one of the major C-C chemokines that has been implicated in liver injury. The C-C chemokine receptor, CCR2, has been identified as the primary receptor that mediates monocyte chemoattractant protein-1 (MCP-1) responses in the mouse. Accordingly, the present study addressed the role of CCR2 in mice acutely challenged with acetaminophen (APAP). Mice genetically deficient in CCR2 (CCR2−/−) and their wild-type counterparts (CCR2+/+) were fasted for 10 hours before receiving an intraperitoneal injection of APAP (300 mg/kg). Liver and serum samples were removed from both groups of mice before and at 24 and 48 hours post APAP. Significantly elevated levels of MCP-1 were detected in liver samples from CCR2+/+ and CCR2−/− mice at 24 hours post-APAP. Although CCR2+/+ mice exhibited no liver injury at any time after receiving APAP, CCR2−/− mice exhibited marked evidence of necrotic and TUNEL-positive cells in the liver, particularly at 24 hours post-APAP. Enzyme-linked immunosorbent assay analysis of liver homogenates from both groups of mice at the 24 hours time point revealed that liver tissue from CCR2−/− mice contained significantly greater amounts of immunoreactive IFN-γ and TNF-α. The in vivo immunoneutralization of IFN-γ or TNF-α significantly attenuated APAP-induced liver injury in CCR2−/− mice and increased hepatic IL-13 levels. Taken together, these findings demonstrate that CCR2 expression in the liver provides a hepatoprotective effect through its regulation of cytokine generation during APAP challenge. PMID:10751350

Quantitative structure-activity relationships studies of CCR5 inhibitors and toxicity of aromatic compounds using gene expression programming.

PubMed

Shi, Weimin; Zhang, Xiaoya; Shen, Qi

2010-01-01

Quantitative structure-activity relationship (QSAR) study of chemokine receptor 5 (CCR5) binding affinity of substituted 1-(3,3-diphenylpropyl)-piperidinyl amides and ureas and toxicity of aromatic compounds have been performed. The gene expression programming (GEP) was used to select variables and produce nonlinear QSAR models simultaneously using the selected variables. In our GEP implementation, a simple and convenient method was proposed to infer the K-expression from the number of arguments of the function in a gene, without building the expression tree. The results were compared to those obtained by artificial neural network (ANN) and support vector machine (SVM). It has been demonstrated that the GEP is a useful tool for QSAR modeling. Copyright 2009 Elsevier Masson SAS. All rights reserved.

Intestinal double-positive CD4+CD8+ T cells are highly activated memory cells with an increased capacity to produce cytokines.

PubMed

Pahar, Bapi; Lackner, Andrew A; Veazey, Ronald S

2006-03-01

Peripheral blood and intestinal CD4+CD8+ double-positive (DP) T cells have been described in several species including humans, but their function and immunophenotypic characteristics are still not clearly understood. Here we demonstrate that DP T cells are abundant in the intestinal lamina propria of normal rhesus macaques (Macaca mulatta). Moreover, DP T cells have a memory phenotype and are capable of producing different and/or higher levels of cytokines and chemokines in response to mitogen stimulation compared to CD4+ single-positive T cells. Intestinal DP T cells are also highly activated and have higher expression of CCR5, which makes them preferred targets for simian immunodeficiency virus/HIV infection. Increased levels of CD69, CD25 and HLA-DR, and lower CD62L expression were found on intestinal DP T cells populations compared to CD4+ single-positive T cells. Collectively, these findings demonstrate that intestinal and peripheral blood DP T cells are effector cells and may be important in regulating immune responses, which distinguishes them from the immature DP cells found in the thymus. Finally, these intestinal DP T cells may be important target cells for HIV infection and replication due to their activation, memory phenotype and high expression of CCR5.

Not5-dependent co-translational assembly of Ada2 and Spt20 is essential for functional integrity of SAGA

PubMed Central

Kassem, Sari; Villanyi, Zoltan

2017-01-01

Abstract Acetylation of histones regulates gene expression in eukaryotes. In the yeast Saccharomyces cerevisiae it depends mainly upon the ADA and SAGA histone acetyltransferase complexes for which Gcn5 is the catalytic subunit. Previous screens have determined that global acetylation is reduced in cells lacking subunits of the Ccr4–Not complex, a global regulator of eukaryotic gene expression. In this study we have characterized the functional connection between the Ccr4–Not complex and SAGA. We show that SAGA mRNAs encoding a core set of SAGA subunits are tethered together for co-translational assembly of the encoded proteins. Ccr4–Not subunits bind SAGA mRNAs and promote the co-translational assembly of these subunits. This is needed for integrity of SAGA. In addition, we determine that a glycolytic enzyme, the glyceraldehyde-3-phosphate dehydrogenase Tdh3, a prototypical moonlighting protein, is tethered at this site of Ccr4–Not-dependent co-translational SAGA assembly and functions as a chaperone. PMID:28180299

Enhanced Chemokine Receptor Recycling and Impaired S1P1 Expression Promote Leukemic Cell Infiltration of Lymph Nodes in Chronic Lymphocytic Leukemia.

PubMed

Patrussi, Laura; Capitani, Nagaja; Martini, Veronica; Pizzi, Marco; Trimarco, Valentina; Frezzato, Federica; Marino, Filippo; Semenzato, Gianpietro; Trentin, Livio; Baldari, Cosima T

2015-10-01

Lymphocyte trafficking is orchestrated by chemokine and sphingosine 1-phosphate (S1P) receptors that enable homing and egress from secondary lymphoid organs (SLO). These receptors undergo rapid internalization and plasma membrane recycling to calibrate cellular responses to local chemoattractants. Circulating chronic lymphocytic leukemia (CLL) cells display an abnormal increase in the surface levels of the homing receptors CCR7 and CXCR4 concomitant with low S1P receptor 1 (S1P1) expression. In this study, we investigated the role of receptor recycling on CXCR4/CCR7 surface levels in CLL cells and addressed the impact of quantitative alterations of these receptors and S1P1 on the ability of leukemic cells to accumulate in SLOs. We show that recycling accounts, to a major extent, for the high levels of surface CXCR4/CCR7 on CLL cells. In addition, increased expression of these receptors, together with S1P1 deficiency, is detectable not only in circulating leukemic cells, but also in SLOs of CLL patients with lymphoadenopathy. We further provide evidence that ibrutinib, a Btk inhibitor that promotes mobilization of leukemic cells from SLOs, normalizes the imbalance between CXCR4/CCR7 and S1P1. Taken together, our results highlight the relevance of chemokine and S1P receptor recycling in CLL pathogenesis and clinical outcome. ©2015 American Association for Cancer Research.

Up-regulation of CCL17, CCL22 and CCR4 in drug-induced maculopapular exanthema.

PubMed

Tapia, B; Morel, E; Martín-Díaz, M-A; Díaz, R; Alves-Ferreira, J; Rubio, P; Padial, A; Bellón, T

2007-05-01

Maculopapular exanthema has been reported to be the most frequently drug-induced cutaneous reaction. Although T lymphocytes are involved in the pathomechanism of this disease, little is know about the recruitment of these cells to the skin. The aim of this work is to study the role of the chemokines TARC/CCL17 and MDC/CCL22 in the lymphocyte trafficking to affected skin in drug-induced exanthemas. Real-time PCR was performed to quantify gene expression levels of CCL17, CCL22 and their receptor CCR4 in lesional skin biopsies and in peripheral blood mononuclear cells from patients. CCL27 and CCL22 proteins were detected in the skin by immunochemistry. Protein expression of CCR4 was determined by flow cytometry in peripheral blood lymphocytes. Functional migration assays to CCL17 and CCL22 were assessed to compare the migratory responses of peripheral blood lymphocytes from patients and healthy subjects. CCL17 and CCL22 were up-regulated in maculopapular exanthema-affected skin. CCR4 mRNA levels and protein expression were increased in peripheral blood mononuclear cells during the acute phase of the disease. The increased expression of the receptor was consistent with a higher response of peripheral blood lymphocytes to CCL17 and CCL22 compared with the migratory response in healthy donors. TARC/CCL17 and MDC/CCL22 might cooperate in attracting T lymphocytes to skin in drug-induced maculopapular exanthemas.

Editor's Highlight: CCR2 Regulates Inflammatory Cell Accumulation in the Lung and Tissue Injury following Ozone Exposure.

PubMed

Francis, Mary; Groves, Angela M; Sun, Richard; Cervelli, Jessica A; Choi, Hyejeong; Laskin, Jeffrey D; Laskin, Debra L

2017-02-01

Ozone-induced lung injury is associated with an accumulation of activated macrophages in the lung. Chemokine receptor CCR2 mediates the migration of inflammatory monocytes/macrophages to sites of tissue injury. It is also required for monocyte egress from the bone marrow. In the present studies, we analyzed the role of CCR2 in inflammatory cell trafficking to the lung in response to ozone. Treatment of mice with ozone (0.8 ppm, 3 h) resulted in increases in proinflammatory CCR2 + macrophages in the lung at 24 h, as well as proinflammatory CD11b  +  Ly6C Hi and iNOS +  macrophages at 24 and 48 h. Mannose receptor +  anti-inflammatory macrophages were also observed in the lung 24 and 48 h post-ozone. Loss of CCR2 was associated with reduced numbers of proinflammatory macrophages in the lung and decreased expression of the proinflammatory cytokines, IL-1β and TNFα. Decreases in anti-inflammatory CD11b  +  Ly6C Lo macrophages were also observed in lungs of CCR2 -/- mice treated with ozone, whereas mannose receptor +  macrophage accumulation was delayed; conversely, CX3CL1 and CX3CR1 were upregulated. Changes in lung macrophage subpopulations and inflammatory gene expression in CCR2 -/- mice were correlated with reduced ozone toxicity and oxidative stress, as measured by decreases in bronchoalveolar lavage protein content and reduced lung expression of heme-oxygenase-1, 4-hydroxynonenal and cytochrome b5. These data demonstrate that CCR2 plays a role in both pro- and anti-inflammatory macrophage accumulation in the lung following ozone exposure. The fact that ozone-induced lung injury and oxidative stress are reduced in CCR2 -/- mice suggests more prominent effects on proinflammatory macrophages. © The Author 2016. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Editor’s Highlight: CCR2 Regulates Inflammatory Cell Accumulation in the Lung and Tissue Injury following Ozone Exposure

PubMed Central

Francis, Mary; Groves, Angela M.; Sun, Richard; Cervelli, Jessica A.; Choi, Hyejeong; Laskin, Jeffrey D.; Laskin, Debra L.

2017-01-01

Ozone-induced lung injury is associated with an accumulation of activated macrophages in the lung. Chemokine receptor CCR2 mediates the migration of inflammatory monocytes/macrophages to sites of tissue injury. It is also required for monocyte egress from the bone marrow. In the present studies, we analyzed the role of CCR2 in inflammatory cell trafficking to the lung in response to ozone. Treatment of mice with ozone (0.8 ppm, 3 h) resulted in increases in proinflammatory CCR2+ macrophages in the lung at 24 h, as well as proinflammatory CD11b + Ly6CHi and iNOS+ macrophages at 24 and 48 h. Mannose receptor+ anti-inflammatory macrophages were also observed in the lung 24 and 48 h post-ozone. Loss of CCR2 was associated with reduced numbers of proinflammatory macrophages in the lung and decreased expression of the proinflammatory cytokines, IL-1β and TNFα. Decreases in anti-inflammatory CD11b + Ly6CLo macrophages were also observed in lungs of CCR2−/− mice treated with ozone, whereas mannose receptor+ macrophage accumulation was delayed; conversely, CX3CL1 and CX3CR1 were upregulated. Changes in lung macrophage subpopulations and inflammatory gene expression in CCR2−/− mice were correlated with reduced ozone toxicity and oxidative stress, as measured by decreases in bronchoalveolar lavage protein content and reduced lung expression of heme-oxygenase-1, 4-hydroxynonenal and cytochrome b5. These data demonstrate that CCR2 plays a role in both pro- and anti-inflammatory macrophage accumulation in the lung following ozone exposure. The fact that ozone-induced lung injury and oxidative stress are reduced in CCR2−/− mice suggests more prominent effects on proinflammatory macrophages. PMID:27837169

Characterization of a cinnamoyl-CoA reductase 1 (CCR1) mutant in maize: effects on lignification, fibre development, and global gene expression

PubMed Central

Tamasloukht, Barek; Wong Quai Lam, Mary Sarah-Jane; Martinez, Yves; Tozo, Koffi; Barbier, Odile; Jourda, Cyril; Jauneau, Alain; Borderies, Gisèle; Balzergue, Sandrine; Renou, Jean-Pierre; Huguet, Stéphanie; Martinant, Jean Pierre; Tatout, Christophe; Lapierre, Catherine; Barrière, Yves; Goffner, Deborah; Pichon, Magalie

2011-01-01

Cinnamoyl-CoA reductase (CCR), which catalyses the first committed step of the lignin-specific branch of monolignol biosynthesis, has been extensively characterized in dicot species, but few data are available in monocots. By screening a Mu insertional mutant collection in maize, a mutant in the CCR1 gene was isolated named Zmccr1–. In this mutant, CCR1 gene expression is reduced to 31% of the residual wild-type level. Zmccr1– exhibited enhanced digestibility without compromising plant growth and development. Lignin analysis revealed a slight decrease in lignin content and significant changes in lignin structure. p-Hydroxyphenyl units were strongly decreased and the syringyl/guaiacyl ratio was slightly increased. At the cellular level, alterations in lignin deposition were mainly observed in the walls of the sclerenchymatic fibre cells surrounding the vascular bundles. These cell walls showed little to no staining with phloroglucinol. These histochemical changes were accompanied by an increase in sclerenchyma surface area and an alteration in cell shape. In keeping with this cell type-specific phenotype, transcriptomics performed at an early stage of plant development revealed the down-regulation of genes specifically associated with fibre wall formation. To the present authors’ knowledge, this is the first functional characterization of CCR1 in a grass species. PMID:21493812

The CCL3L1-CCR5 genotype influences the development of AIDS, but not HIV susceptibility or the response to HAART

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bhattacharya, Tanmoy; Stanton, Jennifer; Kim, Eun - Young

2008-01-01

A selective advantage against infectious diseases such as HIV/AIDS is associated with differences in the genes relevant to immunity and virus replication. The CC chemokine receptor 5 (CCR5), the principal coreceptor for HIV, and its chemokine ligands, including CCL3L1, influences the CD4+ target cells susceptibility to infection. The CCL3L1 gene is in a region of segmental duplication on the q-arm of human chromosome 17. Increased numbers of CCL3L1 gene copies that affect the gene expression phenotype might have substantial protective effects. Here we show that the population-specific CCL3L1 gene copy number and the CCR5 {Delta}32 protein-inactivating deletion that categorizes themore » CCL3L1-CCR5 genotype do not influence HIV/AIDS susceptibility or the robustness of immune recovery after the initiation of highly active antiretroviral therapy (HAART).« less

CCL20 and β-defensin-2 induce arrest of human Th17 cells on inflamed endothelium in vitro under flow conditions.

PubMed

Ghannam, Soufiane; Dejou, Cécile; Pedretti, Nathalie; Giot, Jean-Philipe; Dorgham, Karim; Boukhaddaoui, Hassan; Deleuze, Virginie; Bernard, François-Xavier; Jorgensen, Christian; Yssel, Hans; Pène, Jérôme

2011-02-01

CCR6 is a chemokine receptor that is expressed at the cell surface of Th17 cells, an IL-17- and IL-22-secreting population of CD4(+) T cells with antipathogenic, as well as inflammatory, properties. In the current study, we have determined the involvement of CCR6 in human Th17 lymphocyte migration toward inflamed tissue by analyzing the capacity of its ligands to induce arrest of these cells onto inflamed endothelium in vitro under flow conditions. We show that polarized, in situ-differentiated, skin-derived Th17 clones activated via the TCR-CD3 complex produce CCL20 in addition to IL-17 and IL-22. The latter cytokines induce, in a synergic fashion, the production of human β-defensin (hBD)-2, but neither hBD-1 nor hBD-3, by epidermal keratinocytes. Both CCL20 and hBD-2 are capable of inducing the arrest of Th17 cells, but not Th1 or Th2 cells, on HUVEC in an CD54-dependent manner that is CCR6 specific and independent from the expression of CXCR4, reported to be an alternative receptor for hBD-2. In addition, Ag-specific activation induces a transient loss of CCR6 expression, both at the transcriptional and protein level, which occurs with slow kinetics and is not due to endogenous CCL20-mediated internalization of CCR6. Together, these results indicate that Ag-specific activation will initially contribute to CCR6-mediated Th17 cell trafficking toward and sequestration in inflamed tissue, but that it eventually results in a transitory state of nonresponsiveness to further stimulation of these cells with CCR6 ligands, thus permitting their subsequent migration out of the inflamed site.

Increased Transendothelial Transport of CCL3 Is Insufficient to Drive Immune Cell Transmigration through the Blood-Brain Barrier under Inflammatory Conditions In Vitro.

PubMed

De Laere, Maxime; Sousa, Carmelita; Meena, Megha; Buckinx, Roeland; Timmermans, Jean-Pierre; Berneman, Zwi; Cools, Nathalie

2017-01-01

Many neuroinflammatory diseases are characterized by massive immune cell infiltration into the central nervous system. Identifying the underlying mechanisms could aid in the development of therapeutic strategies specifically interfering with inflammatory cell trafficking. To achieve this, we implemented and validated a blood-brain barrier (BBB) model to study chemokine secretion, chemokine transport, and leukocyte trafficking in vitro. In a coculture model consisting of a human cerebral microvascular endothelial cell line and human astrocytes, proinflammatory stimulation downregulated the expression of tight junction proteins, while the expression of adhesion molecules and chemokines was upregulated. Moreover, chemokine transport across BBB cocultures was upregulated, as evidenced by a significantly increased concentration of the inflammatory chemokine CCL3 at the luminal side following proinflammatory stimulation. CCL3 transport occurred independently of the chemokine receptors CCR1 and CCR5, albeit that migrated cells displayed increased expression of CCR1 and CCR5. However, overall leukocyte transmigration was reduced in inflammatory conditions, although higher numbers of leukocytes adhered to activated endothelial cells. Altogether, our findings demonstrate that prominent barrier activation following proinflammatory stimulation is insufficient to drive immune cell recruitment, suggesting that additional traffic cues are crucial to mediate the increased immune cell infiltration seen in vivo during neuroinflammation.

CCR2-dependent Gr1high monocytes promote kidney injury in shiga toxin-induced hemolytic uremic syndrome in mice.

PubMed

Pohl, Judith-Mira; Volke, Julia K; Thiebes, Stephanie; Brenzel, Alexandra; Fuchs, Kerstin; Beziere, Nicolas; Ehrlichmann, Walter; Pichler, Bernd J; Squire, Anthony; Gueler, Faikah; Engel, Daniel R

2018-06-01

The hemolytic uremic syndrome (HUS) is a life-threatening disease of the kidney that is induced by shiga toxin-producing E.coli. Major changes in the monocytic compartment and in CCR2-binding chemokines have been observed. However, the specific contribution of CCR2-dependent Gr1 high monocytes is unknown. To investigate the impact of these monocytes during HUS, we injected a combination of LPS and shiga toxin into mice. We observed an impaired kidney function and elevated levels of the CCR2-binding chemokine CCL2 after shiga toxin/LPS- injection, thus suggesting Gr1 high monocyte infiltration into the kidney. Indeed, the number of Gr1 high monocytes was strongly increased one day after HUS induction. Moreover, these cells expressed high levels of CD11b suggesting activation after tissue entry. Non-invasive PET-MR imaging revealed kidney injury mainly in the kidney cortex and this damage coincided with the detection of Gr1 high monocytes. Lack of Gr1 high monocytes in Ccr2-deficient animals reduced neutrophil gelatinase-associated lipocalin and blood urea nitrogen levels. Moreover, the survival of Ccr2-deficient animals was significantly improved. Conclusively, this study demonstrates that CCR2-dependent Gr1 high monocytes contribute to the kidney injury during HUS and targeting these cells is beneficial during this disease. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Role of Eotaxin-1 (CCL11) and CC chemokine receptor 3 (CCR3) in bleomycin-induced lung injury and fibrosis.

PubMed

Huaux, Francois; Gharaee-Kermani, M; Liu, Tianju; Morel, Valérie; McGarry, Bridget; Ullenbruch, Matt; Kunkel, Steven L; Wang, Jun; Xing, Zhou; Phan, Sem H

2005-12-01

Eotaxin-1/CCL11 and its receptor CCR3 are involved in recruitment of eosinophils to diverse tissues, but their role in eosinophil recruitment in pulmonary fibrosis is unclear. The present study examined the pulmonary expression of CCL11 and CCR3 during bleomycin (blm)-induced lung injury and determined their importance in the recruitment of inflammatory cells and the development of lung fibrosis. In mice, blm induced a marked pulmonary expression of CCL11 and CCR3. Immunostaining for CCR3 revealed that this receptor was not only expressed by eosinophils but also by neutrophils. CCL11-deficient (CCL11(-/-)) mice developed significantly reduced pulmonary fibrosis. Expression of profibrotic cytokines such as transforming growth factor-beta1 was diminished in the absence of CCL11. Furthermore, increased lung expression of CCL11 significantly enhanced blm-induced lung fibrosis and production of profibrotic cytokines. These effects were also associated with an increase of eosinophil and neutrophil pulmonary infiltration. In contrast, mice treated with neutralizing CCR3 antibodies developed significantly reduced pulmonary fibrosis, eosinophilia, neutrophilia, and expression of profibrotic cytokines. Together, these data suggest that CCL11 and CCR3 are important in the pulmonary recruitment of granulocytes and play significant pathogenic roles in blm-induced lung fibrosis.

Role of Eotaxin-1 (CCL11) and CC Chemokine Receptor 3 (CCR3) in Bleomycin-Induced Lung Injury and Fibrosis

PubMed Central

Huaux, Francois; Gharaee-Kermani, M.; Liu, Tianju; Morel, Valérie; McGarry, Bridget; Ullenbruch, Matt; Kunkel, Steven L.; Wang, Jun; Xing, Zhou; Phan, Sem H.

2005-01-01

Eotaxin-1/CCL11 and its receptor CCR3 are involved in recruitment of eosinophils to diverse tissues, but their role in eosinophil recruitment in pulmonary fibrosis is unclear. The present study examined the pulmonary expression of CCL11 and CCR3 during bleomycin (blm)-induced lung injury and determined their importance in the recruitment of inflammatory cells and the development of lung fibrosis. In mice, blm induced a marked pulmonary expression of CCL11 and CCR3. Immunostaining for CCR3 revealed that this receptor was not only expressed by eosinophils but also by neutrophils. CCL11-deficient (CCL11−/−) mice developed significantly reduced pulmonary fibrosis. Expression of profibrotic cytokines such as transforming growth factor-β1 was diminished in the absence of CCL11. Furthermore, increased lung expression of CCL11 significantly enhanced blm-induced lung fibrosis and production of profibrotic cytokines. These effects were also associated with an increase of eosinophil and neutrophil pulmonary infiltration. In contrast, mice treated with neutralizing CCR3 antibodies developed significantly reduced pulmonary fibrosis, eosinophilia, neutrophilia, and expression of profibrotic cytokines. Together, these data suggest that CCL11 and CCR3 are important in the pulmonary recruitment of granulocytes and play significant pathogenic roles in blm-induced lung fibrosis. PMID:16314464

The Effect of Post-Resistance Exercise Amino Acids on Plasma MCP-1 and CCR2 Expression

PubMed Central

Wells, Adam J.; Hoffman, Jay R.; Jajtner, Adam R.; Varanoske, Alyssa N.; Church, David D.; Gonzalez, Adam M.; Townsend, Jeremy R.; Boone, Carleigh H.; Baker, Kayla M.; Beyer, Kyle S.; Mangine, Gerald T.; Oliveira, Leonardo P.; Fukuda, David H.; Stout, Jeffrey R.

2016-01-01

The recruitment and infiltration of classical monocytes into damaged muscle is critical for optimal tissue remodeling. This study examined the effects of an amino acid supplement on classical monocyte recruitment following an acute bout of lower body resistance exercise. Ten resistance-trained men (24.7 ± 3.4 years; 90.1 ± 11.3 kg; 176.0 ± 4.9 cm) ingested supplement (SUPP) or placebo (PL) immediately post-exercise in a randomized, cross-over design. Blood samples were obtained at baseline (BL), immediately (IP), 30-min (30P), 1-h (1H), 2-h (2H), and 5-h (5H) post-exercise to assess plasma concentrations of monocyte chemoattractant protein 1 (MCP-1), myoglobin, cortisol and insulin concentrations; and expressions of C-C chemokine receptor-2 (CCR2), and macrophage-1 antigen (CD11b) on classical monocytes. Magnitude-based inferences were used to provide inferences on the true effects of SUPP compared to PL. Changes in myoglobin, cortisol, and insulin concentrations were similar between treatments. Compared to PL, plasma MCP-1 was “very likely greater” (98.1% likelihood effect) in SUPP at 2H. CCR2 expression was “likely greater” at IP (84.9% likelihood effect), “likely greater” at 1H (87.7% likelihood effect), “very likely greater” at 2H (97.0% likelihood effect), and “likely greater” at 5H (90.1% likelihood effect) in SUPP, compared to PL. Ingestion of SUPP did not influence CD11b expression. Ingestion of an amino acid supplement immediately post-exercise appears to help maintain plasma MCP-1 concentrations and augment CCR2 expression in resistance trained men. PMID:27384580

CCR2-V64I genetic polymorphism: a possible involvement in HER2+ breast cancer.

PubMed

Banin-Hirata, Bruna Karina; Losi-Guembarovski, Roberta; Oda, Julie Massayo Maeda; de Oliveira, Carlos Eduardo Coral; Campos, Clodoaldo Zago; Mazzuco, Tânia Longo; Borelli, Sueli Donizete; Ceribelli, Jesus Roberto; Watanabe, Maria Angelica Ehara

2016-05-01

Many tumor cells express chemokines and chemokine receptors, and these molecules can affect both tumor progression and anti-tumor immune response. Genetic polymorphisms of some chemokine receptors were found to be closely related to malignant tumors, especially in metastasis process, including breast cancer (BC). Considering this, it was investigated a possible role for CCR2-V64I (C-C chemokine receptor 2) and CCR5-Δ32 (C-C chemokine receptor 5) genetic variants in BC context. Patients were divided into subgroups according to immunohistochemical profile of estrogen (ER) and progesterone (PR) receptors and the human epidermal growth factor receptor 2 (HER2) overexpression. No significant associations were found in relation to susceptibility (CCR2-V64I: OR 1.32; 95 % CI 0.57-3.06; CCR5-∆32: OR 1.04; 95 % CI 0.60-1.81), clinical outcome (tumor size, lymph nodes commitment and/or distant metastasis, TNM staging and nuclear grade) or therapeutic response (recurrence and survival). However, it was found a significant correlation between CCR2-V64I allelic variant and HER2 immunohistochemical positive samples (p = 0.026). All in all, we demonstrate, for the first time, a positive correlation between CCR2 receptor gene polymorphism and a subgroup of BC related to poor prognosis, which deserves further investigation in larger samples for validation.

Simultaneous Down-Regulation of Caffeic/5-Hydroxy Ferulic Acid-O-Methyltransferase I and Cinnamoyl-Coenzyme A Reductase in the Progeny from a Cross between Tobacco Lines Homozygous for Each Transgene. Consequences for Plant Development and Lignin Synthesis1

PubMed Central

Pinçon, Gaelle; Chabannes, Matthieu; Lapierre, Catherine; Pollet, Brigitte; Ruel, Katia; Joseleau, Jean-Paul; Boudet, Alain M.; Legrand, Michel

2001-01-01

Inhibition of specific lignin biosynthetic steps by antisense strategy has previously been shown to alter lignin content and/or structure. In this work, homozygous tobacco (Nicotiana tabacum) lines transformed with cinnamoyl-coenzyme A reductase (CCR) or caffeic acid/5-hydroxy ferulic acid-O-methyltransferase I (COMT I) antisense sequences have been crossed and enzyme activities, lignin synthesis, and cell wall structure of the progeny have been analyzed. In single transformed parents, CCR inhibition did not affect COMT I expression, whereas marked increases in CCR activity were observed in COMT I antisense plants, suggesting potential cross talk between some genes of the pathway. In the progeny, both CCR and COMT I activities were shown to be markedly decreased due to the simultaneous repression of the two genes. In these double transformants, the lignin profiles were dependent on the relative extent of down-regulation of each individual enzyme. For the siblings issued from a strongly repressed antisense CCR parent, the lignin patterns mimicked the patterns obtained in single transformants with a reduced CCR activity. In contrast, the specific lignin profile of COMT I repression could not be detected in double transformed siblings. By transmission electron microscopy some cell wall loosening was detected in the antisense CCR parent but not in the antisense COMT I parent. In double transformants, immunolabeling of non-condensed guaiacyl-syringyl units was weaker and revealed changes in epitope distribution that specifically affected vessels. Our results more widely highlight the impact of culture conditions on phenotypes and gene expression of transformed plants. PMID:11351078

CCL11 promotes angiogenic activity by activating the PI3K/Akt pathway in HUVECs.

PubMed

Park, Jun Young; Kang, Yeo Wool; Choi, Byung Young; Yang, Young Chul; Cho, Byung Pil; Cho, Won Gil

2017-08-01

CCR3, the receptor for CCL11, is expressed on the surface of immune cells and even on non-immune cells. CCL11-CCR3 interactions can promote cell migration and proliferation. In this study, we investigated the effect of CCL11 on angiogenesis in HUVECs and also examined the molecular mechanisms of this process. We found that CCL11 induced mRNA transcription and protein expression of CCR3 in HUVECs. Moreover, the scratch wound healing assay and MTS proliferation assay both demonstrated that CCL11 promotes endothelial cell migration and induces weak proliferation. CCL11 directly induced microvessel sprouting from the rat aortic ring; these effects occurred earlier and to a greater extent than with VEGF stimulation. Furthermore, CCL11-induced phosphorylation of Akt was abolished by PI3K inhibitors. siRNA-mediated knockdown of CCR3 led to a significant reduction of PI3K phosphorylation. However, the phosphorylation levels of ERK1/2 were not changed, even after CCL11 treatment. Cumulatively, our data suggest that the CCL11-CCR3 interaction mainly activates PI3K/Akt signal transduction pathway in HUVECs.

Localization of HIV-1 co-receptors CCR5 and CXCR4 in the brain of children with AIDS.

PubMed Central

Vallat, A. V.; De Girolami, U.; He, J.; Mhashilkar, A.; Marasco, W.; Shi, B.; Gray, F.; Bell, J.; Keohane, C.; Smith, T. W.; Gabuzda, D.

1998-01-01

The chemokine receptors CCR5 and CXCR4 are co-receptors together with CD4 for human immunodeficiency virus (HIV)-1 entry into target cells. Macrophage-tropic HIV-1 viruses use CCR5 as a co-receptor, whereas T-cell-line tropic viruses use CXCR4. HIV-1 infects the brain and causes a progressive encephalopathy in 20 to 30% of infected children and adults. Most of the HIV-1-infected cells in the brain are macrophages and microglia. We examined expression of CCR5 and CXCR4 in brain tissue from 20 pediatric acquired immune deficiency syndrome (AIDS) patients in relation to neuropathological consequences of HIV-1 infection. The overall frequency of CCR5-positive perivascular mononuclear cells and macrophages was increased in the brains of children with severe HIV-1 encephalitis (HIVE) compared with children with mild HIVE or non-AIDS controls, whereas the frequency of CXCR4-positive perivascular cells did not correlate with disease severity. CCR5- and CXCR4-positive macrophages and microglia were detected in inflammatory lesions in the brain of children with severe HIVE. In addition, CXCR4 was detected in a subpopulation of neurons in autopsy brain tissue and primary human brain cultures. Similar findings were demonstrated in the brain of adult AIDS patients and controls. These findings suggest that CCR5-positive mononuclear cells, macrophages, and microglia contribute to disease progression in the central nervous system of children and adults with AIDS by serving as targets for virus replication. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 8 PMID:9422534

CCL19/CCR7 contributes to the pathogenesis of endometriosis via PI3K/Akt pathway by regulating the proliferation and invasion of ESCs.

PubMed

Diao, Ruiying; Wei, Weixia; Zhao, Jinghui; Tian, Fuying; Cai, Xueyong; Duan, Yong-Gang

2017-11-01

The level of CCL19 increased in the peritoneal fluid of women with endometriosis, but the precise mechanism of CCL19/CCR7 in the pathogenesis of endometriosis remains unknown. ELISA and immunohistochemistry were performed to analyze CCL19/CCR7 expressions in peritoneal fluid and endometrium from women with endometriosis (n = 38) and controls (n = 32). Cell proliferation and transwell invasion assays were applied to detect proliferation and invasion of human endometrial stromal cells (ESCs). Expressions of Bcl2, MMP2, MMP9, and p-AKT/AKT were analyzed by Western blot. Peritoneal fluid concentration of CCL19 in patients with endometriosis was higher than that in controls. Those patients with moderate/severe endometriosis had significantly higher peritoneal fluid concentrations of CCL19 compared to those with minimal/mild endometriosis. Higher CCL19 and CCR7 were found in the endometrium with endometriosis compared to control. CCL19 significantly enhanced ESC proliferation and invasion through CCR7 via activating PI3K/Akt signal pathways. CCL19/CCR7 interaction significantly enhanced phosphorylation of Akt, Bcl2, MMP2, and MMP9 in ESCs. These data indicate CCL19/CCR7 contributes to proliferation and invasion of ESCs, which are conducive to the pathogenesis of endometriosis through activating PI3K/Akt pathway. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Mapping Interaction Sites on Human Chemokine Receptors by Deep Mutational Scanning.

PubMed

Heredia, Jeremiah D; Park, Jihye; Brubaker, Riley J; Szymanski, Steven K; Gill, Kevin S; Procko, Erik

2018-06-01

Chemokine receptors CXCR4 and CCR5 regulate WBC trafficking and are engaged by the HIV-1 envelope glycoprotein gp120 during infection. We combine a selection of human CXCR4 and CCR5 libraries comprising nearly all of ∼7000 single amino acid substitutions with deep sequencing to define sequence-activity landscapes for surface expression and ligand interactions. After consideration of sequence constraints for surface expression, known interaction sites with HIV-1-blocking Abs were appropriately identified as conserved residues following library sorting for Ab binding, validating the use of deep mutational scanning to map functional interaction sites in G protein-coupled receptors. Chemokine CXCL12 was found to interact with residues extending asymmetrically into the CXCR4 ligand-binding cavity, similar to the binding surface of CXCR4 recognized by an antagonistic viral chemokine previously observed crystallographically. CXCR4 mutations distal from the chemokine binding site were identified that enhance chemokine recognition. This included disruptive mutations in the G protein-coupling site that diminished calcium mobilization, as well as conservative mutations to a membrane-exposed site (CXCR4 residues H79 2.45 and W161 4.50 ) that increased ligand binding without loss of signaling. Compared with CXCR4-CXCL12 interactions, CCR5 residues conserved for gp120 (HIV-1 BaL strain) interactions map to a more expansive surface, mimicking how the cognate chemokine CCL5 makes contacts across the entire CCR5 binding cavity. Acidic substitutions in the CCR5 N terminus and extracellular loops enhanced gp120 binding. This study demonstrates how comprehensive mutational scanning can define functional interaction sites on receptors, and novel mutations that enhance receptor activities can be found simultaneously. Copyright © 2018 by The American Association of Immunologists, Inc.

Effect of Female Genital Schistosomiasis and Anti-Schistosomal Treatment on Monocytes, CD4+ T-Cells and CCR5 Expression in the Female Genital Tract

PubMed Central

Kleppa, Elisabeth; Ramsuran, Veron; Zulu, Siphosenkosi; Karlsen, Gunn Hege; Bere, Alfred; Passmore, Jo-Ann S.; Ndhlovu, Patricia; Lillebø, Kristine; Holmen, Sigve D.; Onsrud, Mathias; Gundersen, Svein Gunnar; Taylor, Myra; Kjetland, Eyrun F.; Ndung’u, Thumbi

2014-01-01

Background Schistosoma haematobium is a waterborne parasite that may cause female genital schistosomiasis (FGS), characterized by genital mucosal lesions. There is clinical and epidemiological evidence for a relationship between FGS and HIV. We investigated the impact of FGS on HIV target cell density and expression of the HIV co-receptor CCR5 in blood and cervical cytobrush samples. Furthermore we evaluated the effect of anti-schistosomal treatment on these cell populations. Design The study followed a case-control design with post treatment follow-up, nested in an on-going field study on FGS. Methods Blood and cervical cytobrush samples were collected from FGS negative and positive women for flow cytometry analyses. Urine samples were investigated for schistosome ova by microscopy and polymerase chain reaction (PCR). Results FGS was associated with a higher frequency of CD14+ cells (monocytes) in blood (11.5% in FGS+ vs. 2.2% in FGS-, p = 0.042). Frequencies of CD4+ cells expressing CCR5 were higher in blood samples from FGS+ than from FGS- women (4.7% vs. 1.5%, p = 0.018). The CD14+ cell population decreased significantly in both compartments after anti-schistosomal treatment (p = 0.043). Although the frequency of CD4+ cells did not change after treatment, frequencies of CCR5 expression by CD4+ cells decreased significantly in both compartments (from 3.4% to 0.5% in blood, p = 0.036; and from 42.4% to 5.6% in genital samples, p = 0.025). Conclusions The results support the hypothesis that FGS may increase the risk of HIV acquisition, not only through damage of the mucosal epithelial barrier, but also by affecting HIV target cell populations, and that anti-schistosomal treatment can modify this. PMID:24896815

Host and Viral Factors in HIV-Mediated Bystander Apoptosis

PubMed Central

Garg, Himanshu; Joshi, Anjali

2017-01-01

Human immunodeficiency virus (HIV) infections lead to a progressive loss of CD4 T cells primarily via the process of apoptosis. With a limited number of infected cells and vastly disproportionate apoptosis in HIV infected patients, it is believed that apoptosis of uninfected bystander cells plays a significant role in this process. Disease progression in HIV infected individuals is highly variable suggesting that both host and viral factors may influence HIV mediated apoptosis. Amongst the viral factors, the role of Envelope (Env) glycoprotein in bystander apoptosis is well documented. Recent evidence on the variability in apoptosis induction by primary patient derived Envs underscores the role of Env glycoprotein in HIV disease. Amongst the host factors, the role of C-C Chemokine Receptor type 5 (CCR5), a coreceptor for HIV Env, is also becoming increasingly evident. Polymorphisms in the CCR5 gene and promoter affect CCR5 cell surface expression and correlate with both apoptosis and CD4 loss. Finally, chronic immune activation in HIV infections induces multiple defects in the immune system and has recently been shown to accelerate HIV Env mediated CD4 apoptosis. Consequently, those factors that affect CCR5 expression and/or immune activation in turn indirectly regulate HIV mediated apoptosis making this phenomenon both complex and multifactorial. This review explores the complex role of various host and viral factors in determining HIV mediated bystander apoptosis. PMID:28829402

CCR-2 neutralization augments murine fresh BMC activation by Staphylococcus aureus via two distinct mechanisms: at the level of ROS production and cytokine response.

PubMed

Nandi, Ajeya; Bishayi, Biswadev

2017-05-01

CCR-2 signaling regulates recruitment of monocytes from the bone marrow into the bloodstream and then to sites of infection. We sought to determine whether CCL-2/CCR-2 signaling is involved in the killing of Staphylococcus aureus by murine bone marrow cells (BMCs). The intermittent link of reactive oxygen species (ROS)-NF-κB/p38-MAPK-mediated CCL-2 production in CCR-2 signaling prompted us to determine whether neutralization of CCR-2 augments the response of murine fresh BMCs (FBMCs) after S. aureus infection. It was observed that anti-CCR-2 Ab-treated FBMCs released fewer ROS on encountering S. aureus infection than CCR-2 non-neutralized FBMCs, also correlating with reduced killing of S. aureus in CCR-2 neutralized FBMCs. Staphylococcal catalase and SOD were also found to play a role in protecting S. aureus from the ROS-mediated killing of FBMC. S. aureus infection of CCR-2 intact FBMCs pre-treated with either NF-κB or p-38-MAPK blocker induced less CCL-2, suggesting that NF-κB or p-38-MAPK is required for CCL-2 production by FBMCs. Moreover, blocking of CCR-2 along with NF-κB or p-38-MAPK resulted in elevated CCL-2 production and reduced CCR-2 expression. Inhibition of CCR-2 impairs the response of murine BMCs to S. aureus infection by attenuation ROS production and modulating the cytokine response.

Anibamine and its Analogues as Novel Anti-Prostate Cancer Agents

DTIC Science & Technology

2009-06-01

expression of CCR5 and CCL5 was quantitated by SYBR-based Real-time PCR. The U6 gene was used as internal control . cDNA was synthesized using the iScript...15 Conclusion The major focus of the research pro ject will be the syntheses of the ligands we designed as chem okine receptor CCR5 antagonists with...provide useful information and insights for both basic research and drug design and hence are widely welcome by the science community. More specifically

CD4 is expressed on a heterogeneous subset of hematopoietic progenitors, which persistently harbor CXCR4 and CCR5-tropic HIV proviral genomes in vivo.

PubMed

Sebastian, Nadia T; Zaikos, Thomas D; Terry, Valeri; Taschuk, Frances; McNamara, Lucy A; Onafuwa-Nuga, Adewunmi; Yucha, Ryan; Signer, Robert A J; Riddell, James; Bixby, Dale; Markowitz, Norman; Morrison, Sean J; Collins, Kathleen L

2017-07-01

Latent HIV infection of long-lived cells is a barrier to viral clearance. Hematopoietic stem and progenitor cells are a heterogeneous population of cells, some of which are long-lived. CXCR4-tropic HIVs infect a broad range of HSPC subtypes, including hematopoietic stem cells, which are multi-potent and long-lived. However, CCR5-tropic HIV infection is limited to more differentiated progenitor cells with life spans that are less well understood. Consistent with emerging data that restricted progenitor cells can be long-lived, we detected persistent HIV in restricted HSPC populations from optimally treated people. Further, genotypic and phenotypic analysis of amplified env alleles from donor samples indicated that both CXCR4- and CCR5-tropic viruses persisted in HSPCs. RNA profiling confirmed expression of HIV receptor RNA in a pattern that was consistent with in vitro and in vivo results. In addition, we characterized a CD4high HSPC sub-population that was preferentially targeted by a variety of CXCR4- and CCR5-tropic HIVs in vitro. Finally, we present strong evidence that HIV proviral genomes of both tropisms can be transmitted to CD4-negative daughter cells of multiple lineages in vivo. In some cases, the transmitted proviral genomes contained signature deletions that inactivated the virus, eliminating the possibility that coincidental infection explains the results. These data support a model in which both stem and non-stem cell progenitors serve as persistent reservoirs for CXCR4- and CCR5-tropic HIV proviral genomes that can be passed to daughter cells.

Evolution of the Human Immunodeficiency Virus Type 1 Envelope during Infection Reveals Molecular Corollaries of Specificity for Coreceptor Utilization and AIDS Pathogenesis

PubMed Central

Hu, Qin-xue; Barry, Ashley Perkins; Wang, Zi-xuan; Connolly, Shanon M.; Peiper, Stephen C.; Greenberg, Michael L.

2000-01-01

The evolution of human immunodeficiency virus type 1 infection is associated with a shift in the target cell population, driven by variability in coreceptor utilization resulting from diversity in env. To elucidate the potential consequences of these changes for Env-mediated fusion over the course of AIDS, we examined the biological properties of serial viral isolates and determined coreceptor utilization by the products of env cloned from two individuals, followed from the detection of seroconversion throughout the course of their infection. One had a typical course, and the other had an accelerated progression. Early isolates were non-syncytium inducing, and the corresponding Env exclusively utilized CCR5, whereas Env from late phases of infection showed restricted utilization of CXCR4 in both patients. Env from subject SC24, who had a standard progression, demonstrated multitropism, manifested by utilization of CCR3, CXCR4, and CCR5 in the intervening period. In contrast, Env from patient SC51, who experienced early conversion to the syncytium-inducing phenotype, developed dualtropic coreceptor utilization of CCR5 and CXCR4. Genetic analysis of env from each isolate revealed that those with an X4 phenotype formed a distinct subcluster within each subject. Analysis of chimeras constructed from R5 and multispecific env from patient SC24 demonstrated that while the V3 domain played a dominant role in determining coreceptor utilization, sequences in the V4–V5 region also contributed to the latter phenotype. Immunoprecipitation experiments confirmed that the hybrid Env proteins were expressed at similar levels. These experiments demonstrate that progression from the R5 to X4 phenotype may occur through a multi- or dual-tropic intermediate and that multiple domains contribute to this process. PMID:11090186

Phenotype Variation in Human Immunodeficiency virus Type 1 Transmission and Disease Progression

PubMed Central

Cavarelli, Mariangela; Scarlatti, Gabriella

2009-01-01

Human immunodeficiency virus type I (HIV-1) infects target cells through interaction with the CD4 molecule and chemokine receptors, mainly CCR5 and CXCR4. Viral isolates can be phenotypically classified based on the co-receptor they utilize to infect target cells. Thus, R5 and X4 virus use respectively CCR5 and CXCR4, whereas R5X4 virus can use either CCR5 or CXCR4. This review describes the central role played by co-receptor expression and usage for HIV-1 cell tropism, transmission and pathogenesis. We discuss various hypotheses proposed to explain the preferential transmission of R5 viruses and the mechanisms driving the change of HIV-1 co-receptor usage in the course of infection. Recent insights in the intrinsic variability of R5 viruses and their role in influencing disease progression in both adults and children are also discussed. PMID:19893208

Phenotype variation in human immunodeficiency virus type 1 transmission and disease progression.

PubMed

Cavarelli, Mariangela; Scarlatti, Gabriella

2009-01-01

Human immunodeficiency virus type I (HIV-1) infects target cells through interaction with the CD4 molecule and chemokine receptors, mainly CCR5 and CXCR4. Viral isolates can be phenotypically classified based on the co-receptor they utilize to infect target cells. Thus, R5 and X4 virus use respectively CCR5 and CXCR4, whereas R5X4 virus can use either CCR5 or CXCR4. This review describes the central role played by co-receptor expression and usage for HIV-1 cell tropism, transmission and pathogenesis. We discuss various hypotheses proposed to explain the preferential transmission of R5 viruses and the mechanisms driving the change of HIV-1 co-receptor usage in the course of infection. Recent insights in the intrinsic variability of R5 viruses and their role in influencing disease progression in both adults and children are also discussed.

Role of CXCR4 in Cell-Cell Fusion and Infection of Monocyte-Derived Macrophages by Primary Human Immunodeficiency Virus Type 1 (HIV-1) Strains: Two Distinct Mechanisms of HIV-1 Dual Tropism

PubMed Central

Yi, Yanjie; Isaacs, Stuart N.; Williams, Darlisha A.; Frank, Ian; Schols, Dominique; De Clercq, Erik; Kolson, Dennis L.; Collman, Ronald G.

1999-01-01

Dual-tropic human immunodeficiency virus type 1 (HIV-1) strains infect both primary macrophages and transformed T-cell lines. Prototype T-cell line-tropic (T-tropic) strains use CXCR4 as their principal entry coreceptor (X4 strains), while macrophagetropic (M-tropic) strains use CCR5 (R5 strains). Prototype dual tropic strains use both coreceptors (R5X4 strains). Recently, CXCR4 expressed on macrophages was found to support infection by certain HIV-1 isolates, including the dual-tropic R5X4 strain 89.6, but not by T-tropic X4 prototypes like 3B. To better understand the cellular basis for dual tropism, we analyzed the macrophage coreceptors used for Env-mediated cell-cell fusion as well as infection by several dual-tropic HIV-1 isolates. Like 89.6, the R5X4 strain DH12 fused with and infected both wild-type and CCR5-negative macrophages. The CXCR4-specific inhibitor AMD3100 blocked DH12 fusion and infection in macrophages that lacked CCR5 but not in wild-type macrophages. This finding indicates two independent entry pathways in macrophages for DH12, CCR5 and CXCR4. Three primary isolates that use CXCR4 but not CCR5 (tybe, UG021, and UG024) replicated efficiently in macrophages regardless of whether CCR5 was present, and AMD3100 blocking of CXCR4 prevented infection in both CCR5 negative and wild-type macrophages. Fusion mediated by UG021 and UG024 Envs in both wild-type and CCR5-deficient macrophages was also blocked by AMD3100. Therefore, these isolates use CXCR4 exclusively for entry into macrophages. These results confirm that macrophage CXCR4 can be used for fusion and infection by primary HIV-1 isolates and indicate that CXCR4 may be the sole macrophage coreceptor for some strains. Thus, dual tropism can result from two distinct mechanisms: utilization of both CCR5 and CXCR4 on macrophages and T-cell lines, respectively (dual-tropic R5X4), or the ability to efficiently utilize CXCR4 on both macrophages and T-cell lines (dual-tropic X4). PMID:10438797

CC Chemokine Receptor 5: The Interface of Host Immunity and Cancer

PubMed Central

de Oliveira, Carlos Eduardo Coral; Oda, Julie Massayo Maeda; Losi Guembarovski, Roberta; de Oliveira, Karen Brajão; Ariza, Carolina Batista; Neto, Jamil Soni; Banin Hirata, Bruna Karina; Watanabe, Maria Angelica Ehara

2014-01-01

Solid tumors are embedded in a stromal microenvironment consisting of immune cells, such as macrophages and lymphocytes, as well as nonimmune cells, such as endothelial cells and fibroblasts. Chemokines are a type of small secreted chemotactic cytokine and together with their receptors play key roles in the immune defense. Critically, they regulate cancer cellular migration and also contribute to their proliferation and survival. The CCR5 chemokine receptor is involved in leucocytes chemotaxis to sites of inflammation and plays an important role in the macrophages, T cells, and monocytes recruitment. Additionally, CCR5 may have an indirect effect on cancer progression by controlling the antitumor immune response, since it has been demonstrated that its expression could promote tumor growth and contribute to tumor metastasis, in different types of malignant tumors. Furthermore, it was demonstrated that a CCR5 antagonist may inhibit tumor growth, consisting of a possible therapeutic target. In this context, the present review focuses on the establishment of CCR5 within the interface of host immunity, tumor microenvironment, and its potential as a targeting to immunotherapy. PMID:24591756

CCR5 Signal Transduction in Macrophages by Human Immunodeficiency Virus and Simian Immunodeficiency Virus Envelopes

PubMed Central

Arthos, James; Rubbert, Andrea; Rabin, Ronald L.; Cicala, Claudia; Machado, Elizabeth; Wildt, Kathryne; Hanbach, Meredith; Steenbeke, Tavis D.; Swofford, Ruth; Farber, Joshua M.; Fauci, Anthony S.

2000-01-01

The capacity of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) envelopes to transduce signals through chemokine coreceptors on macrophages was examined by measuring the ability of recombinant envelope proteins to mobilize intracellular calcium stores. Both HIV and SIV envelopes mobilized calcium via interactions with CCR5. The kinetics of these responses were similar to those observed when macrophages were treated with MIP-1β. Distinct differences in the capacity of envelopes to mediate calcium mobilization were observed. Envelopes derived from viruses capable of replicating in macrophages mobilized relatively high levels of calcium, while envelopes derived from viruses incapable of replicating in macrophages mobilized relatively low levels of calcium. The failure to efficiently mobilize calcium was not restricted to envelopes derived from CXCR4-utilizing isolates but also included envelopes derived from CCR5-utilizing isolates that fail to replicate in macrophages. We characterized one CCR5-utilizing isolate, 92MW959, which entered macrophages but failed to replicate. A recombinant envelope derived from this virus mobilized low levels of calcium. When macrophages were inoculated with 92MW959 in the presence of MIP-1α, viral replication was observed, indicating that a CC chemokine-mediated signal provided the necessary stimulus to allow the virus to complete its replication cycle. Although the role that envelope-CCR5 signal transduction plays in viral replication is not yet understood, it has been suggested that envelope-mediated signals facilitate early postfusion events in viral replication. The data presented here are consistent with this hypothesis and suggest that the differential capacity of viral envelopes to signal through CCR5 may influence their ability to replicate in macrophages. PMID:10864653

CCR5 signal transduction in macrophages by human immunodeficiency virus and simian immunodeficiency virus envelopes.

PubMed

Arthos, J; Rubbert, A; Rabin, R L; Cicala, C; Machado, E; Wildt, K; Hanbach, M; Steenbeke, T D; Swofford, R; Farber, J M; Fauci, A S

2000-07-01

The capacity of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) envelopes to transduce signals through chemokine coreceptors on macrophages was examined by measuring the ability of recombinant envelope proteins to mobilize intracellular calcium stores. Both HIV and SIV envelopes mobilized calcium via interactions with CCR5. The kinetics of these responses were similar to those observed when macrophages were treated with MIP-1beta. Distinct differences in the capacity of envelopes to mediate calcium mobilization were observed. Envelopes derived from viruses capable of replicating in macrophages mobilized relatively high levels of calcium, while envelopes derived from viruses incapable of replicating in macrophages mobilized relatively low levels of calcium. The failure to efficiently mobilize calcium was not restricted to envelopes derived from CXCR4-utilizing isolates but also included envelopes derived from CCR5-utilizing isolates that fail to replicate in macrophages. We characterized one CCR5-utilizing isolate, 92MW959, which entered macrophages but failed to replicate. A recombinant envelope derived from this virus mobilized low levels of calcium. When macrophages were inoculated with 92MW959 in the presence of MIP-1alpha, viral replication was observed, indicating that a CC chemokine-mediated signal provided the necessary stimulus to allow the virus to complete its replication cycle. Although the role that envelope-CCR5 signal transduction plays in viral replication is not yet understood, it has been suggested that envelope-mediated signals facilitate early postfusion events in viral replication. The data presented here are consistent with this hypothesis and suggest that the differential capacity of viral envelopes to signal through CCR5 may influence their ability to replicate in macrophages.

Increased expression of chemokine receptor CCR3 and its ligands in ulcerative colitis: the role of colonic epithelial cells in in vitro studies.

PubMed

Manousou, P; Kolios, G; Valatas, V; Drygiannakis, I; Bourikas, L; Pyrovolaki, K; Koutroubakis, I; Papadaki, H A; Kouroumalis, E

2010-11-01

Human colonic epithelial cells express T helper type 1 (Th1)-associated chemoattractants, yet little is known about the production of Th2-associated chemoattractants. CCL11/eotaxin-1, CCL24/eotaxin-2 and CCL26/eotaxin-3 are known to attract CCR3-expressing, Th2-polarized lymphocytes. We studied constitutive and inflammation-induced expression and production of CCR3 together with its ligands in the colon and peripheral blood of patients with inflammatory bowel disease (IBD) by flow cytometry, reverse transcription–polymerase chain reaction (RT–PCR) and enzyme-linked immunosorbent assay (ELISA). We further defined the regulated expression of these chemokines by RT–PCR and ELISA using cultured human epithelial cell lines. A higher fraction of peripheral T lymphocytes were found to be positive for CCR3 in patients with ulcerative colitis (UC) compared to Crohn’s disease (CD), while almost no CCR3(+) T cells were found in normal controls (NC). Similarly, higher and more frequent expression of CCR3 was observed in colonic biopsies from patients with UC, regardless of the disease activity, when compared to CD or NCs. Serum CCL11/eotaxin-1 was increased significantly in UC (306 ± 87 pg/ml) and less so in CD (257 ± 43 pg/ml), whereas CCL24/eotaxin-2, and CCL26/eotaxin-3 were increased only in UC. Colonic expression of the three chemokines was minimal in NCs but high in inflammatory bowel diseases (especially UC) and was independent of disease activity. Th2, and to a lesser extent Th1, cytokines were able to induce expression and production of all three eotaxins from colonic epithelial cells in culture. CCR3 and ligands over-expression would appear to be a characteristic of UC. The production of CCR3 ligands by human colonic epithelial cells suggests further that epithelium can play a role in modulating pathological T cell-mediated mucosal inflammation.

Precursor Forms of Vitamin D Reduce HIV-1 Infection In Vitro.

PubMed

Aguilar-Jimenez, Wbeimar; Villegas-Ospina, Simon; Gonzalez, Sandra; Zapata, Wildeman; Saulle, Irma; Garziano, Micaela; Biasin, Mara; Clerici, Mario; Rugeles, Maria T

2016-12-15

Although the anti-HIV-1 effects of vitamin D (VitD) have been reported, mechanisms behind such protection remain largely unexplored. The effects of two precursor forms (cholecalciferol/calciol at 0.01, 1 and 100 nM and calcidiol at 100 and 250 nM) on HIV-1 infection, immune activation, and gene expression were analyzed in vitro in cells of Colombian and Italian healthy donors. We quantified levels of released p24 by enzyme-linked immunosorbent assay, of intracellular p24 and cell-surface expression of CD38 and HLA-DR by flow cytometry, and mRNA expression of antiviral and immunoregulatory genes by real-time reverse transcription-polymerase chain reaction. Cholecalciferol decreased the frequency of HIV-1-infected p24CD4 T cells and levels of p24 in supernatants in a dose-dependent manner. Moreover, the CD4CD38HLA-DR and CD4CD38HLA-DR subpopulations were more susceptible to infection but displayed the greatest cholecalciferol-induced decreases in infection rate by an X4-tropic strain. Likewise, cholecalciferol at its highest concentration decreased the frequency of CD38HLA-DR but not of CD38HLA-DR T-cell subsets. Analyzing the effects of calcidiol, the main VitD source for immune cells and an R5-tropic strain as the most frequently transmitted virus, a reduction in HIV-1 productive infection was also observed. In addition, an increase in mRNA expression of APOBEC3G and PI3 and a reduction of TRIM22 and CCR5 expression, this latter positively correlated with p24 levels, was noted. VitD reduces HIV-1 infection in T cells possibly by inducing antiviral gene expression, reducing the viral co-receptor CCR5 and, at least at the highest cholecalciferol concentration, by promoting an HIV-1-restrictive CD38HLA-DR immunophenotype.

Capillary arterialization requires the bone-marrow-derived cell (BMC)-specific expression of chemokine (C-C motif) receptor-2, but BMCs do not transdifferentiate into microvascular smooth muscle.

PubMed

Nickerson, Meghan M; Burke, Caitlin W; Meisner, Joshua K; Shuptrine, Casey W; Song, Ji; Price, Richard J

2009-01-01

Chemokine (C-C motif) receptor-2 (CCR2) regulates arteriogenesis and angiogenesis, facilitating the MCP-1-dependent recruitment of growth factor-secreting bone marrow-derived cells (BMCs). Here, we tested the hypothesis that the BMC-specific expression of CCR2 is also required for new arteriole formation via capillary arterialization. Following non-ischemic saphenous artery occlusion, we measured the following in gracilis muscles: monocyte chemotactic protein-1 (MCP-1) in wild-type (WT) C57Bl/6J mice by ELISA, and capillary arterialization in WT-WT and CCR2(-/-)-WT (donor-host) bone marrow chimeric mice, as well as BMC transdifferentiation in EGFP(+)-WT mice, by smooth muscle (SM) alpha-actin immunochemistry. MCP-1 levels were significantly elevated 1 day after occlusion in WT mice. In WT-WT mice at day 7, compared to sham controls, arterial occlusion induced a 34% increase in arteriole length density, a 46% increase in SM alpha-actin(+) vessels, and a 45% increase in the fraction of vessels coated with SM alpha-actin, indicating significant capillary arterialization. However, in CCR2(-/-)-WT mice, no differences were observed between arterial occlusion and sham surgery. In EGFP(+)-WT mice, EGFP and SM alpha-actin never colocalized. We conclude that BMC-specific CCR2 expression is required for skeletal muscle capillary arterialization following arterial occlusion; however, BMCs do not transdifferentiate into smooth muscle.

Effects of treatment with Maraviroc a CCR5 inhibitor on a human hepatic stellate cell line.

PubMed

Coppola, Nicola; Perna, Angelica; Lucariello, Angela; Martini, Salvatore; Macera, Margherita; Carleo, Maria A; Guerra, Germano; Esposito, Vincenzo; De Luca, Antonio

2018-08-01

After an acute liver damage, tissue regeneration repairs lesions with degradation of deposed fibrotic material, while mechanisms of tissue restoration are persistently activated following several repeated injuries, inducing deposition of extracellular matrix. (ECM). Factors responsible for ECM remodeling have been identified in a pathway involving a family of zinc-dependent enzyme matrix metalloproteinases (MMPs), together with tissue inhibitor of metalloproteinases (TIMPs). Recent experimental models suggested a role of CCR5 receptor in the genesis of liver fibrosis. Drawing from these background we decided to evaluate the effects of the treatment with the CCR5 inhibitor Maraviroc on LX-2, a human hepatic stellate cell line (HSC). Treatment with Maraviroc resulted in a block in S phase of LX-2 cells with increased expression levels of cyclin D1 and p21 while the expression of p53 was reduced. Treatment with Maraviroc was also able to block the accumulation of fibrillar collagens and extracellular matrix proteins (ECM), as demonstrated by the decrease of specific markers as Collagen type I, α-SMA, and TGF-β1. In addition we observed a down regulation of both metalloproteins (MMP-2, MMP-9), used for the degradation of the extracellular matrix and their inhibitors (TIMP-1, TIMP-2). The identification of a compound that may modulate the dynamic of liver fibrosis could be crucial in all chronic liver diseases. Maraviroc could play an important role because, in addition to its own anti-HIV activity, it could reduce the release of pro-inflammatory citokynes implicated in liver fibrogenesis. © 2018 Wiley Periodicals, Inc.

Protection of macaques from vaginal SHIV challenge by an orally delivered CCR5 inhibitor.

PubMed

Veazey, Ronald S; Springer, Martin S; Marx, Preston A; Dufour, Jason; Klasse, Per Johan; Moore, John P

2005-12-01

Pre-exposure oral prophylaxis with antiviral drugs is a potential method for preventing transmission of human immunodeficiency virus type 1 (HIV-1). We show that oral delivery of CMPD167, a small molecule that binds to the CCR5 coreceptor, for 10-14 d can protect a substantial proportion of macaques from vaginal infection with a CCR5-using virus (SHIV-162P3). The macaques that became infected despite receiving CMPD167 had reduced plasma viremia levels during the earliest stages of infection.

Intestinal IFN-γ-producing type 1 regulatory T cells coexpress CCR5 and programmed cell death protein 1 and downregulate IL-10 in the inflamed guts of patients with inflammatory bowel disease.

PubMed

Alfen, Johanna Sophie; Larghi, Paola; Facciotti, Federica; Gagliani, Nicola; Bosotti, Roberto; Paroni, Moira; Maglie, Stefano; Gruarin, Paola; Vasco, Chiara Maria; Ranzani, Valeria; Frusteri, Cristina; Iseppon, Andrea; Moro, Monica; Crosti, Maria Cristina; Gatti, Stefano; Pagani, Massimiliano; Caprioli, Flavio; Abrignani, Sergio; Flavell, Richard A; Geginat, Jens

2018-01-31

IL-10 is an anti-inflammatory cytokine required for intestinal immune homeostasis. It mediates suppression of T-cell responses by type 1 regulatory T (T R 1) cells but is also produced by CD25 + regulatory T (Treg) cells. We aimed to identify and characterize human intestinal T R 1 cells and to investigate whether they are a relevant cellular source of IL-10 in patients with inflammatory bowel diseases (IBDs). CD4 + T cells isolated from the intestinal lamina propria of human subjects and mice were analyzed for phenotype, cytokine production, and suppressive capacities. Intracellular IL-10 expression by CD4 + T-cell subsets in the inflamed guts of patients with IBD (Crohn disease or ulcerative colitis) was compared with that in cells from noninflamed control subjects. Finally, the effects of proinflammatory cytokines on T-cell IL-10 expression were analyzed, and IL-1β and IL-23 responsiveness was assessed. Intestinal T R 1 cells could be identified by coexpression of CCR5 and programmed cell death protein 1 (PD-1) in human subjects and mice. CCR5 + PD-1 + T R 1 cells expressed IFN-γ and efficiently suppressed T-cell proliferation and transfer colitis. Intestinal IFN-γ + T R 1 cells, but not IL-7 receptor-positive T H cells or CD25 + Treg cells, showed lower IL-10 expression in patients with IBDs. T R 1 cells were responsive to IL-23, and IFN-γ + T R 1 cells downregulated IL-10 with IL-1β and IL-23. Conversely, CD25 + Treg cells expressed higher levels of IL-1 receptor but showed stable IL-10 expression. We provide the first ex vivo characterization of human intestinal T R 1 cells. Selective downregulation of IL-10 by IFN-γ + T R 1 cells in response to proinflammatory cytokines is likely to drive excessive intestinal inflammation in patients with IBDs. Copyright © 2018 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

Characterization of human immunodeficiency virus type 1 populations containing CXCR4-using variants from recently infected individuals.

PubMed

Huang, Wei; Toma, Jonathan; Stawiski, Eric; Fransen, Signe; Wrin, Terri; Parkin, Neil; Whitcomb, Jeannette M; Coakley, Eoin; Hecht, Frederick M; Deeks, Steven G; Gandhi, Rajesh T; Eshleman, Susan H; Petropoulos, Christos J

2009-08-01

We screened 150 individuals from two recent seroconverter cohorts and found that six (4%) had CXCR4-using viruses. Clonal analysis of these six individuals, along with a seventh individual identified during clinical care as a recent seroconverter, revealed the presence of both X4- and dual-tropic variants in these recently infected adults. The ability of individual CXCR4-using variants to infect cells expressing CD4/CXCR4 or CD4/CCR5 varied dramatically. These data demonstrate that virus populations in some newly infected individuals can consist of either heterogeneous populations containing both CXCR4-using and CCR5-tropic viruses, or homogeneous populations containing only CXCR4-using viruses. The presence of CXCR4-using viruses at early stages of infection suggests that testing for viral tropism before using CCR5 antagonists may be important even in persons with known recent infection. The presence of CXCR4-using viruses in a subset of newly infected individuals could impact the efficacies of vaccine and microbicide strategies that target CCR5-tropic viruses.

Characterization of Human Immunodeficiency Virus Type 1 Populations Containing CXCR4-Using Variants from Recently Infected Individuals

PubMed Central

Toma, Jonathan; Stawiski, Eric; Fransen, Signe; Wrin, Terri; Parkin, Neil; Whitcomb, Jeannette M.; Coakley, Eoin; Hecht, Frederick M.; Deeks, Steven G.; Gandhi, Rajesh T.; Eshleman, Susan H.; Petropoulos, Christos J.

2009-01-01

Abstract We screened 150 individuals from two recent seroconverter cohorts and found that six (4%) had CXCR4-using viruses. Clonal analysis of these six individuals, along with a seventh individual identified during clinical care as a recent seroconverter, revealed the presence of both X4- and dual-tropic variants in these recently infected adults. The ability of individual CXCR4-using variants to infect cells expressing CD4/CXCR4 or CD4/CCR5 varied dramatically. These data demonstrate that virus populations in some newly infected individuals can consist of either heterogeneous populations containing both CXCR4-using and CCR5-tropic viruses, or homogeneous populations containing only CXCR4-using viruses. The presence of CXCR4-using viruses at early stages of infection suggests that testing for viral tropism before using CCR5 antagonists may be important even in persons with known recent infection. The presence of CXCR4-using viruses in a subset of newly infected individuals could impact the efficacies of vaccine and microbicide strategies that target CCR5-tropic viruses. PMID:19678765

CCR5-edited gene therapies for HIV cure: Closing the door to viral entry.

PubMed

Haworth, Kevin G; Peterson, Christopher W; Kiem, Hans-Peter

2017-11-01

Human immunodeficiency virus (HIV) was first reported and characterized more than three decades ago. Once thought of as a death sentence, HIV infection has become a chronically manageable disease. However, it is estimated that a staggering 0.8% of the world's population is infected with HIV, with more than 1 million deaths reported in 2015 alone. Despite the development of effective anti-retroviral drugs, a permanent cure has only been documented in one patient to date. In 2007, an HIV-positive patient received a bone marrow transplant to treat his leukemia from an individual who was homozygous for a mutation in the CCR5 gene. This mutation, known as CCR5Δ32, prevents HIV replication by inhibiting the early stage of viral entry into cells, resulting in resistance to infection from the majority of HIV isolates. More than 10 years after his last dose of anti-retroviral therapy, the transplant recipient remains free of replication-competent virus. Multiple groups are now attempting to replicate this success through the use of other CCR5-negative donor cell sources. Additionally, developments in the use of lentiviral vectors and targeted nucleases have opened the doors of precision medicine and enabled new treatment methodologies to combat HIV infection through targeted ablation or down-regulation of CCR5 expression. Here, we review historical cases of CCR5-edited cell-based therapies, current clinical trials and future benefits and challenges associated with this technology. Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Chemokine Receptor Ccr1 Drives Neutrophil-Mediated Kidney Immunopathology and Mortality in Invasive Candidiasis

PubMed Central

Lionakis, Michail S.; Swamydas, Muthulekha; Wan, Wuzhou; Richard Lee, Chyi-Chia; Cohen, Jeffrey I.; Scheinberg, Phillip; Gao, Ji-Liang; Murphy, Philip M.

2012-01-01

Invasive candidiasis is the 4th leading cause of nosocomial bloodstream infection in the US with mortality that exceeds 40% despite administration of antifungal therapy; neutropenia is a major risk factor for poor outcome after invasive candidiasis. In a fatal mouse model of invasive candidiasis that mimics human bloodstream-derived invasive candidiasis, the most highly infected organ is the kidney and neutrophils are the major cellular mediators of host defense; however, factors regulating neutrophil recruitment have not been previously defined. Here we show that mice lacking chemokine receptor Ccr1, which is widely expressed on leukocytes, had selectively impaired accumulation of neutrophils in the kidney limited to the late phase of the time course of the model; surprisingly, this was associated with improved renal function and survival without affecting tissue fungal burden. Consistent with this, neutrophils from wild-type mice in blood and kidney switched from Ccr1lo to Ccr1high at late time-points post-infection, when Ccr1 ligands were produced at high levels in the kidney and were chemotactic for kidney neutrophils ex vivo. Further, when a 1∶1 mixture of Ccr1+/+ and Ccr1−/− donor neutrophils was adoptively transferred intravenously into Candida-infected Ccr1+/+ recipient mice, neutrophil trafficking into the kidney was significantly skewed toward Ccr1+/+ cells. Thus, neutrophil Ccr1 amplifies late renal immunopathology and increases mortality in invasive candidiasis by mediating excessive recruitment of neutrophils from the blood to the target organ. PMID:22916017

[Molecular mechanisms and relationship of M2-polarized macrophages with early response in multiple myeloma].

PubMed

Chen, X Y; Sun, R X; Zhang, W Y; Liu, T; Zheng, Y H; Wu, Y

2017-06-14

Objective: To investigate the relationship between M2-polarized macrophages and early response in multiple myeloma and its molecular mechanism. Methods: Two hundred and forty bone marrow biopsy tissue were collected and M2-polarized macrophages were stained by anti-CD163 monoclonal antibody. In vitro M2-polarized macrophages were derived from human peripheral blood mononuclear cell or THP-1 cells and identified by flow cytometry. Two myeloma cell lines RPMI 8226 and U266 were co-cultured with M2 macrophages using a transwell system. We measured myeloma cells proliferation through CCK-8 method and the pro-inflammatory cytokines expression (TNF-α and IL-6) by ELISA. Real time PCR was applied to measure chemokines (CCL2 and CCL3) , chemokine receptors (CCR2, CCR5) , VEGF and their receptors. In addition, flow cytometry was used to analyze the apoptosis of myeloma cells induced by dexamethasone. Results: ①Patients with high percentage of M2 macrophage involvement in bone marrow showed poorer response (23.9% versus 73.0%, χ (2)=60.31, P <0.001). ② In vitro the proliferation of RPMI 8226 cells ( P =0.005 at 24 h, P =0.020 at 36 h) or U266 myeloma cells ( P = 0.030 at 24h, P =0.020 at 36h) co-cultured with M2-polarized macrophages was higher than control group. ③In vitro the apoptotic rate of RPMI 8226 cells (29.0% versus 71.0%, t =4.97, P =0.008) or U266 myeloma cells (24.9% versus 67.7%, t =6.99, P =0.002) co-cultured with M2-polarized macrophages was lower than control group. ④ In vitro M2-polarized macrophages promoted myeloma cells secreting higher level of IL-6, TNF-α and higher expression of CCL2, CCL3, CCR2, CCR5, VEGFA, VEGFR-1,-2 compared with the non-macrophage co-culture system. Conclusion: M2-polarized macrophages promote myeloma cells proliferation and inhibit apoptosis through a very complex mechanism involving pro-inflammatory cytokines IL-6 and TNF-α, chemokines and related receptors such as CCL2, CCL3, CCR2, CCR3, and VEGF as well as related VEGFR.

Fusion of CCL21 non-migratory active breast epithelial and breast cancer cells give rise to CCL21 migratory active tumor hybrid cell lines.

PubMed

Berndt, Benjamin; Haverkampf, Sonja; Reith, Georg; Keil, Silvia; Niggemann, Bernd; Zänker, Kurt S; Dittmar, Thomas

2013-01-01

The biological phenomenon of cell fusion has been linked to tumor progression because several data provided evidence that fusion of tumor cells and normal cells gave rise to hybrid cell lines exhibiting novel properties, such as increased metastatogenic capacity and an enhanced drug resistance. Here we investigated M13HS hybrid cell lines, derived from spontaneous fusion events between M13SV1-EGFP-Neo breast epithelial cells exhibiting stem cell characteristics and HS578T-Hyg breast cancer cells, concerning CCL21/CCR7 signaling. Western Blot analysis showed that all cell lines varied in their CCR7 expression levels as well as differed in the induction and kinetics of CCR7 specific signal transduction cascades. Flow cytometry-based calcium measurements revealed that a CCL21 induced calcium influx was solely detected in M13HS hybrid cell lines. Cell migration demonstrated that only M13HS hybrid cell lines, but not parental derivatives, responded to CCL21 stimulation with an increased migratory activity. Knockdown of CCR7 expression by siRNA completely abrogated the CCL21 induced migration of hybrid cell lines indicating the necessity of CCL21/CCR7 signaling. Because the CCL21/CCR7 axis has been linked to metastatic spreading of breast cancer to lymph nodes we conclude from our data that cell fusion could be a mechanism explaining the origin of metastatic cancer (hybrid) cells.

Fusion of CCL21 Non-Migratory Active Breast Epithelial and Breast Cancer Cells Give Rise to CCL21 Migratory Active Tumor Hybrid Cell Lines

PubMed Central

Reith, Georg; Keil, Silvia; Niggemann, Bernd; Zänker, Kurt S.; Dittmar, Thomas

2013-01-01

The biological phenomenon of cell fusion has been linked to tumor progression because several data provided evidence that fusion of tumor cells and normal cells gave rise to hybrid cell lines exhibiting novel properties, such as increased metastatogenic capacity and an enhanced drug resistance. Here we investigated M13HS hybrid cell lines, derived from spontaneous fusion events between M13SV1-EGFP-Neo breast epithelial cells exhibiting stem cell characteristics and HS578T-Hyg breast cancer cells, concerning CCL21/CCR7 signaling. Western Blot analysis showed that all cell lines varied in their CCR7 expression levels as well as differed in the induction and kinetics of CCR7 specific signal transduction cascades. Flow cytometry-based calcium measurements revealed that a CCL21 induced calcium influx was solely detected in M13HS hybrid cell lines. Cell migration demonstrated that only M13HS hybrid cell lines, but not parental derivatives, responded to CCL21 stimulation with an increased migratory activity. Knockdown of CCR7 expression by siRNA completely abrogated the CCL21 induced migration of hybrid cell lines indicating the necessity of CCL21/CCR7 signaling. Because the CCL21/CCR7 axis has been linked to metastatic spreading of breast cancer to lymph nodes we conclude from our data that cell fusion could be a mechanism explaining the origin of metastatic cancer (hybrid) cells. PMID:23667660

Genetic diversity and linkage disequilibrium in the chemokine receptor CCR2-CCR5 region among individuals and populations

PubMed Central

Lawhorn, Collene; Yuferov, Vadim; Randesi, Matthew; Ho, Ann; Morgello, Susan; Kreek, Mary Jeanne; Levran, Orna

2013-01-01

Background Chemokine receptors CCR2 and CCR5 play a key role in immune and inflammatory responses and have been associated with several diseases, including AIDS. In order to comprehend health disparities it is important to understand the nature of genetic variation in specific genes of interest in different populations. Current studies of the CCR2 and CCR5 receptor genes are primarily focused on the CCR5-Δ32, and CCR2-V64I SNPs. Methods Sanger sequencing was used to sequence the regions containing 16 SNPs in the adjacent CCR2 and CCR5 genes (including CCR5-Δ32, and CCR2-V64I) in 249 subjects of African, European and Hispanic ancestry. Linkage disequilibrium (LD) and haplotypes were determined using Haploview. Results The data revealed large differences in allele frequencies of several SNPs and LD patterns among the ethnic groups, including SNPs that were restricted to Africans or Europeans. Seven known CCR5 haplotypes and six novel CCR2 haplotypes were identified. A rare case of an HIV+ subject with the CCR5-Δ32/Δ32 was identified. Conclusions These data demonstrate a LD between CCR2 and CCR5 at several loci and provide new information about CCR2 that contributes to our understanding of its population-specific genetic variability. The data indicate that in addition to CCR5-Δ32 and CCR2-V64I, other SNPs and haplotypes may be important genetic determinants of disease and should be investigated. PMID:24011637

Up-regulation of CC chemokine receptor 6 on tonsillar T cells and its induction by in vitro stimulation with α-streptococci in patients with pustulosis palmaris et plantaris

PubMed Central

Yoshizaki, T; Bandoh, N; Ueda, S; Nozawa, H; Goto, T; Kishibe, K; Takahara, M; Harabuchi, Y

2009-01-01

Pustulosis palmaris et plantaris (PPP) is a tonsil-related disease; tonsillectomy is somewhat effective in treating the condition. However, the aetiological association between the tonsils and PPP has not yet been elucidated fully. Recently, some chemokines and chemokine receptors, including CC chemokine receptor (CCR) 4, CCR6 and CX chemokine receptor (CXCR) 3, have been reported to play important roles in the development of psoriasis, a disease related closely to PPP. In this study, we found that CCR6 expression on both tonsillar and peripheral blood T cells was up-regulated more intensively in PPP patients than in non-PPP patients (P < 0·001 for both), but CCR4 and CXCR3 expressions were not. In vitro stimulation with α-streptococcal antigen enhanced CCR6 expression significantly on tonsillar T cells in PPP patients (P < 0·05), but this was not observed in non-PPP patients. The chemotactic response of tonsillar T cells to the CCR6 ligand CC chemokine ligand (CCL) 20 was significantly higher in PPP patients than in non-PPP patients (P < 0·05). The percentage of CCR6-positive peripheral blood T cells decreased after tonsillectomy in PPP patients (P < 0·01); this decrease correlated with an improvement of skin lesions (P < 0·05, r = −0·63). The numbers of CCR6-positive cells and the expression of CCL20 were increased significantly in pathological lesions compared with non-pathological lesions in PPP skin (P < 0·01, P < 0·05 respectively). These results suggest that a novel immune response to α-streptococci may enhance CCR6 expression on T cells in tonsils and that CCR6-positive T cells may move to peripheral blood circulation, resulting in recruitment to target skin lesions expressing CCL20 in PPP patients. This may be one of the key roles in pathogenesis of the tonsil-related disease PPP. PMID:19659772

Simian Immunodeficiency Virus SIVagm Efficiently Utilizes Non-CCR5 Entry Pathways in African Green Monkey Lymphocytes: Potential Role for GPR15 and CXCR6 as Viral Coreceptors.

PubMed

Riddick, Nadeene E; Wu, Fan; Matsuda, Kenta; Whitted, Sonya; Ourmanov, Ilnour; Goldstein, Simoy; Goeken, Robert M; Plishka, Ronald J; Buckler-White, Alicia; Brenchley, Jason M; Hirsch, Vanessa M

2015-12-09

African green monkeys (AGM) are natural hosts of simian immunodeficiency virus (SIV), and infection in these animals is generally nonpathogenic, whereas infection of nonnatural hosts, such as rhesus macaques (RM), is commonly pathogenic. CCR5 has been described as the primary entry coreceptor for SIV in vivo, while human-derived CXCR6 and GPR15 also appear to be used in vitro. However, sooty mangabeys that are genetically deficient in CCR5 due to an out-of-frame deletion are infectible with SIVsmm, indicating that SIVsmm can use alternative coreceptors in vivo. In this study, we examined the CCR5 dependence of SIV strains derived from vervet AGM (SIVagmVer) and the ability of AGM-derived GPR15 and CXCR6 to serve as potential entry coreceptors. We found that SIVagmVer replicated efficiently in AGM and RM peripheral blood mononuclear cells (PBMC) in the presence of the CCR5 antagonist maraviroc, despite the fact that maraviroc was capable of blocking the CCR5-tropic strains SIVmac239, SIVsmE543-3, and simian-human immunodeficiency virus SHIV-AD8 in RM PBMC. We also found that AGM CXCR6 and AGM GPR15, to a lesser extent, supported entry of pseudotype viruses bearing SIVagm envelopes, including SIVagm transmitted/founder envelopes. Lastly, we found that CCR5, GPR15, and CXCR6 mRNAs were detected in AGM and RM memory CD4(+) T cells. These results suggest that GPR15 and CXCR6 are expressed on AGM CD4(+) T cells and are potential alternative coreceptors for SIVagm use in vivo. These data suggest that the use of non-CCR5 entry pathways may be a common feature of SIV replication in natural host species, with the potential to contribute to nonpathogenicity in these animals. African green monkeys (AGM) are natural hosts of SIV, and infection in these animals generally does not cause AIDS, whereas SIV-infected rhesus macaques (RM) typically develop AIDS. Although it has been reported that SIV generally uses CD4 and CCR5 to enter target cells in vivo, other molecules, such as GPR15 and CXCR6, also function as SIV coreceptors in vitro. In this study, we investigated whether SIV from vervet AGM can use non-CCR5 entry pathways, as has been observed in sooty mangabeys. We found that SIVagmVer efficiently replicated in AGM and RM peripheral blood mononuclear cells in the presence of the CCR5 antagonist maraviroc, suggesting that non-CCR5 entry pathways can support SIVagm entry. We found that AGM-derived GPR15 and CXCR6 support SIVagmVer entry in vitro and may serve as entry coreceptors for SIVagm in vivo, since their mRNAs were detected in AGM memory CD4(+) T cells, the preferred target cells of SIV. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Simian Immunodeficiency Virus SIVagm Efficiently Utilizes Non-CCR5 Entry Pathways in African Green Monkey Lymphocytes: Potential Role for GPR15 and CXCR6 as Viral Coreceptors

PubMed Central

Riddick, Nadeene E.; Wu, Fan; Matsuda, Kenta; Whitted, Sonya; Ourmanov, Ilnour; Goldstein, Simoy; Goeken, Robert M.; Plishka, Ronald J.; Buckler-White, Alicia; Brenchley, Jason M.

2015-01-01

ABSTRACT African green monkeys (AGM) are natural hosts of simian immunodeficiency virus (SIV), and infection in these animals is generally nonpathogenic, whereas infection of nonnatural hosts, such as rhesus macaques (RM), is commonly pathogenic. CCR5 has been described as the primary entry coreceptor for SIV in vivo, while human-derived CXCR6 and GPR15 also appear to be used in vitro. However, sooty mangabeys that are genetically deficient in CCR5 due to an out-of-frame deletion are infectible with SIVsmm, indicating that SIVsmm can use alternative coreceptors in vivo. In this study, we examined the CCR5 dependence of SIV strains derived from vervet AGM (SIVagmVer) and the ability of AGM-derived GPR15 and CXCR6 to serve as potential entry coreceptors. We found that SIVagmVer replicated efficiently in AGM and RM peripheral blood mononuclear cells (PBMC) in the presence of the CCR5 antagonist maraviroc, despite the fact that maraviroc was capable of blocking the CCR5-tropic strains SIVmac239, SIVsmE543-3, and simian-human immunodeficiency virus SHIV-AD8 in RM PBMC. We also found that AGM CXCR6 and AGM GPR15, to a lesser extent, supported entry of pseudotype viruses bearing SIVagm envelopes, including SIVagm transmitted/founder envelopes. Lastly, we found that CCR5, GPR15, and CXCR6 mRNAs were detected in AGM and RM memory CD4+ T cells. These results suggest that GPR15 and CXCR6 are expressed on AGM CD4+ T cells and are potential alternative coreceptors for SIVagm use in vivo. These data suggest that the use of non-CCR5 entry pathways may be a common feature of SIV replication in natural host species, with the potential to contribute to nonpathogenicity in these animals. IMPORTANCE African green monkeys (AGM) are natural hosts of SIV, and infection in these animals generally does not cause AIDS, whereas SIV-infected rhesus macaques (RM) typically develop AIDS. Although it has been reported that SIV generally uses CD4 and CCR5 to enter target cells in vivo, other molecules, such as GPR15 and CXCR6, also function as SIV coreceptors in vitro. In this study, we investigated whether SIV from vervet AGM can use non-CCR5 entry pathways, as has been observed in sooty mangabeys. We found that SIVagmVer efficiently replicated in AGM and RM peripheral blood mononuclear cells in the presence of the CCR5 antagonist maraviroc, suggesting that non-CCR5 entry pathways can support SIVagm entry. We found that AGM-derived GPR15 and CXCR6 support SIVagmVer entry in vitro and may serve as entry coreceptors for SIVagm in vivo, since their mRNAs were detected in AGM memory CD4+ T cells, the preferred target cells of SIV. PMID:26656714

Single-color laser ranging with a cube-corner-retroreflector array

NASA Technical Reports Server (NTRS)

Song, G. Hugh

1987-01-01

Lidar cross section of some typical types of cube-corner retroreflectors (CCRs) having a three corner mirror system is investigated for the case that the CCR is tilted from the normal illumination axis. Analytic expressions for the effective aperture area for the two typical window types (circular and hexagonal) of CCRs are obtained for the case that the CCR is tilted. The range of incidence angle in which only the total reflection occurs at all three uncoated corner mirrors has been found to vary considerably with the orientation of CCR and the refractive index of the CCR prism. The analytical expression for the far-field diffraction pattern of a tilted CCR is obtained by taking different polarization transformation of the six sectors of the effective reflecting aperture into account. This expression is essential when evaluating the lidar cross section of a moving CCR which is tilted in general. Formulas for the angles defining the six sectors have also been obtained.

Loss of SMAD4 Promotes Lung Metastasis of Colorectal Cancer by Accumulation of CCR1+ Tumor-Associated Neutrophils through CCL15-CCR1 Axis.

PubMed

Yamamoto, Takamasa; Kawada, Kenji; Itatani, Yoshiro; Inamoto, Susumu; Okamura, Ryosuke; Iwamoto, Masayoshi; Miyamoto, Ei; Chen-Yoshikawa, Toyofumi F; Hirai, Hideyo; Hasegawa, Suguru; Date, Hiroshi; Taketo, Makoto M; Sakai, Yoshiharu

2017-02-01

We have reported loss of SMAD4 promotes expression of CCL15 from colorectal cancer to recruit CCR1 + myeloid cells through the CCL15-CCR1 axis, which contributes to invasion and liver metastasis. However, the molecular mechanism of lung metastasis is yet to be elucidated. Our purpose is to determine whether similar mechanism is involved in the lung metastasis of colorectal cancer. In a mouse model, we examined whether SMAD4 could affect the metastatic activity of colorectal cancer cells to the lung through the CCL15-CCR1 axis. We immunohistochemically analyzed expression of SMAD4, CCL15, and CCR1 with 107 clinical specimens of colorectal cancer lung metastases. We also characterized the CCR1 + myeloid cells using several cell-type-specific markers. In a mouse model, CCL15 secreted from SMAD4-deficient colorectal cancer cells recruited CCR1 + cells, promoting their metastatic activities to the lung. Immunohistochemical analysis of lung metastases from colorectal cancer patients revealed that CCL15 expression was significantly correlated with loss of SMAD4, and that CCL15-positive metastases recruited approximately 1.9 times more numbers of CCR1 + cells than CCL15-negative metastases. Importantly, patients with CCL15-positive metastases showed a significantly shorter relapse-free survival (RFS) than those with CCL15-negative metastases, and multivariate analysis indicated that CCL15 expression was an independent predictor of shorter RFS. Immunofluorescent staining showed that most CCR1 + cells around lung metastases were tumor-associated neutrophil, although a minor fraction was granulocytic myeloid-derived suppressor cell. CCL15-CCR1 axis may be a therapeutic target to prevent colorectal cancer lung metastasis. CCL15 can be a biomarker indicating poor prognosis of colorectal cancer patients with lung metastases. Clin Cancer Res; 23(3); 833-44. ©2016 AACR. ©2016 American Association for Cancer Research.

Chemokine Receptor Ccr6 Deficiency Alters Hepatic Inflammatory Cell Recruitment and Promotes Liver Inflammation and Fibrosis

PubMed Central

Blaya, Delia; Morales-Ibanez, Oriol; Coll, Mar; Millán, Cristina; Altamirano, José; Arroyo, Vicente; Caballería, Joan; Bataller, Ramón; Ginès, Pere; Sancho-Bru, Pau

2015-01-01

Chronic liver diseases are characterized by a sustained inflammatory response in which chemokines and chemokine-receptors orchestrate inflammatory cell recruitment. In this study we investigated the role of the chemokine receptor CCR6 in acute and chronic liver injury. In the absence of liver injury Ccr6 -/- mice presented a higher number of hepatic macrophages and increased expression of pro-inflammatory cytokines and M1 markers Tnf-α, Il6 and Mcp1. Inflammation and cell recruitment were increased after carbon tetrachloride-induced acute liver injury in Ccr6 -/- mice. Moreover, chronic liver injury by carbon tetrachloride in Ccr6 -/- mice was associated with enhanced inflammation and fibrosis, altered macrophage recruitment, enhanced CD4+ cells and a reduction in Th17 (CD4+IL17+) and mature dendritic (MHCII+CD11c+) cells recruitment. Clodronate depletion of macrophages in Ccr6 -/- mice resulted in a reduction of hepatic pro-inflammatory and pro-fibrogenic markers in the absence and after liver injury. Finally, increased CCR6 hepatic expression in patients with alcoholic hepatitis was found to correlate with liver expression of CCL20 and severity of liver disease. In conclusion, CCR6 deficiency affects hepatic inflammatory cell recruitment resulting in the promotion of hepatic inflammation and fibrosis. PMID:26691857

TALEN-mediated generation and genetic correction of disease-specific human induced pluripotent stem cells.

PubMed

Ramalingam, Sivaprakash; Annaluru, Narayana; Kandavelou, Karthikeyan; Chandrasegaran, Srinivasan

2014-01-01

Generation and precise genetic correction of patient-derived hiPSCs have great potential in regenerative medicine. Such targeted genetic manipulations can now be achieved using gene-editing nucleases. Here, we report generation of cystic fibrosis (CF) and Gaucher's disease (GD) hiPSCs respectively from CF (homozygous for CFTRΔF508 mutation) and Type II GD [homozygous for β-glucocerebrosidase (GBA) 1448T>C mutation] patient fibroblasts, using CCR5- specific TALENs. Site-specific addition of loxP-flanked Oct4/Sox2/Klf4/Lin28/Nanog/eGFP gene cassette at the endogenous CCR5 site of patient-derived disease-specific primary fibroblasts induced reprogramming, giving rise to both monoallele (heterozygous) and biallele CCR5-modified hiPSCs. Subsequent excision of the donor cassette was done by treating CCR5-modified CF and GD hiPSCs with Cre. We also demonstrate site-specific correction of sickle cell disease (SCD) mutations at the endogenous HBB locus of patient-specific hiPSCs [TNC1 line that is homozygous for mutated β- globin alleles (βS/βS)], using HBB-specific TALENs. SCD-corrected hiPSC lines showed gene conversion of the mutated βS to the wild-type βA in one of the HBB alleles, while the other allele remained a mutant phenotype. After excision of the loxP-flanked DNA cassette from the SCD-corrected hiPSC lines using Cre, we obtained secondary heterozygous βS/βA hiPSCs, which express the wild-type (βA) transcript to 30-40% level as compared to uncorrected (βS/βS) SCD hiPSCs when differentiated into erythroid cells. Furthermore, we also show that TALEN-mediated generation and genetic correction of disease-specific hiPSCs did not induce any off-target mutations at closely related sites.

Cloning and expression analysis of a novel G-protein-coupled receptor selectively expressed on granulocytes.

PubMed

Yousefi, S; Cooper, P R; Potter, S L; Mueck, B; Jarai, G

2001-06-01

The migration of neutrophils into sites of acute and chronic inflammation is mediated by chemokines. We used degenerate-primer reverse transcriptase-polymerase chain reaction (RT-PCR) to analyze chemokine receptor expression in neutrophils and identify novel receptors. RNA was isolated from human peripheral blood neutrophils and from neutrophils that had been stimulated for 5 h with granulocyte-macrophage colony-stimulating factor or by coculturing with primary human bronchial epithelial cells. Amplification products were cloned, and clone redundancy was determined. Seven known G-protein-coupled receptors were identified among 38 clones-CCR1, CCR4, CXCR1, CXCR2, CXCR4, HM63, and FPR1-as well as a novel gene, EX33. The full-length EX33 clone was obtained, and an in silico approach was used to identify the putative murine homologue. The EX33 gene encodes a 396-amino-acid protein with limited sequence identity to known receptors. Expression studies of several known chemokine receptors and EX33 revealed that resting neutrophils expressed higher levels of CXCRs and EX33 compared with activated neutrophils. Northern blot experiments revealed that EX33 is expressed mainly in bone marrow, lung, and peripheral blood leukocytes. Using RT-PCR analysis, we showed more abundant expression of EX33 in neutrophils and eosinophils, in comparison with that in T- or B-lymphocytes, indicating cell-specific expression among leukocytes.

PSC-RANTES Blocks R5 Human Immunodeficiency Virus Infection of Langerhans Cells Isolated from Individuals with a Variety of CCR5 Diplotypes

PubMed Central

Kawamura, Tatsuyoshi; Bruce, Shannon E.; Abraha, Awet; Sugaya, Makoto; Hartley, Oliver; Offord, Robin E.; Arts, Eric J.; Zimmerman, Peter A.; Blauvelt, Andrew

2004-01-01

Topical microbicides that effectively block interactions between CCR5+ immature Langerhans cells (LC) residing within genital epithelia and R5 human immunodeficiency virus (HIV) may decrease sexual transmission of HIV. Here, we investigated the ability of synthetic RANTES analogues (AOP-, NNY-, and PSC-RANTES) to block R5 HIV infection of human immature LC by using a skin explant model. In initial experiments using activated peripheral blood mononuclear cells, each analogue compound demonstrated marked antiviral activity against two R5 HIV isolates. Next, we found that 20-min preincubation of skin explants with each RANTES analogue blocked R5 HIV infection of LC in a dose-dependent manner (1 to 100 nM) and that PSC-RANTES was the most potent of these compounds. Similarly, preincubation of LC with each analogue was able to block LC-mediated infection of cocultured CD4+ T cells. Competition experiments between primary R5 and X4 HIV isolates showed blocking of R5 HIV by PSC-RANTES and no evidence of increased propagation of X4 HIV, data that are consistent with the specificity of PSC-RANTES for CCR5 and the CCR5+ CXCR4− phenotype of immature LC. Finally, when CCR5 genetic polymorphism data were integrated with results from the in vitro LC infection studies, PSC-RANTES was found to be equally effective in inhibiting R5 HIV in LC isolated from individuals with CCR5 diplotypes known to be associated with low, intermediate, and high cell surface levels of CCR5. In summary, PSC-RANTES is a potent inhibitor of R5 HIV infection in immature LC, suggesting that it may be useful as a topical microbicide to block sexual transmission of HIV. PMID:15220435

Infiltration of myeloid cells in the pregnant uterus is affected by heme oxygenase-1.

PubMed

Zhao, Hui; Kalish, Flora; Wong, Ronald J; Stevenson, David K

2017-01-01

Infiltrating myeloid cells in pregnant uteri play critical roles in the establishment of the placenta and maintenance of normal pregnancies. Their recruitment and proliferation are primarily mediated by the interactions of cytokines and chemokines secreted locally with their corresponding receptors. Heme oxygenase-1 (HO-1) has various physiologic properties that contribute to placental vascular development, with deficiencies in HO-1 associated with pregnancy disorders. Here, we investigated the effect of HO-1 on myeloid cell infiltration into pregnant uteri using a partial HO-1-deficient (Het, HO-1 +/- ) mouse model. With the use of flow cytometry, HO-1 was found predominantly expressed in circulating and uterine myeloid cells, specifically neutrophils and monocytes/macrophages. In pregnant Het uteri, the numbers of neutrophils and monocytes/macrophages were significantly reduced compared with pregnant wild-type (WT; HO-1 +/+ ) uteri. With the use of BrdU in vivo assays, HO-1 deficiency did not affect cell proliferation or blood cell populations. With the use of PCR arrays, gene expression of cytokines (Csf1, Csf3), chemokines (Ccl1, Ccl2, Ccl6, Ccl8, Ccl11, Ccl12, Cxcl4, Cxcl9, Cxcl12), and their receptors (Ccr1, Ccr2, Ccr3, Ccr5) were also reduced significantly in Het compared with pregnant WT uteri. Moreover, with the use of flow cytometry, myeloid CSF1R and CCR2 expression in blood and uteri from both pregnant and nonpregnant mice was characterized, and a deficiency in HO-1 significantly reduced CCR2 expression in infiltrating uterine monocytes/macrophages and dendritic cells (DCs). These data reveal that HO-1 regulates not only cytokine/chemokine production in pregnant uteri but also myeloid cell receptor numbers, suggesting a role of HO-1 in the recruitment and maintenance of myeloid cells in pregnant uteri and subsequent effects on placental vascular formation. © Society for Leukocyte Biology.

Increase in chemokines CXCL10 and CCL2 in blood from pigs infected with high compared to low virulence African swine fever virus isolates.

PubMed

Fishbourne, Emma; Hutet, Evelyne; Abrams, Charles; Cariolet, Roland; Le Potier, Marie-Frédérique; Takamatsu, Haru-H; Dixon, Linda K

2013-10-01

Modulation of the expression of chemokines and chemokine receptors in whole blood was compared following infection of pigs with high and low virulence isolates of African swine fever virus. Levels of mRNAs for CCL2, CCL3L1, CCL4, CXCL10, CCR1 and CCR5 were significantly increased in at least one time point following infection in two experiments and CCL5, CCR9 and CXCR4 mRNA were significantly increased in one of the experiments. The results showed that greatest fold increases in mRNAs for CXCL10 and CCL2 were observed following infection of pigs. CXCL10 mRNA was increased by up to 15 fold in infected compared to uninfected pigs. CXCL10 protein was also detected in serum from pigs infected with the high virulence Benin 97/1 isolate. Levels of CCL2 mRNA were increased in pigs infected with high virulence Benin 97/1 isolate compared to low virulence OURT88/3 isolate and this correlated with an increase of greater than 30 fold in levels of CCL2 protein detected in serum from pigs infected with this isolate. An increase in overall chemotaxis active compounds in defibrinated plasma samples from Benin 97/1 infected pigs was observed at 3 days post-infection (dpi) and a decrease by 7 dpi as measured by chemotaxis assay using normal pig leucocytes in vitro. Increased levels of CXCL10 may either contribute to the activation of lymphocyte priming toward the Th1 phenotype or induction of T lymphocyte apoptosis. Increased levels of CCL2, a chemoattractant for macrophages, may result in increased recruitment of monocytes from bone marrow thus increasing the pool of cells susceptible to infection.

Stromal CCR6 drives tumor growth in a murine transplantable colon cancer through recruitment of tumor-promoting macrophages

PubMed Central

Nandi, Bisweswar; Shapiro, Mia; Samur, Mehmet K.; Pai, Christine; Frank, Natasha Y.; Yoon, Charles; Prabhala, Rao H.; Munshi, Nikhil C.; Gold, Jason S.

2016-01-01

ABSTRACT Interactions between the inflammatory chemokine CCL20 and its receptor CCR6 have been implicated in promoting colon cancer; however, the mechanisms behind this effect are poorly understood. We have previously demonstrated that deficiency of CCR6 is associated with decreased tumor macrophage accumulation in a model of sporadic intestinal tumorigenesis. In this study, we aimed to determine the role of stromal CCR6 expression in a murine syngeneic transplantable colon cancer model. We show that deficiency of host CCR6 is associated with decreased growth of syngeneic CCR6-expressing colon cancers. Colon cancers adoptively transplanted into CCR6-deficient mice have decreased tumor-associated macrophages without alterations in the number of monocytes in blood or bone marrow. CCL20, the unique ligand for CCR6, promotes migration of monocytes in vitro and promotes accumulation of macrophages in vivo. Depletion of tumor-associated macrophages decreases the growth of tumors in the transplantable tumor model. Macrophages infiltrating the colon cancers in this model secrete the inflammatory mediators CCL2, IL-1α, IL-6 and TNFα. Ccl2, Il1α and Il6 are consequently downregulated in tumors from CCR6-deficient mice. CCL2, IL-1α and IL-6 also promote proliferation of colon cancer cells, linking the decreased macrophage migration into tumors mediated by CCL20–CCR6 interactions to the delay in tumor growth in CCR6-deficient hosts. The relevance of these findings in human colon cancer is demonstrated through correlation of CCR6 expression with that of the macrophage marker CD163 as well as that of CCL2, IL1α and TNFα. Our findings support the exploration of targeting the CCL20–CCR6 pathway for the treatment of colon cancer. PMID:27622061

Resistance of chemokine receptor 6-deficient mice to Yersinia enterocolitica infection: evidence of defective M-cell formation in vivo.

PubMed

Westphal, Sabine; Lügering, Andreas; von Wedel, Julia; von Eiff, Christof; Maaser, Christian; Spahn, Thomas; Heusipp, Gerhard; Schmidt, M Alexander; Herbst, Hermann; Williams, Ifor R; Domschke, Wolfram; Kucharzik, Torsten

2008-03-01

M cells, specialized cells within Peyer's patches (PPs), are reduced in number in chemokine receptor 6 (CCR6)-deficient mice. The pathogenic microorganism Yersinia enterocolitica exploits M cells for the purpose of mucosal tissue invasion exclusively through PPs. The aim of this study was to evaluate the course of yersiniosis in CCR6-deficient mice and to investigate whether these mice might be used as an in vivo model to determine M-cell function. After oral challenge with Y. enterocolitica, control mice suffered from lethal septic infection whereas CCR6-deficient mice showed very limited symptoms of infection. Immunohistochemical analysis demonstrated PP invasion by Y. enterocolitica in control mice whereas no bacteria could be found in CCR6-deficient mice. In addition, a significant induction of proinflammatory cytokines could be found in control mice whereas proinflammatory cytokine levels in CCR6-deficient mice remained unchanged. In contrast, intraperitoneal infection resulted in severe systemic yersiniosis in both mouse groups. Abrogated oral Y. enterocolitica infection in CCR6-deficient mice demonstrates the importance of CCR6 expression in the physiological and pathological immune responses generated within PPs by influencing M-cell differentiation, underscoring the important role of M cells in the process of microbial uptake. CCR6-deficient mice may therefore represent a suitable model for the study of M-cell function in vivo.

A rare subset of skin-tropic regulatory T cells expressing Il10/Gzmb inhibits the cutaneous immune response.

PubMed

Ikebuchi, Ryoyo; Teraguchi, Shunsuke; Vandenbon, Alexis; Honda, Tetsuya; Shand, Francis H W; Nakanishi, Yasutaka; Watanabe, Takeshi; Tomura, Michio

2016-10-19

Foxp3 + regulatory T cells (Tregs) migrating from the skin to the draining lymph node (dLN) have a strong immunosuppressive effect on the cutaneous immune response. However, the subpopulations responsible for their inhibitory function remain unclear. We investigated single-cell gene expression heterogeneity in Tregs from the dLN of inflamed skin in a contact hypersensitivity model. The immunosuppressive genes Ctla4 and Tgfb1 were expressed in the majority of Tregs. Although Il10-expressing Tregs were rare, unexpectedly, the majority of Il10-expressing Tregs co-expressed Gzmb and displayed Th1-skewing. Single-cell profiling revealed that CD43 + CCR5 + Tregs represented the main subset within the Il10/Gzmb-expressing cell population in the dLN. Moreover, CD43 + CCR5 + CXCR3 - Tregs expressed skin-tropic chemokine receptors, were preferentially retained in inflamed skin and downregulated the cutaneous immune response. The identification of a rare Treg subset co-expressing multiple immunosuppressive molecules and having tissue-remaining capacity offers a novel strategy for the control of skin inflammatory responses.

Altered Peripheral Blood Monocyte Phenotype and Function in Chronic Liver Disease: Implications for Hepatic Recruitment and Systemic Inflammation.

PubMed

Gadd, Victoria L; Patel, Preya J; Jose, Sara; Horsfall, Leigh; Powell, Elizabeth E; Irvine, Katharine M

2016-01-01

Liver and systemic inflammatory factors influence monocyte phenotype and function, which has implications for hepatic recruitment and subsequent inflammatory and fibrogenic responses, as well as host defence. Peripheral blood monocyte surface marker (CD14, CD16, CD163, CSF1R, CCR2, CCR4, CCR5, CXCR3, CXCR4, CX3CR1, HLA-DR, CD62L, SIGLEC-1) expression and capacity for phagocytosis, oxidative burst and LPS-stimulated TNF production were assessed in patients with hepatitis C (HCV) (n = 39) or non-alcoholic fatty liver disease (NAFLD) (n = 34) (classified as non-advanced disease, compensated cirrhosis and decompensated cirrhosis) and healthy controls (n = 11) by flow cytometry. The selected markers exhibited similar monocyte-subset-specific expression patterns between patients and controls. Monocyte phenotypic signatures differed between NAFLD and HCV patients, with an increased proportion of CD16+ non-classical monocytes in NAFLD, but increased expression of CXCR3 and CXCR4 in HCV. In both cohorts, monocyte CCR2 expression was reduced and CCR4 elevated over controls. CD62L expression was specifically elevated in patients with decompensated cirrhosis and positively correlated with the model-for-end-stage-liver-disease score. Functionally, monocytes from patients with decompensated cirrhosis had equal phagocytic capacity, but displayed features of dysfunction, characterised by lower HLA-DR expression and blunted oxidative responses. Lower monocyte TNF production in response to LPS stimulation correlated with time to death in 7 (46%) of the decompensated patients who died within 8 months of recruitment. Chronic HCV and NAFLD differentially affect circulating monocyte phenotype, suggesting specific injury-induced signals may contribute to hepatic monocyte recruitment and systemic activation state. Monocyte function, however, was similarly impaired in patients with both HCV and NAFLD, particularly in advanced disease, which likely contributes to the increased susceptibility to infection in these patients.

Cross-Desensitization of CCR1, But Not CCR2, Following Activation of the Formyl Peptide Receptor (FPR1)1

PubMed Central

Bednar, Filip; Song, Changcheng; Bardi, Giuseppe; Cornwell, William; Rogers, Thomas J.

2014-01-01

The cross-regulation of G protein-coupled receptors (GPCRs) plays an important role in the immune response. Studies from several laboratories have suggested that a hierarchy of sensitivities to cross-desensitization exists for the chemoattractant GPCRs. We carried out experiments to study the capacity of the formyl peptide receptor-1 (FPR1) to desensitize chemokine receptors CCR1 and CCR2. Our results show that activation of FPR1 resulted in the desensitization and partial internalization of CCR1, but not CCR2, in both primary human monocytes and HEK293 cells co-expressing CCR1, CCR2, and FPR1 (HR1R2F cells). The desensitization of CCR1 by FPR1 stimulation was not due to the simple depletion of the Ca2+ stores, but was dependent on activation of PKC. Furthermore, we found that the cross-desensitization of CCR1 by FPR1 was associated with CCR1 phosphorylation, and moderate reduction of CCR1 cell surface expression. In contrast, CCR2 was not phosphorylated or internalized following FPR1 activation. Additional studies showed that optimal cross-talk between FPR1 and CCR1 were dependent on the functional activity of PKCβ. These results provide a mechanistic basis for the capacity of certain GPCR ligands to exert rapid and selective cross-inactivation of other chemoattractant receptors, and suggest that FPR1 is able to exert “traffic control” in the migration of inflammatory cells by rapidly inhibiting the cell responses to potentially “low priority” chemoattractants such as CCR1 agonists without inhibiting the response to “higher priority” CCR2 chemoattractants. PMID:24778447

Gene expression profiles in the bovine corpus luteum (CL) during the estrous cycle and pregnancy: Possible roles of chemokines in regulating CL function during pregnancy

PubMed Central

SAKUMOTO, Ryosuke; HAYASHI, Ken-Go; HOSOE, Misa; IGA, Kosuke; KIZAKI, Keiichiro; OKUDA, Kiyoshi

2014-01-01

To determine functional differences between the corpus luteum (CL) of the estrous cycle and pregnancy in cows, gene expression profiles were compared using a 15 K bovine oligo DNA microarray. In the pregnant CL at days 20–25, 40–45 and 150–160, the expressions of 138, 265 and 455 genes differed by a factor of > 2-fold (P < 0.05) from their expressions in the cyclic CL (days 10–12 of the estrous cycle). Messenger RNA expressions of chemokines (eotaxin, lymphotactin and ENA-78) and their receptors (CCR3, XCR1 and CXCR2) were validated by quantitative real-time PCR. Transcripts of eotaxin were more abundant in the CL at days 40–45 and 150–160 of pregnancy than in the cyclic CL (P < 0.01). In contrast, the mRNA expressions of lymphotactin, ENA-78 and XCR1 were lower in the CL of pregnancy (P < 0.05). Messenger RNAs of CCR3 and CXCR2 were similarly detected both in the cyclic and pregnant CL. Tissue protein levels of eotaxin were significantly higher in the CL at days 150–160 of pregnancy than in the CL at other stages, whereas the lymphotactin protein levels in the CL at days 20–25 of pregnancy were lower (P < 0.05). Immunohistochemical staining showed that CCR3 was expressed in the luteal cells and that XCR1 was expressed in both the luteal cells and endothelial cells. Collectively, the different gene expression profiles may contribute to functional differences between the cyclic and pregnant CL, and chemokines including eotaxin and lymphotactin may regulate CL function during pregnancy in cows. PMID:25382605

Eotaxin/CCL11 in idiopathic retroperitoneal fibrosis.

PubMed

Mangieri, Domenica; Corradi, Domenico; Martorana, Davide; Malerba, Giovanni; Palmisano, Alessandra; Libri, Irene; Bartoli, Veronica; Carnevali, Maria L; Goldoni, Matteo; Govoni, Paolo; Alinovi, Rossella; Buzio, Carlo; Vaglio, Augusto

2012-10-01

Idiopathic retroperitoneal fibrosis (IRF) is a rare fibro-inflammatory disorder characterized by a periaortic tissue which often encases the ureters causing acute renal failure. IRF histology shows fibrosis and a chronic inflammatory infiltrate with frequent tissue eosinophilia. We assessed a panel of molecules promoting eosinophilia and fibrosis in IRF patients and performed an immunogenetic study. Serum levels of eotaxin/CCL11, regulated and normal T-cell expressed and secreted (RANTES), granulocyte colony-stimulating factor (G-CSF), interleukin (IL)-5, platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) were measured using a multiplex assay in 24 newly diagnosed, untreated IRF patients and 14 healthy controls. Retroperitoneal biopsies (available in 8/24 patients) were histologically evaluated to assess eosinophil infiltration, whereas mast cells (MCs) were identified by immunohistochemical analysis for human tryptase. Immunohistochemistry for eotaxin/CCL11 and its receptor CCR3 was also performed. Six single nucleotide polymorphisms (SNPs) within the CCL11 gene (rs6505403, rs1860184, rs4795896, rs17735961, rs16969415 and rs17809012) were investigated in 142 IRF patients and 214 healthy controls. Serum levels of eotaxin/CCL11 were higher in IRF patients than in controls (P = 0.009). Eotaxin/CCL11 drives tissue infiltration of eosinophils and MCs, which can promote fibrosis. Eosinophilic infiltration was prominent (>5 cells/hpf) in five (62.5%) cases, and abundant tryptase-positive MCs were found in all cases; notably, MCs were in a degranulating state. Immunohistochemistry showed that CCL11 was highly produced by infiltrating mononuclear cells and that its receptor CCR3 was expressed by infiltrating eosinophils, MCs, lymphocytes and fibroblasts. None of the tested CCL11 SNPs showed disease association, but the TTCCAT haplotype was significantly associated with IRF (P = 0.0005). These findings suggest that the eotaxin/CCL11-CCR3 axis is active in IRF and may contribute to its pathogenesis; the TTCCAT haplotype within the CCL11 gene is significantly associated with IRF.

The CC chemokine eotaxin/CCL11 has a selective profibrogenic effect on human lung fibroblasts.

PubMed

Puxeddu, Ilaria; Bader, Reem; Piliponsky, Adrian Martin; Reich, Reuven; Levi-Schaffer, Francesca; Berkman, Neville

2006-01-01

Eotaxin/CCL11 plays an important role in asthma. It acts through the chemokine receptor CCR3 expressed on hematopoietic and nonhematopoietic cells in the lung. To determine whether eotaxin/CCL11 modulates lung and bronchial fibroblast properties and thereby might contribute to airway remodeling. CCR3 expression was characterized on a lung fibroblast line (MRC-5; flow cytometry, fluorescent microscopy, RT-PCR, and Northern blotting), on primary bronchial fibroblasts (flow cytometry), and on fibroblasts in human lung tissue (confocal laser microscopy). The effects of eotaxin/CCL11 on lung fibroblast migration (Boyden chamber), proliferation (tritiated thymidine incorporation), alpha-smooth muscle actin expression (ELISA), 3-dimensional collagen gel contraction (floating gel), pro-alpha1(I) collagen mRNA (Northern blotting), total collagen synthesis (tritiated proline incorporation), matrix metalloproteinase activity (gelatin zymography), and TGF-beta(1) release (ELISA) were evaluated. The contribution of eotaxin/CCL11/CCR3 binding on lung fibroblasts was also investigated by neutralizing experiments. CCR3 is constitutively expressed in cultured lung and primary bronchial fibroblasts and colocalizes with specific surface markers for human fibroblasts in lung tissue. Eotaxin/CCL11 selectively modulates fibroblast activities by increasing their proliferation, matrix metalloproteinase 2 activity, and collagen synthesis but not their differentiation into myofibroblasts, contractility in collagen gel, or TGF-beta(1) release. Eotaxin/CCL11 enhances migration of lung fibroblasts in response to nonspecific chemoattractants, and this effect is completely inhibited by anti-CCR3-neutralizing antibodies. These data demonstrate that eotaxin/CCL11 has a direct and selective profibrogenic effect on lung and bronchial fibroblasts, providing a novel mechanism whereby eotaxin/CCL11 can participate in airway remodeling in asthma.

A polymorphism in CCR1/CCR3 is associated with narcolepsy.

PubMed

Toyoda, Hiromi; Miyagawa, Taku; Koike, Asako; Kanbayashi, Takashi; Imanishi, Aya; Sagawa, Yohei; Kotorii, Nozomu; Kotorii, Tatayu; Hashizume, Yuji; Ogi, Kimihiro; Hiejima, Hiroshi; Kamei, Yuichi; Hida, Akiko; Miyamoto, Masayuki; Imai, Makoto; Fujimura, Yota; Tamura, Yoshiyuki; Ikegami, Azusa; Wada, Yamato; Moriya, Shunpei; Furuya, Hirokazu; Takeuchi, Masaki; Kirino, Yohei; Meguro, Akira; Remmers, Elaine F; Kawamura, Yoshiya; Otowa, Takeshi; Miyashita, Akinori; Kashiwase, Koichi; Khor, Seik-Soon; Yamasaki, Maria; Kuwano, Ryozo; Sasaki, Tsukasa; Ishigooka, Jun; Kuroda, Kenji; Kume, Kazuhiko; Chiba, Shigeru; Yamada, Naoto; Okawa, Masako; Hirata, Koichi; Mizuki, Nobuhisa; Uchimura, Naohisa; Shimizu, Tetsuo; Inoue, Yuichi; Honda, Yutaka; Mishima, Kazuo; Honda, Makoto; Tokunaga, Katsushi

2015-10-01

Etiology of narcolepsy-cataplexy involves multiple genetic and environmental factors. While the human leukocyte antigen (HLA)-DRB1*15:01-DQB1*06:02 haplotype is strongly associated with narcolepsy, it is not sufficient for disease development. To identify additional, non-HLA susceptibility genes, we conducted a genome-wide association study (GWAS) using Japanese samples. An initial sample set comprising 409 cases and 1562 controls was used for the GWAS of 525,196 single nucleotide polymorphisms (SNPs) located outside the HLA region. An independent sample set comprising 240 cases and 869 controls was then genotyped at 37 SNPs identified in the GWAS. We found that narcolepsy was associated with a SNP in the promoter region of chemokine (C-C motif) receptor 1 (CCR1) (rs3181077, P=1.6×10(-5), odds ratio [OR]=1.86). This rs3181077 association was replicated with the independent sample set (P=0.032, OR=1.36). We measured mRNA levels of candidate genes in peripheral blood samples of 38 cases and 37 controls. CCR1 and CCR3 mRNA levels were significantly lower in patients than in healthy controls, and CCR1 mRNA levels were associated with rs3181077 genotypes. In vitro chemotaxis assays were also performed to measure monocyte migration. We observed that monocytes from carriers of the rs3181077 risk allele had lower migration indices with a CCR1 ligand. CCR1 and CCR3 are newly discovered susceptibility genes for narcolepsy. These results highlight the potential role of CCR genes in narcolepsy and support the hypothesis that patients with narcolepsy have impaired immune function. Copyright © 2015 Elsevier Inc. All rights reserved.

Genetic modification of mesenchymal stem cells overexpressing CCR1 increases cell viability, migration, engraftment, and capillary density in the injured myocardium.

PubMed

Huang, Jing; Zhang, Zhiping; Guo, Jian; Ni, Aiguo; Deb, Arjun; Zhang, Lunan; Mirotsou, Maria; Pratt, Richard E; Dzau, Victor J

2010-06-11

Although mesenchymal stem cell (MSC) transplantation has been shown to promote cardiac repair in acute myocardial injury in vivo, its overall restorative capacity appears to be restricted mainly because of poor cell viability and low engraftment in the ischemic myocardium. Specific chemokines are upregulated in the infarcted myocardium. However the expression levels of the corresponding chemokine receptors (eg, CCR1, CXCR2) in MSCs are very low. We hypothesized that this discordance may account for the poor MSC engraftment and survival. To determine whether overexpression of CCR1 or CXCR2 chemokine receptors in MSCs augments their cell survival, migration and engraftment after injection in the infarcted myocardium. Overexpression of CCR1, but not CXCR2, dramatically increased chemokine-induced murine MSC migration and protected MSC from apoptosis in vitro. Moreover, when MSCs were injected intramyocardially one hour after coronary artery ligation, CCR1-MSCs accumulated in the infarcted myocardium at significantly higher levels than control-MSCs or CXCR2-MSCs 3 days postmyocardial infarction (MI). CCR1-MSC-injected hearts exhibited a significant reduction in infarct size, reduced cardiomyocytes apoptosis and increased capillary density in injured myocardium 3 days after MI. Furthermore, intramyocardial injection of CCR1-MSCs prevented cardiac remodeling and restored cardiac function 4 weeks after MI. Our results demonstrate the in vitro and in vivo salutary effects of genetic modification of stem cells. Specifically, overexpression of chemokine receptor enhances the migration, survival and engraftment of MSCs, and may provide a new therapeutic strategy for the injured myocardium.

The Chemokine CCL4 (MIP-1β) Evokes Antinociceptive Effects in Mice: a Role for CD4+ Lymphocytes and Met-Enkephalin.

PubMed

García-Domínguez, Mario; Lastra, Ana; Folgueras, Alicia R; Cernuda-Cernuda, Rafael; Fernández-García, María Teresa; Hidalgo, Agustín; Menéndez, Luis; Baamonde, Ana

2018-06-15

In the present study, we characterize the antinociceptive effects produced by the chemokine CCL4 in mice. The intraplantar administration of very low doses of CCL4 (0.1-3 pg) produced bilateral antinociception assessed by the unilateral hot-plate test (UHP) without evoking chemotactic responses at the injection site. Moreover, the subcutaneous administration of CCL4 (3-100 pg/kg) also yielded bilateral antinociception in the UHP and the paw pressure test and reduced the number of spinal neurons that express Fos protein in response to noxious stimulation. The implication of peripheral CCR5 but not CCR1 in CCL4-evoked antinociception was deduced from the inhibition produced by systemic but not intrathecal, administration of the CCR5 antagonist DAPTA, and the inefficacy of the CCR1 antagonist J113863. Besides, the inhibition observed after subcutaneous but not intrathecal administration of naloxone demonstrated the involvement of peripheral opioids and the efficacy of naltrindole but not cyprodime or nor-binaltorphimine supported the participation of δ-opioid receptors. In accordance, plasma levels of met-enkephalin, but not β-endorphin, were augmented in response to CCL4. Likewise, CCL4-evoked antinociception was blocked by the administration of an anti-met-enk antibody. Leukocyte depletion experiments performed with cyclophosphamide, anti-Ly6G, or anti-CD3 antibodies indicated that the antinociceptive effect evoked by CCL4 depends on circulating T lymphocytes. Double immunofluorescence experiments showed a four times more frequent expression of met-enk in CD4 + than in CD8 + T lymphocytes. CCL4-induced antinociception almost disappeared upon CD4 + , but not CD8 + , lymphocyte depletion with selective antibodies, thus supporting that the release of met-enk from CD4 + lymphocytes underlies the opioid antinociceptive response evoked by CCL4.

CCR7 Plays No Appreciable Role in Trafficking of Central Memory CD4 T Cells to Lymph Nodes

PubMed Central

Lugt, Bryan Vander; Tubo, Noah J.; Nizza, Suzanne T.; Boes, Marianne; Malissen, Bernard; Fuhlbrigge, Robert C.; Kupper, Thomas S.; Campbell, James J.

2013-01-01

CCR7−/− mice exhibit profound anomalies in LN and spleen architecture, which complicates the study of CCR7-mediated T cell trafficking in vivo. To circumvent this problem, we established in vivo models in which WT and CCR7−/− populations coexist within mice possessing normal lymphoid organs, and must compete for developmental niches within the tissues of these mice. Under the conditions we have created in vivo, we find the entry of memory CD4 T cells into LN from the blood to be independent of CCR7. Thus, the central memory CD4 T cells that traffic though LN, which are often defined by their expression of CCR7, do not appear to gain any competitive homing advantage by expressing this receptor. Furthermore, in contrast to cutaneous DC populations, we found that CCR7 deficiency had no appreciable effect on the exit of CD4 T cells from inflamed skin. Finally, we found that WT and CCR7−/− precursors were equally represented within the major thymic subpopulations, despite previous findings that CCR7 plays a role in seeding the thymus from bone marrow-derived T cell precursors. PMID:23935190

HIV type 1 chemokine receptor usage in mother-to-child transmission.

PubMed

Salvatori, F; Scarlatti, G

2001-07-01

To investigate the role of the HIV-1 phenotype in mother-to-child HIV-1 transmission, we evaluated coreceptor usage and replication kinetics in chemokine receptor-expressing U87MG.CD4 cells of primary isolates from 32 HIV-1-infected mothers of Italian origin, none under preventive antiretroviral therapy, and from their infected infants. Five of 15 mothers of infected children and 2 of 17 mothers of uninfected children harbored viruses able to use CXCR4 as coreceptor. However, all isolates used CCR5, alone or in association with CXCR4. The replicative capacity in coreceptor-expressing cells of the viral isolates did not differ between the two groups of mothers. All mothers with an R5 virus transmitted a virus with the same coreceptor usage, whereas those four with a multitropic virus transmitted such a virus in one case. Although the presence of a mixed viral population was documented in the mothers, we did not observe transmission solely of X4 viruses. Interestingly, the only child infected with a multitropic virus carried a defective CCR5 allele. Analysis of the env V3 region of the provirus from this child revealed infection with multiple viral variants with a predominance of R5-type over X4-type sequences. These findings show that CCR5 usage of a viral isolate is not a discriminating risk factor for vertical transmission. Furthermore, X4 viruses can be transmitted to the newborn, although less frequently. In particular, we document the transmission of multiple viral variants with different coreceptor usage in a Delta32 CCR5 heterozygous child, and demonstrate that the heterozygous genotype per se does not contribute to the restriction of R5-type virus spread.

Cocaine and sigma-1 receptors modulate HIV infection, chemokine receptors, and the HPA axis in the huPBL-SCID model.

PubMed

Roth, Michael D; Whittaker, Katherine M; Choi, Ruth; Tashkin, Donald P; Baldwin, Gayle Cocita

2005-12-01

Cocaine is associated with an increased risk for, and progression of, clinical disease associated with human immunodeficiency virus (HIV) infection. A human xenograft model, in which human peripheral blood mononuclear cells were implanted into severe combined immunodeficiency mice (huPBL-SCID) and infected with a HIV reporter virus, was used to investigate the biological interactions between cocaine and HIV infection. Systemic administration of cocaine (5 mg/kg/d) significantly increased the percentage of HIV-infected PBL (two- to threefold) and viral load (100- to 300-fold) in huPBL-SCID mice. Despite the capacity for cocaine to increase corticosterone and adrenocorticotropic hormone levels in control mice, the hypothalamic-pituitary-adrenal axis was suppressed in HIV-infected animals, and corticosterone levels were further decreased when animals were exposed to HIV and cocaine. Activating huPBL in vitro in the presence of 10(-8) M cocaine increased expression of CC chemokine receptor 5 (CCR5) and CXC chemokine receptor 4 (CXCR4) coreceptors. Expression of CCR5 was also increased at early time-points in the huPBL-SCID model following systemic exposure to cocaine (54.1+/-9.4% increase over control, P<0.01). This effect preceded the boost in viral infection and waned as HIV infection progressed. Cocaine has been shown to mediate immunosuppressive effects by activating sigma-1 receptors in immune cells in vitro and in vivo. Consistent with these reports, a selective sigma-1 antagonist, BD1047, blocked the effects of cocaine on HIV replication in the huPBL-SCID mouse. Our results suggest that systemic exposure to cocaine can enhance HIV infection in vivo by activating sigma-1 receptors and by modulating the expression of HIV coreceptors.

CC chemokine receptor 4 is required for experimental autoimmune encephalomyelitis by regulating GM-CSF and IL-23 production in dendritic cells

PubMed Central

Poppensieker, Karola; Otte, David-Marian; Schürmann, Britta; Limmer, Andreas; Dresing, Philipp; Drews, Eva; Schumak, Beatrix; Klotz, Luisa; Raasch, Jennifer; Mildner, Alexander; Waisman, Ari; Scheu, Stefanie; Knolle, Percy; Förster, Irmgard; Prinz, Marco; Maier, Wolfgang; Zimmer, Andreas; Alferink, Judith

2012-01-01

Dendritic cells (DCs) are pivotal for the development of experimental autoimmune encephalomyelitis (EAE). However, the mechanisms by which they control disease remain to be determined. This study demonstrates that expression of CC chemokine receptor 4 (CCR4) by DCs is required for EAE induction. CCR4−/− mice presented enhanced resistance to EAE associated with a reduction in IL-23 and GM-CSF expression in the CNS. Restoring CCR4 on myeloid cells in bone marrow chimeras or intracerebral microinjection of CCR4-competent DCs, but not macrophages, restored EAE in CCR4−/− mice, indicating that CCR4+ DCs are cellular mediators of EAE development. Mechanistically, CCR4−/− DCs were less efficient in GM-CSF and IL-23 production and also TH-17 maintenance. Intraspinal IL-23 reconstitution restored EAE in CCR4−/− mice, whereas intracerebral inoculation using IL-23−/− DCs or GM-CSF−/− DCs failed to induce disease. Thus, CCR4-dependent GM-CSF production in DCs required for IL-23 release in these cells is a major component in the development of EAE. Our study identified a unique role for CCR4 in regulating DC function in EAE, harboring therapeutic potential for the treatment of CNS autoimmunity by targeting CCR4 on this specific cell type. PMID:22355103

Specific roles for the Ccr4-Not complex subunits in expression of the genome

PubMed Central

Azzouz, Nowel; Panasenko, Olesya O.; Deluen, Cécile; Hsieh, Julien; Theiler, Grégory; Collart, Martine A.

2009-01-01

In this work we used micro-array experiments to determine the role of each nonessential subunit of the conserved Ccr4-Not complex in the control of gene expression in the yeast Saccharomyces cerevisiae. The study was performed with cells growing exponentially in high glucose and with cells grown to glucose depletion. Specific patterns of gene deregulation were observed upon deletion of any given subunit, revealing the specificity of each subunit's function. Consistently, the purification of the Ccr4-Not complex through Caf40p by tandem affinity purification from wild-type cells or cells lacking individual subunits of the Ccr4-Not complex revealed that each subunit had a particular impact on complex integrity. Furthermore, the micro-arrays revealed that the role of each subunit was specific to the growth conditions. From the study of only two different growth conditions, revealing an impact of the Ccr4-Not complex on more than 85% of all studied genes, we can infer that the Ccr4-Not complex is important for expression of most of the yeast genome. PMID:19155328

Methamphetamine abuse affects gene expression in brain-derived microglia of SIV-infected macaques to enhance inflammation and promote virus targets.

PubMed

Najera, Julia A; Bustamante, Eduardo A; Bortell, Nikki; Morsey, Brenda; Fox, Howard S; Ravasi, Timothy; Marcondes, Maria Cecilia Garibaldi

2016-04-23

Methamphetamine (Meth) abuse is a major health problem linked to the aggravation of HIV- associated complications, especially within the Central Nervous System (CNS). Within the CNS, Meth has the ability to modify the activity/function of innate immune cells and increase brain viral loads. Here, we examined changes in the gene expression profile of neuron-free microglial cell preparations isolated from the brain of macaques infected with the Simian Immunodeficiency Virus (SIV), a model of neuroAIDS, and exposed to Meth. We aimed to identify molecular patterns triggered by Meth that could explain the detection of higher brain viral loads and the development of a pro-inflammatory CNS environment in the brain of infected drug abusers. We found that Meth alone has a strong effect on the transcription of genes associated with immune pathways, particularly inflammation and chemotaxis. Systems analysis led to a strong correlation between Meth exposure and enhancement of molecules associated with chemokines and chemokine receptors, especially CXCR4 and CCR5, which function as co-receptors for viral entry. The increase in CCR5 expression was confirmed in the brain in correlation with increased brain viral load. Meth enhances the availability of CCR5-expressing cells for SIV in the brain, in correlation with increased viral load. This suggests that Meth is an important factor in the susceptibility to the infection and to the aggravated CNS inflammatory pathology associated with SIV in macaques and HIV in humans.

Modulation of gene expression and cell cycle by botryosphaeran, a (1→3)(1→6)-β-d-glucan in human lymphocytes.

PubMed

Malini, Maressa; Souza, Marilesia Ferreira de; Oliveira, Marcelo Tempesta de; Antunes, Lusânia Maria Greggi; Figueiredo, Suely Gomes de; Barbosa, Aneli M; Dekker, Robert F H; Cólus, Ilce Mara de Syllos

2015-01-01

There is growing interest in the anticancer and immunomodulatory potential of fungal β-d-glucans. In the present study, the modulation of gene expression via RT-qPCR and cell cycle kinetics via flow cytometry were assessed in human normal and tumor (Jurkat) lymphocytes after treatment with botryosphaeran (a fungal (1→3)(1→6)-β-d-glucan) from Botryosphaeria rhodina MAMB-05. Cell cultures were treated with botryosphaeran either alone, or in combination with doxorubicin (DXR), in a post-treatment protocol. The expression of genes involved in immunomodulatory processes, apoptosis and cell cycle control, as well as β-d-glucans cell receptors were assessed. Flow cytometry analysis identified tetraploid Jurkat cells in G1 phase when treated with botryosphaeran combined with DXR. This antiproliferative effect in G1 may be associated with down-regulation of the expression of genes involved in the G1 checkpoint. The repression of the CCR5 gene following botryosphaeran treatment, either alone or in combination with DXR, in tumor lymphocytes indicates a possible affinity of this particular (1→3)(1→6)-β-d-glucan for the receptor CCR5. Therefore, botryosphaeran action appears to be involved in the repression of genes related to the G1 phase of the cell cycle and possibly in the interaction of the botryosphaeran, either alone, or in combination with DXR, with the CCR5 receptor. Copyright © 2015 Elsevier B.V. All rights reserved.

CCR6(+) Th cell populations distinguish ACPA positive from ACPA negative rheumatoid arthritis.

PubMed

Paulissen, Sandra M J; van Hamburg, Jan Piet; Davelaar, Nadine; Vroman, Heleen; Hazes, Johanna M W; de Jong, Pascal H P; Lubberts, Erik

2015-11-30

Patients with rheumatoid arthritis (RA) can be separated into two major subpopulations based on the absence or presence of serum anti-citrullinated protein antibodies (ACPAs). The more severe disease course in ACPA(+) RA and differences in treatment outcome between these subpopulations suggest that ACPA(+) and ACPA(-) RA are different disease subsets. The identification of T-helper (Th) cells specifically recognizing citrullinated peptides, combined with the strong association between HLA-DRB1 and ACPA positivity, point toward a pathogenic role of Th cells in ACPA(+) RA. In this context we recently identified a potential pathogenic role for CCR6(+) Th cells in RA. Therefore, we examined whether Th cell population distributions differ by ACPA status. We performed a nested matched case-control study including 27 ACPA(+) and 27 ACPA(-) treatment-naive early RA patients matched for disease activity score in 44 joints, presence of rheumatoid factor, sex, age, duration of complaints and presence of erosions. CD4(+)CD45RO(+) (memory) Th cell distribution profiles from these patients were generated based on differential chemokine receptor expression and related with disease duration. ACPA status was not related to differences in total CD4(+) T cell or memory Th cell proportions. However, ACPA(+) patients had significantly higher proportions of Th cells expressing the chemokine receptors CCR6 and CXCR3. Similar proportions of CCR4(+) and CCR10(+) Th cells were found. Within the CCR6(+) cell population, four Th subpopulations were distinguished based on differential chemokine receptor expression: Th17 (CCR4(+)CCR10(-)), Th17.1 (CXCR3(+)), Th22 (CCR4(+)CCR10(+)) and CCR4/CXCR3 double-positive (DP) cells. In particular, higher proportions of Th22 (p = 0.02), Th17.1 (p = 0.03) and CCR4/CXCR3 DP (p = 0.01) cells were present in ACPA(+) patients. In contrast, ACPA status was not associated with differences in Th1 (CCR6(-)CXCR3(+); p = 0.90), Th2 (CCR6(-)CCR4(+); p = 0.27) and T-regulatory (CD25(hi)FOXP3(+); p = 0.06) cell proportions. Interestingly, CCR6(+) Th cells were inversely correlated with disease duration in ACPA(-) patients (R(2) = -0.35; p < 0.01) but not in ACPA(+) (R(2) < 0.01; p = 0.94) patients. These findings demonstrate that increased peripheral blood CCR6(+) Th cells proportions distinguish ACPA(+) RA from ACPA(-) RA. This suggests that CCR6(+) Th cells are involved in the differences in disease severity and treatment outcome between ACPA(+) and ACPA(-) RA.

Engineering of Ralstonia eutropha for production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from fructose and solid-state properties of the copolymer.

PubMed

Fukui, Toshiaki; Abe, Hideki; Doi, Yoshiharu

2002-01-01

Recombinant Ralstonia eutropha capable of producing poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) copolymer [P(3HB-co-3HHx)] from fructose was engineered by introduction of genes for crotonyl-CoA reductase (CCR) from Streptomyces cinnamonensis (ccrSc) and for PHA synthase and (R)-specific enoyl-CoA hydratase from Aeromonas caviae (phaC-JAc). In this recombinant strain, C6-acyl-CoA intermediates were provided via beta-ketothiolase-mediated elongation of butyryl-CoA, which was generated from crotonyl-CoA by the function of CCR. The recombinant strain could accumulate the copolyester up to 48 wt % of dry cell weight with 1.5 mol % of 3HHx fraction from fructose, when the expression of ccrSc under the control of the PBAD promoter was induced with 0.01% L-arabinose. The absence of L-arabinose or the deletion of ccrSc from the plasmid resulted in accumulation of poly(3-hydroxybutyrate) homopolymer, indicating the critical role of CCR in the formation of the 3-hydroxyhexanoate unit. Higher CCR activity obtained by the addition of a larger amount of L-arabinose did not affect the composition but reduced the intracellular content of the copolyester. The P(3HB-co-1.5 mol % 3HHx) copolyester produced from fructose by the recombinant R. eutropha showed relatively lower melting temperatures (150 degrees C and 161 degrees C) and lower crystallinity (48 +/- 5%) compared to those (175 degrees C and 60 +/- 5%) of P(3HB) homopolymer. It has been found that the incorporation of a small amount (1.5 mol %) of 3HHx units into P(3HB) sequences leads to a remarkable change in the solid-state properties of P(3HB) crystals. The present study demonstrates the potential of the engineered pathway for the production of copolyesters having favorable characteristics from inexpensive carbon resources.

Agonist-induced Endocytosis of CC Chemokine Receptor 5 Is Clathrin Dependent

PubMed Central

Signoret, Nathalie; Hewlett, Lindsay; Wavre, Silène; Pelchen-Matthews, Annegret; Oppermann, Martin; Marsh, Mark

2005-01-01

The signaling activity of several chemokine receptors, including CC chemokine receptor 5 (CCR5), is in part controlled by their internalization, recycling, and/or degradation. For CCR5, agonists such as the chemokine CCL5 induce internalization into early endosomes containing the transferrin receptor, a marker for clathrin-dependent endocytosis, but it has been suggested that CCR5 may also follow clathrin-independent routes of internalization. Here, we present a detailed analysis of the role of clathrin in chemokine-induced CCR5 internalization. Using CCR5-transfected cell lines, immunofluorescence, and electron microscopy, we demonstrate that CCL5 causes the rapid redistribution of scattered cell surface CCR5 into large clusters that are associated with flat clathrin lattices. Invaginated clathrin-coated pits could be seen at the edge of these lattices and, in CCL5-treated cells, these pits contain CCR5. Receptors internalized via clathrin-coated vesicles follow the clathrin-mediated endocytic pathway, and depletion of clathrin with small interfering RNAs inhibits CCL5-induced CCR5 internalization. We found no evidence for CCR5 association with caveolae during agonist-induced internalization. However, sequestration of cholesterol with filipin interferes with agonist binding to CCR5, suggesting that cholesterol and/or lipid raft domains play some role in the events required for CCR5 activation before internalization. PMID:15591129

Defining characteristics of classical Hodgkin lymphoma microenvironment T-helper cells

PubMed Central

Clear, Andrew; Owen, Andrew; Iqbal, Sameena; Lee, Abigail; Matthews, Janet; Wilson, Andrew; Calaminici, Maria; Gribben, John G.

2013-01-01

CD4+ T-helper cells (THs) dominate the classical Hodgkin lymphoma (CHL) microenvironment, but their role is poorly understood. Advances in flow cytometry and immunohistochemistry permit more detailed investigation of this aspect of CHL pathophysiology. To address the hypothesis that the TH-infiltrate, rather than being TH2-enriched, senescent and hypofunctional, is TH1 and activation marker-rich, cytokine-secretory and proliferative, we applied comprehensive flow cytometric immunophenotyping and functional assays of cytokine secretion/proliferation to TH cells from 18 CHL-derived single-cell suspensions (SCSs) compared to reactive lymph nodes (RLNs). CHL-derived TH cells express TH1-associated CXCR3/CCR5 and TNFα/IFNγ/interleukin-2 (IL-2) and less TH2-associated CCR3/CCR4, with no IL-4/IL-13. They lack exhaustion-/suppression-associated PD1, CD57 and terminally differentiated effector memory cells, with more central memory cells, activation-associated partners of Hodgkin Reed Sternberg (HRS) cell-expressed CD30/OX40-L/ICOS-L, and other activation markers. TH cell lines established from CHL and RLN-derived SCSs remain cytokine-secretory. We confirmed and extended these studies using tissue microarray immunohistochemistry (TMA-IHC) from a large CHL tissue bank (n = 122) and demonstrate TH1-associated TBET is abundant in CHL, and TH2-associated CMAF/GATA3 and exhaustion-associated PD1 expressed at significantly lower levels. These molecular insights into the CHL-associated TH offer potential diagnostic, prognostic and pharmacologically modifiable therapeutic targets and do not support the established view of a TH2-enriched, senescent/exhausted, hypofunctional, hypoproliferative infiltrate. PMID:24004665

Defining characteristics of classical Hodgkin lymphoma microenvironment T-helper cells.

PubMed

Greaves, Paul; Clear, Andrew; Owen, Andrew; Iqbal, Sameena; Lee, Abigail; Matthews, Janet; Wilson, Andrew; Calaminici, Maria; Gribben, John G

2013-10-17

CD4(+) T-helper cells (THs) dominate the classical Hodgkin lymphoma (CHL) microenvironment, but their role is poorly understood. Advances in flow cytometry and immunohistochemistry permit more detailed investigation of this aspect of CHL pathophysiology. To address the hypothesis that the TH-infiltrate, rather than being TH2-enriched, senescent and hypofunctional, is TH1 and activation marker-rich, cytokine-secretory and proliferative, we applied comprehensive flow cytometric immunophenotyping and functional assays of cytokine secretion/proliferation to TH cells from 18 CHL-derived single-cell suspensions (SCSs) compared to reactive lymph nodes (RLNs). CHL-derived TH cells express TH1-associated CXCR3/CCR5 and TNFα/IFNγ/interleukin-2 (IL-2) and less TH2-associated CCR3/CCR4, with no IL-4/IL-13. They lack exhaustion-/suppression-associated PD1, CD57 and terminally differentiated effector memory cells, with more central memory cells, activation-associated partners of Hodgkin Reed Sternberg (HRS) cell-expressed CD30/OX40-L/ICOS-L, and other activation markers. TH cell lines established from CHL and RLN-derived SCSs remain cytokine-secretory. We confirmed and extended these studies using tissue microarray immunohistochemistry (TMA-IHC) from a large CHL tissue bank (n = 122) and demonstrate TH1-associated TBET is abundant in CHL, and TH2-associated CMAF/GATA3 and exhaustion-associated PD1 expressed at significantly lower levels. These molecular insights into the CHL-associated TH offer potential diagnostic, prognostic and pharmacologically modifiable therapeutic targets and do not support the established view of a TH2-enriched, senescent/exhausted, hypofunctional, hypoproliferative infiltrate.

Intestinal CCL25 expression is increased in colitis and correlates with inflammatory activity

PubMed Central

Trivedi, Palak J.; Bruns, Tony; Ward, Stephen; Mai, Martina; Schmidt, Carsten; Hirschfield, Gideon M.; Weston, Chris J.; Adams, David H.

2016-01-01

CCL25-mediated activation of CCR9 is critical for mucosal lymphocyte recruitment to the intestine. In immune-mediated liver injury complicating inflammatory bowel disease, intrahepatic activation of this pathway allows mucosal lymphocytes to be recruited to the liver, driving hepatobiliary destruction in primary sclerosing cholangitis (PSC). However, in mice and healthy humans CCL25 expression is restricted to the small bowel, whereas few data exist on activation of this pathway in the inflamed colon despite the vast majority of PSC patients having ulcerative colitis. Herein, we show that colonic CCL25 expression is not only upregulated in patients with active colitis, but strongly correlates with endoscopic Mayo score and mucosal TNFα expression. Moreover, approximately 90% (CD4+) and 30% (CD8+) of tissue-infiltrating T-cells in colitis were identified as CCR9+ effector lymphocytes, compared to <10% of T-cells being CCR9+ in normal colon. Sorted CCR9+ lymphocytes also demonstrated enhanced cellular adhesion to stimulated hepatic sinusoidal endothelium compared with their CCR9– counterparts when under flow. Collectively, these results suggest that CCR9/CCL25 interactions are not only involved in colitis pathogenesis but also correlate with colonic inflammatory burden; further supporting the existence of overlapping mucosal lymphocyte recruitment pathways between the inflamed colon and liver. PMID:26873648

Arteriolar and Venular Remodeling Are Differentially Regulated by Bone Marrow-Derived Cell-Specific CX3CR1 and CCR2 Expression

PubMed Central

Meisner, Joshua K.; Song, Ji; Price, Richard J.

2012-01-01

The chemokine receptors CCR2 and CX3CR1 are critical for the recruitment of “inflammatory” and “resident” monocytes, respectively, subpopulations that differentially affect vascular remodeling in atherosclerosis. Here, we tested the hypothesis that bone marrow-derived cell (BMC)-specific CCR2 and CX3CR1 differentially control venular and arteriolar remodeling. Venular and arteriolar lumenal remodeling were observed by intravital microscopy in mice with either CCR2 or CX3CR1 deficient BMCs after implantation of a dorsal skinfold window chamber, a model in which arterioles and venules lumenally enlarge in wild-type (WT) mice. Arteriolar remodeling was abolished in mice with either CCR2 or CX3CR1-deficient BMCs. In contrast, the loss of CX3CR1 from BMCs, but not CCR2, significantly reduced small venule remodeling compared to WT controls. We conclude that microvascular remodeling is differentially regulated by BMC-expressed chemokine receptors. Both CCR2 and CX3CR1 regulate arteriole growth; however, only BMC-expressed CX3CR1 impacts small venule growth. These findings may provide a basis for additional investigations aimed at determining how patterns of monocyte subpopulation recruitment spatially influence microvascular remodeling. PMID:23029475

Expression and prognostic significance of CCL11/CCR3 in glioblastoma.

PubMed

Tian, Min; Chen, Lina; Ma, Li; Wang, Dandan; Shao, Bin; Wu, Jianyu; Wu, Hangyu; Jin, Yimin

2016-05-31

Glioblastoma (GBM) is the most lethal primary nervous system cancer, but due to its rarity and complexity, its pathogenesis is poorly understood. To identify potential tumorigenic factors in GBM, we screened antibody-based cytokine arrays and found that CCL11 was upregulated. We then demonstrated in vitro that both CCL11 and its receptor, CCR3, were overexpressed and promoted the proliferation, migration and invasion of cancer cells. To examine the clinical significance of CCL11/CCR3, 458 GBM samples were divided into a training cohort with 225 cases and a test cohort containing 233 cases. In the training set, immunohistochemical analysis showed overexpression of CCL11 and CCR3 were correlated with unfavorable overall survival (OS). We further developed a prognostic classifier combining CCL11 and CCR3 expression and Karnofsky performance status (KPS) for predicting one-year survival in GBM patients. Receiver operating characteristic (ROC) analysis demonstrated that this predictor achieved 90.7% sensitivity and 73.4% specificity. These results were validated with the test sample set. Our findings suggest that CCL11-CCR3 binding is involved in the progression of GBM and may prompt a novel therapeutic approach. In addition, CCL11 and CCR3 expression, combined with KPS, may be used as an accurate predictor of one-year survival in GBM patients.

Expression and prognostic significance of CCL11/CCR3 in glioblastoma

PubMed Central

Tian, Min; Chen, Lina; Ma, Li; Wang, Dandan; Shao, Bin; Wu, Jianyu; Wu, Hangyu; Jin, Yimin

2016-01-01

Glioblastoma (GBM) is the most lethal primary nervous system cancer, but due to its rarity and complexity, its pathogenesis is poorly understood. To identify potential tumorigenic factors in GBM, we screened antibody-based cytokine arrays and found that CCL11 was upregulated. We then demonstrated in vitro that both CCL11 and its receptor, CCR3, were overexpressed and promoted the proliferation, migration and invasion of cancer cells. To examine the clinical significance of CCL11/CCR3, 458 GBM samples were divided into a training cohort with 225 cases and a test cohort containing 233 cases. In the training set, immunohistochemical analysis showed overexpression of CCL11 and CCR3 were correlated with unfavorable overall survival (OS). We further developed a prognostic classifier combining CCL11 and CCR3 expression and Karnofsky performance status (KPS) for predicting one-year survival in GBM patients. Receiver operating characteristic (ROC) analysis demonstrated that this predictor achieved 90.7% sensitivity and 73.4% specificity. These results were validated with the test sample set. Our findings suggest that CCL11-CCR3 binding is involved in the progression of GBM and may prompt a novel therapeutic approach. In addition, CCL11 and CCR3 expression, combined with KPS, may be used as an accurate predictor of one-year survival in GBM patients. PMID:27119233

Role of effector cells (CCR7(-)CD27(-)) and effector-memory cells (CCR7(-)CD27(+)) in drug-induced maculopapular exanthema.

PubMed

Fernandez, T D; Torres, M J; Lopez, S; Antunez, C; Gomez, E; Del Prado, M F; Canto, G; Blanca, M; Mayorga, C

2010-01-01

Maculopapular exanthema (MPE) induced by drugs is a T-cell mediated reaction and effector cells may play an important role in its development. We assessed the effector and cutaneous homing phenotype in peripheral blood cells from allergic patients after drug stimulation. This study included 10 patients and 10 controls. The effector phenotype (CCR7(-)CD27(+/-)), chemokine receptors (CCR4 and CCR10), and activation (CD25(low)) and regulatory markers (CD25(high)) were measured by flow cytometry in both peripheral blood mononuclear cells (PBMCs) and CD4-T-lymphocytes. Proliferation was determined by 5-(-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) assay and the migratory capacity by a chemotaxis assay using CCL17 and CCL27. Compared to controls, CCR7(-)CD27(-) cells were increased in patients without (p=0.003) and with drug stimulation (p less than 0.001) and had significantly higher proliferation (p=0.010). CCR10 expression was increased in patients after drug stimulation in total and memory CD27(+) T-cells. Lymphocyte migration with CCL27 was higher in patients with drug stimulation (p=0.048), with a decrease in CCR7(-)CD27(-) (p less than 0.0001) and an increase in CCR7(-)CD27(+) (p=0.017). In patients, CD4-T-lymphocytes were significantly activated after drug stimulation (p less than 0.001). In conclusion, we show that effector memory CD4(+) T-cells (CCR7(-)CD27(+)) respond specifically to the drug responsible for MPE and confirm previous data about the involvement of CCR10 in cell trafficking to the skin.

Influence of CCR5 and CCR2 Genetic Variants in the Resistance/Susceptibility to HIV in Serodiscordant Couples from Colombia

PubMed Central

Zapata, Wildeman; Aguilar-Jiménez, Wbeimar; Pineda-Trujillo, Nicolás; Rojas, Winston; Estrada, Hernando

2013-01-01

Abstract The main genetic factor related to HIV-1 resistance is the CCR5-Δ32 mutation; however, the homozygous genotype is uncommon. The CCR5-Δ32 mutation along with single nucleotide polymorphisms (SNPs) in the CCR5 promoter and the CCR2-V64I mutation have been included in seven human haplogroups (HH) previously associated with resistance/susceptibility to HIV-1 infection and different rates of AIDS progression. Here, we determined the association of the CCR5 promoter SNPs, the CCR5-Δ32 mutation, CCR2-V64I SNP, and HH frequencies with resistance/susceptibility to HIV-1 infection in a cohort of HIV-1-serodiscordant couples from Colombia. Seventy HIV-1-exposed, but seronegative (HESN) individuals, 57 seropositives (SP), and 112 healthy controls (HC) were included. The CCR5-Δ32 mutation and CCR2-V64I SNP were identified by PCR, and the CCR5 promoter SNPs were evaluated by sequencing. None of the individuals exhibited a homozygous Δ32 genotype; the CCR2-I allele was more frequent in HESN (34%) than HC (23%) (p=0.039, OR=1.672). The frequency of the 29G allele was higher in SP than HC (p=0.003, OR=3). HHF2 showed a higher frequency in HC (19%) than SP (9%) (p=0.027), while HHG1 was more frequent in SP (11.1%) than in HC (4.2%) (p=0.019). The AGACCAC-CCR2-I-CCR5 wild-type haplotype showed a higher frequency in SP (14.2%) than in HC (3.7%) (p=0.001). In conclusion, the CCR5-Δ32 allele is not responsible for HIV-1 resistance in this HESN group; however, the CCR2-I allele could be protective, while the 29G allele might increase the likelihood of acquiring HIV-1 infection. HHG1 and the AGACCAC-CCR2-I-CCR5 wild-type haplotype might promote HIV-1 infection while HHF2 might be related to resistance. However, additional studies are required to evaluate the implications of these findings. PMID:24098976

Using a genetic, observational study as a strategy to estimate the potential cost-effectiveness of pharmacological CCR5 blockade in dialysis patients.

PubMed

Muntinghe, Friso L H; Vegter, Stefan; Verduijn, Marion; Boeschoten, Elisabeth W; Dekker, Friedo W; Navis, Gerjan; Postma, Maarten

2011-07-01

Randomized clinical trials are expensive and time consuming. Therefore, strategies are needed to prioritise tracks for drug development. Genetic association studies may provide such a strategy by considering the differences between genotypes as a proxy for a natural, lifelong, randomized at conception, clinical trial. Previously an association with better survival was found in dialysis patients with systemic inflammation carrying a deletion variant of the CC-chemokine receptor 5 (CCR5). We hypothesized that in an analogous manner, pharmacological CCR5 blockade could protect against inflammation-driven mortality and estimated if such a treatment would be cost-effective. A genetic screen and treat strategy was modelled using a decision-analytic Markov model, in which patients were screened for the CCR5 deletion 32 polymorphism and those with the wild type and systemic inflammation were treated with pharmacological CCR5 blockers. Kidney transplantation and mortality rates were calculated using patient level data. Extensive sensitivity analyses were performed. The cost-effectiveness of the genetic screen and treat strategy was &OV0556;18 557 per life year gained and &OV0556;21 896 per quality-adjusted life years gained. Concordance between the genetic association and pharmacological effectiveness was a main driver of cost-effectiveness. Sensitivity analyses showed that even a modest effectiveness of pharmacological CCR5 blockade would result in a treatment strategy that is good value for money. Pharmacological blockade of the CCR5 receptor in inflamed dialysis patients can be incorporated in a potentially cost-effective screen and treat programme. These findings provide formal rationale for clinical studies. This study illustrates the potential of genetic association studies for drug development, as a source of Mendelian randomized evidence from an observational setting.

Ginger Extract Modulates the Expression of Chemokines CCL20 and CCL22 and Their Receptors (CCR6 and CCR4) in the Central Nervous System of Mice with Experimental Autoimmune Encephalomyelitis.

PubMed

Jafarzadeh, Abdollah; Arabi, Zahra; Ahangar-Parvin, Rayhaneh; Mohammadi-Kordkhayli, Marziyeh; Nemati, Maryam

2017-11-01

Background Chemokines facilitate the leukocytes infiltration into the central nervous system (CNS) which is an essential step in the pathogenesis of multiple sclerosis. Ginger has also a broad anti-inflammatory properties. The aim was to evaluate the effects of ginger extract on the expression of CCL20 and CCL22 and their receptors (CCR6 and CCR4, respectively) in experimental autoimmune encephalomyelitis (EAE). Material and Methods Female C57BL/6 mice used for EAE induction by immunization with myelin oligodendroglial glycoprotein. Then, the EAE mice were treated with PBS or ginger extract, from day +3 to +30. At day 31, mice were scarified and the expression of CCL20 and CCL22 and their receptors in the spinal cord measured using real time-PCR. Results The expression of CCL20, CCL22 and CCR4 in the spinal cord of PBS-administrated EAE mice was significantly higher than healthy group (P<0.04, P<0.05 and P<0.02, respectively). In 200- and 300 mg/kg ginger extract-treated EAE mice, the expression of CCL20, CCL22 and CCR4 were significantly reduced as compared with PBS-administrated EAE group (P<0.04, P<0.01 and P<0.002 for 200 mg/kg ginger extract and P<0.01, P<0.005 and P<0.004 for 300 mg/kg ginger extract, respectively). The CCR6 expression in EAE mice treated with 200- or 300 mg/kg ginger extracts was lower than PBS-administrated EAE mice (P<0.01 and P=0.07, respectively). Conclusion Treatment of EAE mice with ginger extract down-regulate the expression of CCL20 and CCL22 and their receptors in EAE mice. The possible therapeutic potential of ginger for treatment of MS can be considered in future investigations. © Georg Thieme Verlag KG Stuttgart · New York.

CCR5 is a suppressor for cortical plasticity and hippocampal learning and memory

PubMed Central

Zhou, Miou; Greenhill, Stuart; Huang, Shan; Silva, Tawnie K; Sano, Yoshitake; Wu, Shumin; Cai, Ying; Nagaoka, Yoshiko; Sehgal, Megha; Cai, Denise J; Lee, Yong-Seok; Fox, Kevin; Silva, Alcino J

2016-01-01

Although the role of CCR5 in immunity and in HIV infection has been studied widely, its role in neuronal plasticity, learning and memory is not understood. Here, we report that decreasing the function of CCR5 increases MAPK/CREB signaling, long-term potentiation (LTP), and hippocampus-dependent memory in mice, while neuronal CCR5 overexpression caused memory deficits. Decreasing CCR5 function in mouse barrel cortex also resulted in enhanced spike timing dependent plasticity and consequently, dramatically accelerated experience-dependent plasticity. These results suggest that CCR5 is a powerful suppressor for plasticity and memory, and CCR5 over-activation by viral proteins may contribute to HIV-associated cognitive deficits. Consistent with this hypothesis, the HIV V3 peptide caused LTP, signaling and memory deficits that were prevented by Ccr5 knockout or knockdown. Overall, our results demonstrate that CCR5 plays an important role in neuroplasticity, learning and memory, and indicate that CCR5 has a role in the cognitive deficits caused by HIV. DOI: http://dx.doi.org/10.7554/eLife.20985.001 PMID:27996938

Aberrant in Vivo T Helper Type 2 Cell Response and Impaired Eosinophil Recruitment in Cc Chemokine Receptor 8 Knockout Mice

PubMed Central

Chensue, Stephen W.; Lukacs, Nicholas W.; Yang, Tong-Yuan; Shang, Xiaozhou; Frait, Kirsten A.; Kunkel, Steven L.; Kung, Ted; Wiekowski, Maria T.; Hedrick, Joseph A.; Cook, Donald N.; Zingoni, Alessandra; Narula, Satwant K.; Zlotnik, Albert; Barrat, Franck J.; O'Garra, Anne; Napolitano, Monica; Lira, Sergio A.

2001-01-01

Chemokine receptors transduce signals important for the function and trafficking of leukocytes. Recently, it has been shown that CC chemokine receptor (CCR)8 is selectively expressed by Th2 subsets, but its functional relevance is unclear. To address the biological role of CCR8, we generated CCR8 deficient (−/−) mice. Here we report defective T helper type 2 (Th2) immune responses in vivo in CCR8−/− mice in models of Schistosoma mansoni soluble egg antigen (SEA)-induced granuloma formation as well as ovalbumin (OVA)- and cockroach antigen (CRA)-induced allergic airway inflammation. In these mice, the response to SEA, OVA, and CRA showed impaired Th2 cytokine production that was associated with aberrant type 2 inflammation displaying a 50 to 80% reduction in eosinophils. In contrast, a prototypical Th1 immune response, elicited by Mycobacteria bovis purified protein derivative (PPD) was unaffected by CCR8 deficiency. Mechanistic analyses indicated that Th2 cells developed normally and that the reduction in eosinophil recruitment was likely due to systemic reduction in interleukin 5. These results indicate an important role for CCR8 in Th2 functional responses in vivo. PMID:11238588

Maraviroc (UK-427,857), a Potent, Orally Bioavailable, and Selective Small-Molecule Inhibitor of Chemokine Receptor CCR5 with Broad-Spectrum Anti-Human Immunodeficiency Virus Type 1 Activity

PubMed Central

Dorr, Patrick; Westby, Mike; Dobbs, Susan; Griffin, Paul; Irvine, Becky; Macartney, Malcolm; Mori, Julie; Rickett, Graham; Smith-Burchnell, Caroline; Napier, Carolyn; Webster, Rob; Armour, Duncan; Price, David; Stammen, Blanda; Wood, Anthony; Perros, Manos

2005-01-01

Maraviroc (UK-427,857) is a selective CCR5 antagonist with potent anti-human immunodeficiency virus type 1 (HIV-1) activity and favorable pharmacological properties. Maraviroc is the product of a medicinal chemistry effort initiated following identification of an imidazopyridine CCR5 ligand from a high-throughput screen of the Pfizer compound file. Maraviroc demonstrated potent antiviral activity against all CCR5-tropic HIV-1 viruses tested, including 43 primary isolates from various clades and diverse geographic origin (geometric mean 90% inhibitory concentration of 2.0 nM). Maraviroc was active against 200 clinically derived HIV-1 envelope-recombinant pseudoviruses, 100 of which were derived from viruses resistant to existing drug classes. There was little difference in the sensitivity of the 200 viruses to maraviroc, as illustrated by the biological cutoff in this assay (= geometric mean plus two standard deviations [SD] of 1.7-fold). The mechanism of action of maraviroc was established using cell-based assays, where it blocked binding of viral envelope, gp120, to CCR5 to prevent the membrane fusion events necessary for viral entry. Maraviroc did not affect CCR5 cell surface levels or associated intracellular signaling, confirming it as a functional antagonist of CCR5. Maraviroc has no detectable in vitro cytotoxicity and is highly selective for CCR5, as confirmed against a wide range of receptors and enzymes, including the hERG ion channel (50% inhibitory concentration, >10 μM), indicating potential for an excellent clinical safety profile. Studies in preclinical in vitro and in vivo models predicted maraviroc to have human pharmacokinetics consistent with once- or twice-daily dosing following oral administration. Clinical trials are ongoing to further investigate the potential of using maraviroc for the treatment of HIV-1 infection and AIDS. PMID:16251317

The mTOR-inhibitor rapamycin mediates proteinuria in nephrotoxic serum nephritis by activating the innate immune response.

PubMed

Kirsch, A H; Riegelbauer, V; Tagwerker, A; Rudnicki, M; Rosenkranz, A R; Eller, K

2012-08-15

Rapamycin (Rapa) is an immunosuppressant used to prevent rejection in recipients of renal transplants. Its clinical use is limited by de novo onset or exacerbation of preexisting proteinuria. In the present study, Rapa administration was started 14 days after induction of murine nephrotoxic serum nephritis (NTS) to study glomerular effects of this mammalian target of rapamycin (mTOR) inhibitor. Glomeruli were laser-microdissected, and real-time PCR was performed to assess effects on glomerular cells and the expression of inflammatory cytokines. Immunohistochemical stainings were performed to confirm mRNA data on the protein level. Compared with nephritic control animals, Rapa-treated mice developed significantly increased albuminuria. This was accompanied by a more prominent glomerular infiltration by CD4(+) T cells and macrophages. Glomerular mRNA expression profiling revealed increased levels of the proinflammatory cytokines interleukin-6 and tumor necrosis factor-α, and the chemokines monocyte chemoattractant protein-1 and macrophage inflammatory protein-1β and their cognate macrophage-associated receptors CCR2 and CCR5 in the Rapa-treated animals. Furthermore, there were elevated glomerular transcription levels of the regulatory T cell phenotype transcription factor Foxp3. No differences in the glomerular expression of the podocyte marker nephrin or the endothelial cell marker CD31 were observed on the mRNA or protein level. In conclusion, our data indicate that Rapa-induced proteinuria in NTS is a result of the activation of the innate immune system rather than a direct toxicity to podocytes or glomerular endothelial cells.

CCR2, CCR5, and CXCL12 variation and HIV/AIDS in Papua New Guinea

PubMed Central

Mehlotra, Rajeev K.; Zikursh, Melinda J. Blood; Stein, Catherine M.; Siba, Peter M.; Zimmerman, Peter A.

2015-01-01

Polymorphisms in chemokine receptors, serving as HIV co-receptors, and their ligands are among the well-known host genetic factors associated with susceptibility to HIV infection and/or disease progression. Papua New Guinea (PNG) has one of the highest adult HIV prevalences in the Asia-Pacific region. However, information regarding the distribution of polymorphisms in chemokine receptor (CCR5, CCR2) and chemokine (CXCL12) genes in PNG is very limited. In this study, we genotyped a total of nine CCR2-CCR5 polymorphisms, including CCR2 190G>A, CCR5 −2459G>A and Δ32, and CXCL12 801G>A in PNG (n = 258), North America (n = 184), and five countries in West Africa (n = 178). Using this data, we determined previously characterized CCR5 haplotypes. In addition, based on the previously reported associations of CCR2 190, CCR5 −2459, CCR5 open reading frame, and CXCL12 801 genotypes with HIV acquisition and/or disease progression, we calculated composite full risk scores, considering both protective as well as susceptibility effects of the CXCL12 801 AA genotype. We observed a very high frequency of the CCR5 −2459A allele (0.98) in the PNG population, which together with the absence of Δ32 resulted in a very high frequency of the HHE haplotype (0.92). These frequencies were significantly higher than in any other population (all P-values < 0.001). Regardless of whether we considered the CXCL12 801 AA genotype protective or susceptible, the risk scores were significantly higher in the PNG population compared with any other population (all P-values < 0.001). The results of this study provide new insights regarding CCR5 variation in the PNG population, and suggest that the collective variation in CCR2, CCR5, and CXCL12 may increase the risk of HIV/AIDS in a large majority of Papua New Guineans. PMID:26397046

The positive correlation of the CCL2-CCR2 axis with the disease activity may indicate the fundamental role in the pathogenesis of oral lichen planus.

PubMed

Yin, Jingfang; Yang, Xi; Zeng, Qi; Yang, Linglan; Cheng, Bin; Tao, Xiaoan

2016-01-01

The important roles of CCL2 and its receptor CCR2 had been reported in a series of inflammatory disorders. However, few studies investigated the potential role of CCL2/CCR2 axis in oral lichen planus (OLP). Therefore, this study aimed to detect the expression of CCL2 and CCR2 in OLP lesions and compare their changes before and after treatment. CCL2 and CCR2 expression was investigated using immunohistochemical staining and real-time RT-PCR in 32 patients with OLP and eight controls. Moreover, changes in their expression after treatment with triamcinolone acetonide were assessed in lesions from three patients. CCL2+ and CCR2+ cells were few in the controls and remarkably increased in the epithelial and subepithelial layers of lesions (n = 32, all P < 0.001). However, the densities of CCL2+ and CCR2+ cells were not significantly different between reticular (n = 12) and erythematous/erosive lesions (n = 20), although they significantly decreased after treatment (627.7 ± 108.2 vs. 258.3 ± 148.3, P = 0.017; 1034.7 ± 74.6 vs. 648 ± 77.6, P = 0.003, respectively). CCL2+/CCR2+ cell numbers were positively correlated with disease activity (correlation coefficient, 0.588; P < 0.001; correlation coefficient, 0.409; P = 0.02, respectively). The results of this study indicated that the CCL2-CCR2 axis was involved in the pathogenesis of OLP and was positively correlated with disease activity. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Structure of CC chemokine receptor 2 with orthosteric and allosteric antagonists

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zheng, Yi; Qin, Ling; Zacarías, Natalia V. Ortiz

CC chemokine receptor 2 (CCR2) is one of 19 members of the chemokine receptor subfamily of human class A G-protein-coupled receptors. CCR2 is expressed on monocytes, immature dendritic cells, and T-cell subpopulations, and mediates their migration towards endogenous CC chemokine ligands such as CCL2 (ref. 1). CCR2 and its ligands are implicated in numerous inflammatory and neurodegenerative diseases2 including atherosclerosis, multiple sclerosis, asthma, neuropathic pain, and diabetic nephropathy, as well as cancer3. These disease associations have motivated numerous preclinical studies and clinical trials4 (see http://www.clinicaltrials.gov) in search of therapies that target the CCR2–chemokine axis. To aid drug discovery efforts5, heremore » we solve a structure of CCR2 in a ternary complex with an orthosteric (BMS-681 (ref. 6)) and allosteric (CCR2-RA-[R]7) antagonist. BMS-681 inhibits chemokine binding by occupying the orthosteric pocket of the receptor in a previously unseen binding mode. CCR2-RA-[R] binds in a novel, highly druggable pocket that is the most intracellular allosteric site observed in class A G-protein-coupled receptors so far; this site spatially overlaps the G-protein-binding site in homologous receptors. CCR2-RA-[R] inhibits CCR2 non-competitively by blocking activation-associated conformational changes and formation of the G-protein-binding interface. The conformational signature of the conserved microswitch residues observed in double-antagonist-bound CCR2 resembles the most inactive G-protein-coupled receptor structures solved so far. Like other protein–protein interactions, receptor–chemokine complexes are considered challenging therapeutic targets for small molecules, and the present structure suggests diverse pocket epitopes that can be exploited to overcome obstacles in drug design.« less

Structure of CC Chemokine Receptor 2 with Orthosteric and Allosteric Antagonists

PubMed Central

Zheng, Yi; Qin, Ling; Ortiz Zacarías, Natalia V.; de Vries, Henk; Han, Gye Won; Gustavsson, Martin; Dabros, Marta; Zhao, Chunxia; Cherney, Robert J.; Carter, Percy; Stamos, Dean; Abagyan, Ruben; Cherezov, Vadim; Stevens, Raymond C.; IJzerman, Adriaan P.; Heitman, Laura H.; Tebben, Andrew; Kufareva, Irina; Handel, Tracy M.

2016-01-01

Summary CC chemokine receptor 2 (CCR2) is one of 19 members of the chemokine receptor subfamily of human Class A G protein-coupled receptors (GPCRs). CCR2 is expressed on monocytes, immature dendritic cells and T cell subpopulations, and mediates their migration towards endogenous CC chemokine ligands such as CCL21. CCR2 and its ligands are implicated in numerous inflammatory and neurodegenerative diseases2 including atherosclerosis, multiple sclerosis, asthma, neuropathic pain, and diabetic nephropathy, as well as cancer3. These disease associations have motivated numerous preclinical studies and clinical trials4 (see ClinicalTrials.gov) in search of therapies that target the CCR2:chemokine axis. To aid drug discovery efforts5, we solved a structure of CCR2 in a ternary complex with an orthosteric (BMS-6816) and allosteric (CCR2-RA-[R]7) antagonist. BMS-681 inhibits chemokine binding by occupying the orthosteric pocket of the receptor in a previously unseen binding mode. CCR2-RA-[R] binds in a novel, highly druggable pocket that is the most intracellular allosteric site observed in Class A GPCRs to date; this site spatially overlaps the G protein-binding site in homologous receptors. CCR2-RA-[R] inhibits CCR2 non-competitively by blocking activation-associated conformational changes and formation of the G protein-binding interface. The conformational signature of the conserved microswitch residues observed in double-antagonist-bound CCR2 resembles the most inactive GPCR structures solved to date. Like other protein:protein interactions, receptor:chemokine complexes are considered challenging therapeutic targets for small molecules, and the present structure suggests diverse pocket epitopes that can be exploited to overcome drug design obstacles. PMID:27926736

CCR5 Disruption in Induced Pluripotent Stem Cells Using CRISPR/Cas9 Provides Selective Resistance of Immune Cells to CCR5-tropic HIV-1 Virus.

PubMed

Kang, HyunJun; Minder, Petra; Park, Mi Ae; Mesquitta, Walatta-Tseyon; Torbett, Bruce E; Slukvin, Igor I

2015-12-15

The chemokine (C-C motif) receptor 5 (CCR5) serves as an HIV-1 co-receptor and is essential for cell infection with CCR5-tropic viruses. Loss of functional receptor protects against HIV infection. Here, we report the successful targeting of CCR5 in GFP-marked human induced pluripotent stem cells (iPSCs) using CRISPR/Cas9 with single and dual guide RNAs (gRNAs). Following CRISPER/Cas9-mediated gene editing using a single gRNA, 12.5% of cell colonies demonstrated CCR5 editing, of which 22.2% showed biallelic editing as determined by a Surveyor nuclease assay and direct sequencing. The use of dual gRNAs significantly increased the efficacy of CCR5 editing to 27% with a biallelic gene alteration frequency of 41%. To ensure the homogeneity of gene editing within cells, we used single cell sorting to establish clonal iPSC lines. Single cell-derived iPSC lines with homozygous CCR5 mutations displayed the typical characteristics of pluripotent stem cells and differentiated efficiently into hematopoietic cells, including macrophages. Although macrophages from both wild-type and CCR5-edited iPSCs supported CXCR4-tropic virus replication, macrophages from CCR5-edited iPSCs were uniquely resistant to CCR5-tropic virus challenge. This study demonstrates the feasibility of applying iPSC technology for the study of the role of CCR5 in HIV infection in vitro, and generation of HIV-resistant cells for potential therapeutic applications.

T helper 17.1 cells associate with multiple sclerosis disease activity: perspectives for early intervention.

PubMed

van Langelaar, Jamie; van der Vuurst de Vries, Roos M; Janssen, Malou; Wierenga-Wolf, Annet F; Spilt, Isis M; Siepman, Theodora A; Dankers, Wendy; Verjans, Georges M G M; de Vries, Helga E; Lubberts, Erik; Hintzen, Rogier Q; van Luijn, Marvin M

2018-05-01

Interleukin-17-expressing CD4+ T helper 17 (Th17) cells are considered as critical regulators of multiple sclerosis disease activity. However, depending on the species and pro-inflammatory milieu, Th17 cells are functionally heterogeneous, consisting of subpopulations that differentially produce interleukin-17, interferon-gamma and granulocyte macrophage colony-stimulating factor. In the current study, we studied distinct effector phenotypes of human Th17 cells and their correlation with disease activity in multiple sclerosis patients. T helper memory populations single- and double-positive for C-C chemokine receptor 6 (CCR6) and CXC chemokine receptor 3 (CXCR3) were functionally assessed in blood and/or cerebrospinal fluid from a total of 59 patients with clinically isolated syndrome, 35 untreated patients and 24 natalizumab-treated patients with relapsing-remitting multiple sclerosis, and nine patients with end-stage multiple sclerosis. Within the clinically isolated syndrome group, 23 patients had a second attack within 1 year and 26 patients did not experience subsequent attacks during a follow-up of >5 years. Low frequencies of T helper 1 (Th1)-like Th17 (CCR6+CXCR3+), and not Th17 (CCR6+CXCR3-) effector memory populations in blood strongly associated with a rapid diagnosis of clinically definite multiple sclerosis. In cerebrospinal fluid of clinically isolated syndrome and relapsing-remitting multiple sclerosis patients, Th1-like Th17 effector memory cells were abundant and showed increased production of interferon-gamma and granulocyte macrophage colony-stimulating factor compared to paired CCR6+ and CCR6-CD8+ T cell populations and their blood equivalents after short-term culturing. Their local enrichment was confirmed ex vivo using cerebrospinal fluid and brain single-cell suspensions. Across all pro-inflammatory T helper cells analysed in relapsing-remitting multiple sclerosis blood, Th1-like Th17 subpopulation T helper 17.1 (Th17.1; CCR6+CXCR3+CCR4-) expressed the highest very late antigen-4 levels and selectively accumulated in natalizumab-treated patients who remained free of clinical relapses. This was not found in patients who experienced relapses during natalizumab treatment. The enhanced potential of Th17.1 cells to infiltrate the central nervous system was supported by their predominance in cerebrospinal fluid of early multiple sclerosis patients and their preferential transmigration across human brain endothelial layers. These findings reveal a dominant contribution of Th1-like Th17 subpopulations, in particular Th17.1 cells, to clinical disease activity and provide a strong rationale for more specific and earlier use of T cell-targeted therapy in multiple sclerosis.

Molecular Gymnastics: Mechanisms of HIV-1 Resistance to CCR5 Antagonists and Impact on Virus Phenotypes.

PubMed

Roche, Michael; Borm, Katharina; Flynn, Jacqueline K; Lewin, Sharon R; Churchill, Melissa J; Gorry, Paul R

2016-01-01

Human immunodeficiency virus type 1 (HIV-1) enters host cells through the binding of its envelope glycoproteins (Env) to the host cell receptor CD4 and then subsequent binding to a chemokine coreceptor, either CCR5 or CXCR4. CCR5 antagonists are a relatively recent class addition to the armamentarium of anti-HIV-1 drugs. These compounds act by binding to a hydrophobic pocket formed by the transmembrane helices of CCR5 and altering the conformation of the extracellular domains, such that they are no longer recognized by Env. Maraviroc is the first drug within this class to be licenced for use in HIV-1 therapy regimens. HIV resistance to CCR5 antagonists occurs either through outgrowth of pre-existing CXCR4-using viruses, or through acquisition of the ability of CCR5-using HIV-1 to use the antagonist bound form of CCR5. In the latter scenario, the mechanism underlying resistance is through complex alterations in the way that resistant Envs engage CCR5. These significant changes are unlikely to occur without consequence to the viral entry phenotype and may also open up new avenues to target CCR5 antagonist resistant viruses. This review discusses the mechanism of action of CCR5 antagonists, how HIV resistance to CCR5 antagonists occurs, and the subsequent effects on Env function.

Analysis of the temporal expression of chemokines and chemokine receptors during experimental granulomatous inflammation: role and expression of MIP-1α and MCP-1

PubMed Central

Carollo, Maria; Hogaboam, Cory M; Kunkel, Stephen L; Delaney, Stephen; Christie, Mark I; Perretti, Mauro

2001-01-01

Chemokine expression and function was monitored in an experimental model of granulomatous tissue formation after injection of croton oil in complete Freund's adjuvant (CO/CFA) into mouse dorsal air-pouches up to 28 days. In the first week, mast cell degranulation and leukocyte influx (mononuclear cell, MNC, and polymorphonuclear cell, PMN) were associated with CXCR2, KC and macrophage inflammatory protein (MIP)-2 mRNA expression, as determined by TaqMan® reverse transcriptase-polymerase chain reaction. KC (∼400 pg mg protein−1, n=12) and MIP-2 (∼800 pg mg protein−1, n=12) proteins peaked at day 7, together with myeloperoxidase (MPO) activity. Highest MIP-1α (>1 ng mg protein−1, n=12) levels were measured at day 3. After day 7, a gradual increase in CCR2 and CCR5 mRNA, monocyte chemoattractant protein (MCP)-1 mRNA and protein expression was measured. MCP-1 protein peaked at day 21 (∼150 pg mg protein−1, n=12) and was predominantly expressed by mast cells. A gradual increase in N-acetyl-β-D-glucosaminidase (NAG) activity (maximal at 28 days) was also measured. An antiserum against MIP-1α did not modify the inflammatory response measured at day 7 (except for a 50% reduction in MIP-1α levels), but provoked a significant increase in MPO, NAG and MCP-1 levels as measured at day 21 (n=6, P<0.05). An antiserum to MCP-1 reduced NAG activity at day 21 but increased MPO activity values (n=8, P<0.05). In conclusion, we have shown that CO/CFA initiates a complex inflammatory reaction in which initial expression of MIP-1α serves a protective role whereas delayed expression of MCP-1 seems to have a genuine pro-inflammatory role. PMID:11704636

Induction of HIV-blocking anti-CCR5 IgA in Peyers's patches without histopathological alterations.

PubMed

Pastori, Claudia; Diomede, Lorenzo; Venuti, Assunta; Fisher, Gregory; Jarvik, Jonathan; Bomsel, Morgane; Sanvito, Francesca; Lopalco, Lucia

2014-04-01

The chemokine receptor CCR5 is essential for HIV infection and is thus a potential target for vaccine development. However, because CCR5 is a host protein, generation of anti-CCR5 antibodies requires the breaking of immune tolerance and thus carries the risk of autoimmune responses. In this study, performed in mice, we compared 3 different immunogens representing surface domains of murine CCR5, 4 different adjuvants, and 13 different immunization protocols, with the goal of eliciting HIV-blocking activity without inducing autoimmune dysfunction. In all cases the CCR5 sequences were presented as fusions to the Flock House virus (FHV) capsid precursor protein. We found that systemic immunization and mucosal boosting elicited CCR5-specific antibodies and achieved consistent priming in Peyer's patches, where most cells showed a phenotype corresponding to activated B cells and secreted high levels of IgA, representing up to one-third of the total HIV-blocking activity. Histopathological analysis revealed mild to moderate chronic inflammation in some tissues but failed in reporting signs of autoimmune dysfunction associated with immunizations. Antisera against immunogens representing the N terminus and extracellular loops 1 and 2 (Nter1 and ECL1 and ECL2) of CCR5 were generated. All showed specific anti-HIV activity, which was stronger in the anti-ECL1 and -ECL2 sera than in the anti-Nter sera. ECL1 and ECL2 antisera induced nearly complete long-lasting CCR5 downregulation of the receptor, and especially, their IgG-depleted fractions prevented HIV infection in neutralization and transcytosis assays. In conclusion, the ECL1 and ECL2 domains could offer a promising path to achieve significant anti-HIV activity in vivo. The study was the first to adopt a systematic strategy to compare the immunogenicities of all extracellular domains of the CCR5 molecule and to set optimal conditions leading to generation of specific antibodies in the mouse model. There were several relevant findings, which could be translated into human trials. (i) Prime (systemic) and boost (mucosal) immunization is the best protocol to induce anti-self antibodies with the expected properties. (ii) Aluminum is the best adjuvant in mice and thus can be easily used in nonhuman primates (NHP) and humans. (iii) The Flock House virus (FHV) system represents a valid delivery system, as the structure is well known and is not pathogenic for humans, and it is possible to introduce constrained regions able to elicit antibodies that recognize conformational epitopes. (iv) The best CCR5 vaccine candidate should include either extracellular loop 1 or 2 (ECL1 or ECL2), but not N terminus domains.

The Monocyte Chemoattractant Protein-1/Cognate CC Chemokine Receptor 2 System Affects Cell Motility in Cultured Human Podocytes

PubMed Central

Burt, Davina; Salvidio, Gennaro; Tarabra, Elena; Barutta, Federica; Pinach, Silvia; Dentelli, Patrizia; Camussi, Giovanni; Perin, Paolo Cavallo; Gruden, Gabriella

2007-01-01

In crescentic glomerulonephritis (GN), monocyte chemoattractant protein-1 (MCP-1) is overexpressed within the glomeruli, and MCP-1 blockade has renoprotective effects. Adult podocytes are in a quiescent state, but acquisition of a migratory/proliferative phenotype has been described in crescentic GN and implicated in crescent formation. The cognate CC chemokine receptor 2 (CCR2), the MCP-1 receptor, is expressed by other cell types besides monocytes and has been implicated in both cell proliferation and migration. We investigated whether MCP-1 binding to CCR2 can induce a migratory/proliferative response in cultured podocytes. MCP-1 binding to CCR2 enhanced podocyte chemotaxis/haptotaxis in a concentration-dependent manner and had a modest effect on cell proliferation. Closure of a wounded podocyte monolayer was delayed by CCR2 blockade, and CCR2 was overexpressed at the wound edge, suggesting a role for CCR2 in driving podocyte migration. Immunohistochemical analysis of kidney biopsies from patients with crescentic GN demonstrated CCR2 expression in both podocytes and cellular crescents, confirming the clinical relevance of our in vitro findings. In conclusion, the MCP-1/CCR2 system is functionally active in podocytes and may be implicated in the migratory events triggered by podocyte injury in crescentic GN and other glomerular diseases. PMID:18055544

The CCR4 Deadenylase Acts with Nanos and Pumilio in the Fine-Tuning of Mei-P26 Expression to Promote Germline Stem Cell Self-Renewal

PubMed Central

Joly, Willy; Chartier, Aymeric; Rojas-Rios, Patricia; Busseau, Isabelle; Simonelig, Martine

2013-01-01

Summary Translational regulation plays an essential role in Drosophila ovarian germline stem cell (GSC) biology. GSC self-renewal requires two translational repressors, Nanos (Nos) and Pumilio (Pum), which repress the expression of differentiation factors in the stem cells. The molecular mechanisms underlying this translational repression remain unknown. Here, we show that the CCR4 deadenylase is required for GSC self-renewal and that Nos and Pum act through its recruitment onto specific mRNAs. We identify mei-P26 mRNA as a direct and major target of Nos/Pum/CCR4 translational repression in the GSCs. mei-P26 encodes a protein of the Trim-NHL tumor suppressor family that has conserved functions in stem cell lineages. We show that fine-tuning Mei-P26 expression by CCR4 plays a key role in GSC self-renewal. These results identify the molecular mechanism of Nos/Pum function in GSC self-renewal and reveal the role of CCR4-NOT-mediated deadenylation in regulating the balance between GSC self-renewal and differentiation. PMID:24286029

The CCR4 deadenylase acts with Nanos and Pumilio in the fine-tuning of Mei-P26 expression to promote germline stem cell self-renewal.

PubMed

Joly, Willy; Chartier, Aymeric; Rojas-Rios, Patricia; Busseau, Isabelle; Simonelig, Martine

2013-01-01

Translational regulation plays an essential role in Drosophila ovarian germline stem cell (GSC) biology. GSC self-renewal requires two translational repressors, Nanos (Nos) and Pumilio (Pum), which repress the expression of differentiation factors in the stem cells. The molecular mechanisms underlying this translational repression remain unknown. Here, we show that the CCR4 deadenylase is required for GSC self-renewal and that Nos and Pum act through its recruitment onto specific mRNAs. We identify mei-P26 mRNA as a direct and major target of Nos/Pum/CCR4 translational repression in the GSCs. mei-P26 encodes a protein of the Trim-NHL tumor suppressor family that has conserved functions in stem cell lineages. We show that fine-tuning Mei-P26 expression by CCR4 plays a key role in GSC self-renewal. These results identify the molecular mechanism of Nos/Pum function in GSC self-renewal and reveal the role of CCR4-NOT-mediated deadenylation in regulating the balance between GSC self-renewal and differentiation.

CCL2 Serum Levels and Adiposity Are Associated with the Polymorphic Phenotypes -2518A on CCL2 and 64ILE on CCR2 in a Mexican Population with Insulin Resistance.

PubMed

Guzmán-Ornelas, Milton-Omar; Petri, Marcelo Heron; Vázquez-Del Mercado, Mónica; Chavarría-Ávila, Efraín; Corona-Meraz, Fernanda-Isadora; Ruíz-Quezada, Sandra-Luz; Madrigal-Ruíz, Perla-Monserrat; Castro-Albarrán, Jorge; Sandoval-García, Flavio; Navarro-Hernández, Rosa-Elena

2016-01-01

Genetic susceptibility has been described in insulin resistance (IR). Chemokine (C-C motif) ligand-2 (CCL2) is overexpressed in white adipose tissue and is the ligand of C-C motif receptor-2 (CCR2). The CCL2 G-2518A polymorphism is known to regulate gene expression, whereas the physiological effects of the CCR2Val64Ile polymorphism are unknown. The aim of the study is to investigate the relationship between these polymorphisms with soluble CCL2 levels (sCCL2), metabolic markers, and adiposity. In a cross-sectional study we included 380 Mexican-Mestizo individuals, classified with IR according to Stern criteria. Polymorphism was identified using PCR-RFLP/sequence-specific primers. Anthropometrics and metabolic markers were measured by routine methods and adipokines and sCCL2 by ELISA. The CCL2 polymorphism was associated with IR (polymorphic A+ phenotype frequencies were 70.9%, 82.6%, in individuals with and without IR, resp.). Phenotype carriers CCL2 (A+) displayed lower body mass and fat indexes, insulin and HOMA-IR, and higher adiponectin levels. Individuals with IR presented higher sCCL2 compared to individuals without IR and was associated with CCR2 (Ile+) phenotype. The double-polymorphic phenotype carriers (A+/Ile+) exhibited higher sCCL2 than double-wild-type phenotype carriers (A-/Ile-). The present findings suggest that sCCL2 production possibly will be associated with the adiposity and polymorphic phenotypes of CCL2 and CCR2, in Mexican-Mestizos with IR.

Gas6 Promotes Inflammatory (CCR2hiCX3CR1lo) Monocyte Recruitment in Venous Thrombosis.

PubMed

Laurance, Sandrine; Bertin, François-René; Ebrahimian, Talin; Kassim, Yusra; Rys, Ryan N; Lehoux, Stéphanie; Lemarié, Catherine A; Blostein, Mark D

2017-07-01

Coagulation and inflammation are inter-related. Gas6 (growth arrest-specific 6) promotes venous thrombosis and participates to inflammation through endothelial-innate immune cell interactions. Innate immune cells can provide the initiating stimulus for venous thrombus development. We hypothesize that Gas6 promotes monocyte recruitment during venous thrombosis. Deep venous thrombosis was induced in wild-type and Gas6-deficient (-/-) mice using 5% FeCl 3 and flow reduction in the inferior vena cava. Total monocyte depletion was achieved by injection of clodronate before deep venous thrombosis. Inflammatory monocytes were depleted using an anti-C-C chemokine receptor type 2 (CCR2) antibody. Similarly, injection of an anti-chemokine ligand 2 (CCL2) antibody induced CCL2 depletion. Flow cytometry and immunofluorescence were used to characterize the monocytes recruited to the thrombus. In vivo, absence of Gas6 was associated with a reduction of monocyte recruitment in both deep venous thrombosis models. Global monocyte depletion by clodronate leads to smaller thrombi in wild-type mice. Compared with wild type, the thrombi from Gas6 -/- mice contain less inflammatory (CCR2 hi CX 3 CR1 lo ) monocytes, consistent with a Gas6-dependent recruitment of this monocyte subset. Correspondingly, selective depletion of CCR2 hi CX 3 CR1 lo monocytes reduced the formation of venous thrombi in wild-type mice demonstrating a predominant role of the inflammatory monocytes in thrombosis. In vitro, the expression of both CCR2 and CCL2 were Gas6 dependent in monocytes and endothelial cells, respectively, impacting monocyte migration. Moreover, Gas6-dependent CCL2 expression and monocyte migration were mediated via JNK (c-Jun N-terminal kinase). This study demonstrates that Gas6 specifically promotes the recruitment of inflammatory CCR2 hi CX 3 CR1 lo monocytes through the regulation of both CCR2 and CCL2 during deep venous thrombosis. © 2017 American Heart Association, Inc.

Selective programming of CCR10+ innate lymphoid cells in skin-draining lymph nodes for cutaneous homeostatic regulation

PubMed Central

Yang, Jie; Hu, Shaomin; Zhao, Luming; Kaplan, Daniel H.; Perdew, Gary H.; Xiong, Na

2016-01-01

Innate lymphoid cells (ILCs) are preferentially localized into barrier tissues where they function in tissue protection but can also contribute to inflammatory diseases. The mechanisms regulating the establishment of ILCs in barrier tissues are poorly understood. Here we show that under steady-state conditions ILCs in skin-draining lymph nodes (sLNs) were continuously activated to acquire regulatory properties and high expression of the chemokine receptor CCR10 for localization into the skin. CCR10+ ILCs promoted the homeostasis of skin-resident T cells and reciprocally, their establishment in the skin required T cell-regulated homeostatic environments. Foxn1-expressing CD207+ dendritic cells were required for the proper generation of CCR10+ ILCs. These observations reveal mechanisms underlying the specific programming and priming of skin-homing CCR10+ ILCs in the sLNs. PMID:26523865

Gene Polymorphisms in the CCL5/CCR5 Pathway as a Genetic Biomarker for Outcome and Hand-Foot Skin Reaction in Metastatic Colorectal Cancer Patients Treated With Regorafenib.

PubMed

Suenaga, Mitsukuni; Schirripa, Marta; Cao, Shu; Zhang, Wu; Yang, Dongyun; Ning, Yan; Cremolini, Chiara; Antoniotti, Carlotta; Borelli, Beatrice; Mashima, Tetsuo; Okazaki, Satoshi; Berger, Martin D; Miyamoto, Yuji; Gopez, Roel; Barzi, Afsaneh; Lonardi, Sara; Yamaguchi, Toshiharu; Falcone, Alfredo; Loupakis, Fotios; Lenz, Heinz-Josef

2018-06-01

The C-C motif chemokine ligand 5/C-C motif chemokine receptor 5 (CCL5/CCR5) pathway has been shown to induce endothelial progenitor cell migration, resulting in increased vascular endothelial growth factor A expression. We hypothesized that genetic polymorphisms in the CCL5/CCR5 pathway predict efficacy and toxicity in patients with metastatic colorectal cancer (mCRC) treated with regorafenib. We analyzed genomic DNA extracted from 229 tumor samples from 2 different cohorts of patients who received regorafenib: an evaluation cohort of 79 Japanese patients and a validation cohort of 150 Italian patients. Single nucleotide polymorphisms of CCL5/CCR5 pathway-related genes were analyzed by PCR-based direct sequencing. CCL4 rs1634517 and CCL3 rs1130371 were associated with progression-free survival in the evaluation cohort (hazard ratio [HR] 1.54, P = .043; HR 1.48, P = .064), and progression-free survival (HR 1.74, P < .001; HR 1.66, P = .002) and overall survival (HR 1.65, P = .004; HR 1.65, P = .004) in the validation cohort. The allelic frequencies of CCL5 single nucleotide polymorphisms varied between the evaluation and validation cohorts (G/G variant in rs2280789, 21.5% vs. 1.3%, P < .001; T/T variant in rs3817655, 22.8% vs. 2.7%, P < .001). In the evaluation cohort, patients with the G/G variant in rs2280789 had a higher incidence of grade 3+ hand-foot skin reaction compared to any A allele (53% vs. 27%, P = .078), and similarly to the T/T variant in rs3817655 compared to any A allele (56% vs. 26%, P = .026). Genetic variants in the CCL5/CCR5 pathway may serve as prognostic markers and may predict severe hand-foot skin reaction in mCRC patients receiving regorafenib therapy. Copyright © 2018 Elsevier Inc. All rights reserved.

Chitin elicits CCL2 from airway epithelial cells and induces CCR2-dependent innate allergic inflammation in the lung

PubMed Central

Roy, René M.; Wüthrich, Marcel; Klein, Bruce S.

2012-01-01

Chitin exposure in the lung induces eosinophilia and alternative activation of macrophages, and is correlated with allergic airway disease. However, the mechanism underlying chitin-induced polarization of macrophages is poorly understood. Here, we show that chitin induces alternative activation of macrophages in vivo, but does not do so directly in vitro. We further show that airway epithelial cells bind chitin in vitro and produce CCL2 in response to chitin both in vitro and in vivo. Supernatants of chitin exposed epithelial cells promoted alternative activation of macrophages in vitro, whereas antibody neutralization of CCL2 in the supernate abolished the alternative activation of macrophages. CCL2 acted redundantly in vivo, but mice lacking the CCL2 receptor, CCR2, showed impaired alternative activation of macrophages in response to chitin, as measured by arginase I, CCL17 and CCL22 expression. Furthermore, CCR2KO mice exposed to chitin had diminished ROS products in the lung, blunted eosinophil and monocyte recruitment, and impaired eosinophil functions as measured by expression of CCL5, IL13 and CCL11. Thus, airway epithelial cells secrete CCL2 in response to chitin and CCR2 signaling mediates chitin-induced alternative activation of macrophages and allergic inflammation in vivo. PMID:22851704

Cytokines Elevated in HIV Elite Controllers Reduce HIV Replication In Vitro and Modulate HIV Restriction Factor Expression

PubMed Central

Jacobs, Evan S.; Abdel-Mohsen, Mohamed; Gibb, Stuart L.; Heitman, John W.; Inglis, Heather C.; Martin, Jeffrey N.; Zhang, Jinbing; Kaidarova, Zhanna; Deng, Xutao; Wu, Shiquan; Anastos, Kathryn; Crystal, Howard; Villacres, Maria C.; Young, Mary; Greenblatt, Ruth M.; Landay, Alan L.; Gange, Stephen J.; Deeks, Steven G.; Golub, Elizabeth T.; Pillai, Satish K.

2017-01-01

ABSTRACT A subset of HIV-infected individuals termed elite controllers (ECs) maintain CD4+ T cell counts and control viral replication in the absence of antiretroviral therapy (ART). Systemic cytokine responses may differentiate ECs from subjects with uncontrolled viral replication or from those who require ART to suppress viral replication. We measured 87 cytokines in four groups of women: 73 ECs, 42 with pharmacologically suppressed viremia (ART), 42 with uncontrolled viral replication (noncontrollers [NCs]), and 48 HIV-uninfected (NEG) subjects. Four cytokines were elevated in ECs but not NCs or ART subjects: CCL14, CCL21, CCL27, and XCL1. In addition, median stromal cell-derived factor-1 (SDF-1) levels were 43% higher in ECs than in NCs. The combination of the five cytokines suppressed R5 and X4 virus replication in resting CD4+ T cells, and individually SDF-1β, CCL14, and CCL27 suppressed R5 virus replication, while SDF-1β, CCL21, and CCL14 suppressed X4 virus replication. Functional studies revealed that the combination of the five cytokines upregulated CD69 and CCR5 and downregulated CXCR4 and CCR7 on CD4+ T cells. The CD69 and CXCR4 effects were driven by SDF-1, while CCL21 downregulated CCR7. The combination of the EC-associated cytokines induced expression of the anti-HIV host restriction factors IFITM1 and IFITM2 and suppressed expression of RNase L and SAMHD1. These results identify a set of cytokines that are elevated in ECs and define their effects on cellular activation, HIV coreceptor expression, and innate restriction factor expression. This cytokine pattern may be a signature characteristic of HIV-1 elite control, potentially important for HIV therapeutic and curative strategies. IMPORTANCE Approximately 1% of people infected with HIV control virus replication without taking antiviral medications. These subjects, termed elite controllers (ECs), are known to have stronger immune responses targeting HIV than the typical HIV-infected subject, but the exact mechanisms of how their immune responses control infection are not known. In this study, we identified five soluble immune signaling molecules (cytokines) in the blood that were higher in ECs than in subjects with typical chronic HIV infection. We demonstrated that these cytokines can activate CD4+ T cells, the target cells for HIV infection. Furthermore, these five EC-associated cytokines could change expression levels of intrinsic resistance factors, or molecules inside the target cell that fight HIV infection. This study is significant in that it identified cytokines elevated in subjects with a good immune response against HIV and defined potential mechanisms as to how these cytokines could induce resistance to the virus in target cells. PMID:28053103

Cytokines Elevated in HIV Elite Controllers Reduce HIV Replication In Vitro and Modulate HIV Restriction Factor Expression.

PubMed

Jacobs, Evan S; Keating, Sheila M; Abdel-Mohsen, Mohamed; Gibb, Stuart L; Heitman, John W; Inglis, Heather C; Martin, Jeffrey N; Zhang, Jinbing; Kaidarova, Zhanna; Deng, Xutao; Wu, Shiquan; Anastos, Kathryn; Crystal, Howard; Villacres, Maria C; Young, Mary; Greenblatt, Ruth M; Landay, Alan L; Gange, Stephen J; Deeks, Steven G; Golub, Elizabeth T; Pillai, Satish K; Norris, Philip J

2017-03-15

A subset of HIV-infected individuals termed elite controllers (ECs) maintain CD4 + T cell counts and control viral replication in the absence of antiretroviral therapy (ART). Systemic cytokine responses may differentiate ECs from subjects with uncontrolled viral replication or from those who require ART to suppress viral replication. We measured 87 cytokines in four groups of women: 73 ECs, 42 with pharmacologically suppressed viremia (ART), 42 with uncontrolled viral replication (noncontrollers [NCs]), and 48 HIV-uninfected (NEG) subjects. Four cytokines were elevated in ECs but not NCs or ART subjects: CCL14, CCL21, CCL27, and XCL1. In addition, median stromal cell-derived factor-1 (SDF-1) levels were 43% higher in ECs than in NCs. The combination of the five cytokines suppressed R5 and X4 virus replication in resting CD4 + T cells, and individually SDF-1β, CCL14, and CCL27 suppressed R5 virus replication, while SDF-1β, CCL21, and CCL14 suppressed X4 virus replication. Functional studies revealed that the combination of the five cytokines upregulated CD69 and CCR5 and downregulated CXCR4 and CCR7 on CD4 + T cells. The CD69 and CXCR4 effects were driven by SDF-1, while CCL21 downregulated CCR7. The combination of the EC-associated cytokines induced expression of the anti-HIV host restriction factors IFITM1 and IFITM2 and suppressed expression of RNase L and SAMHD1. These results identify a set of cytokines that are elevated in ECs and define their effects on cellular activation, HIV coreceptor expression, and innate restriction factor expression. This cytokine pattern may be a signature characteristic of HIV-1 elite control, potentially important for HIV therapeutic and curative strategies. IMPORTANCE Approximately 1% of people infected with HIV control virus replication without taking antiviral medications. These subjects, termed elite controllers (ECs), are known to have stronger immune responses targeting HIV than the typical HIV-infected subject, but the exact mechanisms of how their immune responses control infection are not known. In this study, we identified five soluble immune signaling molecules (cytokines) in the blood that were higher in ECs than in subjects with typical chronic HIV infection. We demonstrated that these cytokines can activate CD4 + T cells, the target cells for HIV infection. Furthermore, these five EC-associated cytokines could change expression levels of intrinsic resistance factors, or molecules inside the target cell that fight HIV infection. This study is significant in that it identified cytokines elevated in subjects with a good immune response against HIV and defined potential mechanisms as to how these cytokines could induce resistance to the virus in target cells. Copyright © 2017 American Society for Microbiology.

Colonic eosinophilic inflammation in experimental colitis is mediated by Ly6Chigh CCR2+ inflammatory monocyte/macrophage-derived CCL11

PubMed Central

Waddell, Amanda; Ahrens, Richard; Steinbrecher, Kris; Donovan, Burke; Rothenberg, Marc E.; Munitz, Ariel; Hogan, Simon P.

2011-01-01

Recent genome-wide association studies of pediatric IBD have implicated the 17q12 loci, which contains the eosinophil specific chemokine gene CCL11, with early-onset IBD susceptibility. In the present study, we employed a murine model of experimental colitis to define the molecular pathways that regulate CCL11 expression in the chronic intestinal inflammation and pathophysiology of experimental colitis. Bone marrow chimera experiments showed that hematopoietic cell-derived CCL11 is sufficient for CCL11-mediated colonic eosinophilic inflammation. We show that DSS treatment promotes the recruitment of F4/80+CD11b+CCR2+Ly6Chigh inflammatory monocytes into the colon. F4/80+CD11b+CCR2+Ly6Chigh monocytes express CCL11, and their recruitment positively correlated with colonic eosinophilic inflammation. Phenotypic analysis of purified Ly6Chigh intestinal inflammatory MΦs revealed that these cells express both M1- and M2-associated genes, including Il6, Ccl4 and Cxcl2, and Arg1, Chi3l3, Ccl11 and IL-10, respectively. Attenuation of DSS-induced F4/80+CD11b+CCR2+Ly6Chigh monocyte recruitment to the colon in CCR2−/− mice was associated with decreased colonic CCL11 expression, eosinophilic inflammation and DSS-induced histopathology. These studies identify a mechanism for DSS-induced colonic eosinophilia mediated by Ly6ChighCCR2+ inflammatory monocyte/MΦ-derived CCL11. PMID:21498668

Bioinformatics, interaction network analysis, and neural networks to characterize gene expression of radicular cyst and periapical granuloma.

PubMed

Poswar, Fabiano de Oliveira; Farias, Lucyana Conceição; Fraga, Carlos Alberto de Carvalho; Bambirra, Wilson; Brito-Júnior, Manoel; Sousa-Neto, Manoel Damião; Santos, Sérgio Henrique Souza; de Paula, Alfredo Maurício Batista; D'Angelo, Marcos Flávio Silveira Vasconcelos; Guimarães, André Luiz Sena

2015-06-01

Bioinformatics has emerged as an important tool to analyze the large amount of data generated by research in different diseases. In this study, gene expression for radicular cysts (RCs) and periapical granulomas (PGs) was characterized based on a leader gene approach. A validated bioinformatics algorithm was applied to identify leader genes for RCs and PGs. Genes related to RCs and PGs were first identified in PubMed, GenBank, GeneAtlas, and GeneCards databases. The Web-available STRING software (The European Molecular Biology Laboratory [EMBL], Heidelberg, Baden-Württemberg, Germany) was used in order to build the interaction map among the identified genes by a significance score named weighted number of links. Based on the weighted number of links, genes were clustered using k-means. The genes in the highest cluster were considered leader genes. Multilayer perceptron neural network analysis was used as a complementary supplement for gene classification. For RCs, the suggested leader genes were TP53 and EP300, whereas PGs were associated with IL2RG, CCL2, CCL4, CCL5, CCR1, CCR3, and CCR5 genes. Our data revealed different gene expression for RCs and PGs, suggesting that not only the inflammatory nature but also other biological processes might differentiate RCs and PGs. Copyright © 2015 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Mode of coreceptor use by R5 HIV type 1 correlates with disease stage: a study of paired plasma and cerebrospinal fluid isolates.

PubMed

Karlsson, Ulf; Antonsson, Liselotte; Repits, Johanna; Medstrand, Patrik; Owman, Christer; Kidd-Ljunggren, Karin; Hagberg, Lars; Svennerholm, Bo; Jansson, Marianne; Gisslén, Magnus; Ljungberg, Bengt

2009-12-01

Through the use of chimeric CXCR4/CCR5 receptors we have previously shown that CCR5-tropic (R5) HIV-1 isolates acquire a more flexible receptor use over time, and that this links to a reduced viral susceptibility to inhibition by the CCR5 ligand RANTES. These findings may have relevance with regards to the efficacy of antiretroviral compounds that target CCR5/virus interactions. Compartmentalized discrepancies in coreceptor use may occur, which could also affect the efficacy of these compounds at specific anatomical sites, such as within the CNS. In this cross-sectional study we have used wild-type CCR5 and CXCR4 as well as chimeric CXCR4/CCR5 receptors to characterize coreceptor use by paired plasma and cerebrospinal fluid (CSF) isolates from 28 HIV-1-infected individuals. Furthermore, selected R5 isolates, with varying chimeric receptor use, were tested for sensitivity to inhibition by the CCR5 antagonist TAK-779. Discordant CSF/plasma virus coreceptor use was found in 10/28 patients. Low CD4+ T cell counts correlated strongly with a more flexible mode of R5 virus CCR5 usage, as disclosed by an increased ability to utilize chimeric CXCR4/CCR5 receptors, specifically receptor FC-2. Importantly, an elevated ability to utilize chimeric receptors correlated with a reduced susceptibility to inhibition by TAK-779. Our findings show that a discordant CSF and plasma virus coreceptor use is not uncommon. Furthermore, we provide support for an emerging paradigm, where the acquisition of a more flexible mode of CCR5 usage is a key event in R5 virus pathogenesis. This may, in turn, negatively impact the efficacy of CCR5 antagonist treatment in late stage HIV-1 disease.

CCL26/eotaxin-3 is more effective to induce the migration of eosinophils of asthmatics than CCL11/eotaxin-1 and CCL24/eotaxin-2.

PubMed

Provost, Véronique; Larose, Marie-Chantal; Langlois, Anick; Rola-Pleszczynski, Marek; Flamand, Nicolas; Laviolette, Michel

2013-08-01

CCL11, CCL24, and CCL26 are chemokines involved in the recruitment of eosinophils into tissues and mainly activate CCR3. Whereas the genomic or pharmacological inhibition of CCR3 prevents the development of experimental asthma in rodents, it only impairs the recruitment of eosinophils by ∼40% in humans. As humans, but not rodents, express CCL26, we investigated the impact of CCL11, CCL24, and CCL26 on human eosinophils recruitment and evaluated the involvement of CCR3. The migration of eosinophils of healthy volunteers was similar for the three eotaxins. Eosinophils of mild asthmatics had a greater response to CCL11 and a much greater response to CCL26. Whereas all eotaxins induced the migration of eosinophil of asthmatics from 0 to 6 h, CCL26 triggered a second phase of migration between 12 and 18 h. Given that the CCR3 antagonists SB 328437 and SB 297006 inhibited the 5-oxo-eicosatetraenoate-induced migration of eosinophils and that the CCR3 antagonist UCB 35625 was not specific for CCR3, CCR3 blockade was performed with the CCR3 mAb. This antibody completely blocked the effect of all eotaxins on eosinophils of healthy subjects and the effect of CCL24 on the eosinophils of asthmatics. Interestingly, CCR3 blockade did not affect the second migration phase induced by CCL26 on eosinophils of asthmatics. In conclusion, CCL26 is a more effective chemoattractant than CCL11 and CCL24 for eosinophils of asthmatics. The mechanism of this greater efficiency is not yet defined. However, these results suggest that CCL26 may play a unique and important role in the recruitment of eosinophils in persistent asthma.

CCL17 and CCL22/CCR4 signaling is a strong candidate for novel targeted therapy against nasal natural killer/T-cell lymphoma

PubMed Central

Kumai, Takumi; Kobayashi, Hiroya; Komabayashi, Yuki; Ueda, Seigo; Kishibe, Kan; Ohkuri, Takayuki; Takahara, Miki; Celis, Esteban; Harabuchi, Yasuaki

2015-01-01

Nasal natural killer/T-cell lymphoma (NNKTL) is associated with Epstein–Barr virus and has a poor prognosis because of local invasion and/or multiple dissemination. Various chemokines play a role in tumor proliferation and invasion, and chemokine receptors including the C-C chemokine receptor 4 (CCR4) are recognized as potential targets for treating hematologic malignancies. The aim of the present study was to determine whether specific chemokines are produced by NNKTL. We compared chemokine expression patterns in culture supernatants of NNKTL cell lines with those of other lymphoma or leukemia cell lines using chemokine protein array and ELISA. Chemokine (C-C motif) ligand (CCL) 17 and CCL22 were highly produced by NNKTL cell lines as compared to the other cell lines. In addition, CCL17 and CCL22 were readily observed in the sera of NNKTL patients. The levels of these chemokines were significantly higher in patients than in healthy controls. Furthermore, we detected the expression of CCR4 (the receptor for CCL17 and CCL22) on the surface of NNKTL cell lines and in tissues of NNKTL patients. Anti-CCR4 monoclonal antibody (mAb) efficiently induced antibody-dependent cellular cytotoxicity mediated by natural killer cells against NNKTL cell lines. Our results suggest that CCL17 and CCL22 may be important factors in the development of NNKTL and open up the possibility of immunotherapy of this lymphoma using anti-CCR4 mAb. PMID:25754123

Natural Infection of a Homozygous Δ24 CCR5 Red-capped Mangabey with an R2b-Tropic Simian Immunodeficiency Virus

PubMed Central

Chen, Zhiwei; Kwon, Douglas; Jin, Zhanqun; Monard, Simon; Telfer, Paul; Jones, Morris S.; Lu, Chang Y.; Aguilar, Roberto F.; Ho, David D.; Marx, Preston A.

1998-01-01

A homozygous 24-bp deletion (Δ24) was found in the CC chemokine receptor 5 (CCR5) of 11 out of 15 red-capped mangabeys (RCMs), Cercocebus torquatus torquatus, both in Africa and in an American zoo. The CCR5 Δ24 defect encompassed eight amino acids in frame in the fourth transmembrane region. Unexpectedly, RCM-009, one of 11 homozygotes (Δ24CCR5/ Δ24CCR5), was found to be naturally infected with a divergent simian immunodeficiency virus (SIV) strain, which was not R5-tropic, but used CCR2b (R2b) as its major coreceptor. SIVrcmGab1 was the only R2b-tropic SIV among other divergent SIVs tested. Cells transfected with the Δ24 CCR5 did not support entry of R5-tropic SIVmac, SIVcpz, SIVmne, HIV-2, or HIV-1, and were also inactive in signal transduction mediated by β-chemokines. At 86.6%, the Δ24 allelic frequency was significantly higher than that of the 32-bp deletion found in humans. The Δ24 frequency was 4.1% in 34 sooty mangabeys (SMs), a geographically isolated subspecies that was naturally infected with R5-tropic SIV. Finding identical deletions in two mangabey subspecies separated for 10,000 years or more dates the Δ24 CCR5 deletion as ancient. However, the source of the selective pressure for the high rate of CCR5 deletion in RCMs remains to be determined. The high allelic frequency of the Δ24 CCR5 in RCMs, in comparison to that of SMs, suggests that R2b-tropism may have been acquired by SIVrcm, as an adaptation to CCR5 genetic defects appeared in its host. PMID:9841919

Characterization and analysis of CCR and CAD gene families at the whole-genome level for lignin synthesis of stone cells in pear (Pyrus bretschneideri) fruit

PubMed Central

Cheng, Xi; Li, Manli; Li, Dahui; Zhang, Jinyun; Jin, Qing; Sheng, Lingling; Lin, Yi

2017-01-01

ABSTRACT The content of stone cells has significant effects on the flavour and quality of pear fruit. Previous research suggested that lignin deposition is closely related to stone cell formation. In the lignin biosynthetic pathway, cinnamoyl-CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD), dehydrogenase/reductase family members, catalyse the last two steps in monolignol synthesis. However, there is little knowledge of the characteristics of the CCR and CAD families in pear and their involvement in lignin synthesis of stone cells. In this study, 31 CCRs and 26 CADs were identified in the pear genome. Phylogenetic trees for CCRs and CADs were constructed; key amino acid residues were analysed, and three-dimensional structures were predicted. Using quantitative real-time polymerase chain reaction (qRT-PCR), PbCAD2, PbCCR1, -2 and -3 were identified as participating in lignin synthesis of stone cells in pear fruit. Subcellular localization analysis showed that the expressed proteins (PbCAD2, PbCCR1, -2 and -3) are found in the cytoplasm or at the cell membrane. These results reveal the evolutionary features of the CCR and CAD families in pear as well as the genes responsible for regulation of lignin synthesis and stone cell development in pear fruit. PMID:29141952

Reduced toll-like receptor 4 and substance P gene expression is associated with airway bacterial colonization in children.

PubMed

Grissell, Terry V; Chang, Anne B; Gibson, Peter G

2007-04-01

Neuro-immune interactions are increasingly relevant to human health and disease. The neuropeptide Substance P also has antibacterial activity and bears similarities to the innate immune antibacterial defensins. This suggests possible co-regulation of neuropeptide and innate immune mediators. In this study, non-bronchoscopic bronchoalveolar lavage (BAL) was performed on 69 children. BAL was examined for cellular profile, microbiology (bacteria, virus) and gene expression for TLRs 2, 3, 4; chemokine receptors (CCR3, CCR5, CXCR1); neurotrophins and neurokinin genes (TAC1, TAC3, CGRP, NGF). In children with bacterial colonization (n=10) there was an airway inflammatory response with increased BAL neutrophils, IL-8 protein, and CXCR1 expression. Substance P (TAC1) and TLR4 RNA expression were reduced in children with bacterial colonization. TLR3 mRNA was increased in 7.2% (n=5) children with rhinovirus, and there was a non-significant trend to increased TLR2. There is evidence for co-regulation of neurokinin (TAC1) and TLR4 gene expression in airway cells from children with airway bacterial colonization and their reduced expression may be associated with an impaired bacterial clearance. (c) 2007 Wiley-Liss, Inc.

Renal creatinine handling in very old patients with chronic renal disease.

PubMed

Musso, Carlos G; Michelángelo, Hernán; Vilas, Manuel; Martinez, Bernardo; Bonetto, Alberto; Jauregui, Ricardo; Algranati, Luis

2011-09-01

Renal creatinine handling is basically the result of its glomerular filtration and proximal tubular secretion. However, creatinine reabsorption has been documented in certain conditions, such as premature babies, newborns, and healthy elderly people. Additionally, it is known that there is an increase in the proportion of secreted creatinine in chronic renal disease. In this paper, we report our studies on the characteristic reabsorption pattern of creatinine in the elderly with chronic renal disease. We studied twenty-seven volunteers with chronic kidney disease, eleven of whom were young and the rest were very old (age > 75 years old). We measured creatinine clearance without (Ccr) and with cimetidine (CcrWC) and Ccr/CcrWC ratio from each volunteer, in timed urine samples. Then, Ccr, CcrWC, and Ccr/CcrWC ratio were compared between young and very old people in two chronic kidney disease subgroups: stages II-III and stages IV-V. Statistical analysis was performed applying a non-parametric test (Wilcoxon). We observed a tendency towards a lower Ccr/CcrWC ratio in the very old stage II-III group compared with the young one: 1 (0.96-1.26) (very old) vs 1.3 (1.1-1.5) (young), P = 0.09, on the contrary, there was no significant difference in Ccr/CcrWC ratio between very old and young person with stage IV-V CKD: 1.66 (1.41-2.21) (young) vs 1.77 (1.1-2.7) (young), P = NS. Creatinine secretion pattern in very old patients with advanced chronic renal disease is similar to that observed in young ones with similar level of CKD.

Fitness and Competitive Ability of Botrytis cinerea Isolates with Resistance to Multiple Chemical Classes of Fungicides.

PubMed

Chen, S N; Luo, C X; Hu, M J; Schnabel, G

2016-09-01

Resistance to multiple chemical classes of fungicides in Botrytis cinerea isolates from eastern United States strawberry fields is common and strategies to control them are needed. In this study, we compared fitness and competitive ability of eight sensitive isolates (S), eight isolates resistant to five or six chemical classes of fungicides but not to phenylpyrroles (5CCR), and eight isolates resistant to six or seven chemical classes including phenylpyrroles (6CCR/MDR1h). The latter included the MDR1h phenotype due to overexpression of atrB based on Δ497V/L in mrr1. The 6CCR/MDR1h isolates grew more slowly at 4°C on potato dextrose agar, and both 5CCR and 6CCR/MDR1h isolates were hypersensitive to osmotic stress compared with S isolates. In contrast, no differences were found in oxidative sensitivity, aggressiveness, and spore production in vivo, and sclerotia production and viability in vitro. In competition experiments, the 5CCR and 6CCR/MDR1h isolates were both outcompeted by S isolates and 6CCR/MDR1h isolates were outcompeted by 5CCR isolates in the absence of fungicide pressure. Under selective pressure of a fludioxonil/pyraclostrobin rotation, the 6CCR/MDR1h isolates dominated after coinoculation with 5CCR and S isolates. The competitive disadvantage of 5CCR and especially 6CCR/MDR1h isolates suggest that, in the absence of fungicide selection pressure, S isolates may reduce inoculum potential of multifungicide-resistant isolates under field conditions.

Effects of CCR5-delta32 and CCR2-64I alleles on disease progression of perinatally HIV-1-infected children: an international meta-analysis.

PubMed

Ioannidis, John P A; Contopoulos-Ioannidis, Despina G; Rosenberg, Philip S; Goedert, James J; De Rossi, Anita; Espanol, Teresa; Frenkel, Lisa; Mayaux, Marie-Jeanne; Newell, Marie-Louise; Pahwa, Savita G; Rousseau, Christine; Scarlatti, Gabriella; Sei, Shizuko; Sen, Luisa; O'Brien, Thomas R

2003-07-25

Among perinatally infected children, the effects of certain alleles of the CCR5 and CCR2 genes on the rate of disease progression remain unclear. We addressed the effects of CCR5-delta32 and CCR2-64I in an international meta-analysis. Genotype data were contributed from 10 studies with 1317 HIV-1-infected children (7263 person-years of follow-up). Time-to-event analyses were performed stratified by study and racial group. Endpoints included progression to clinical AIDS, death, and death after the diagnosis of clinical AIDS. The time-dependence of the genetic effects was specifically investigated. There was large heterogeneity in the observed rates of disease progression between different cohorts. For progression to clinical AIDS, both CCR5-delta32 and CCR2-64I showed overall non-significant trends for protection [hazard ratios 0.84, 95% confidence interval (CI) 0.58-1.23; and 0.87, 95% CI 0.67-1.14, respectively]. However, analyses of survival showed statistically significant time-dependence. No deaths occurred among CCR5-delta32 carriers in the first 3 years of life, whereas there was no protective effect (hazard ratio 0.95; 95% CI 0.43-2.10) in later years (P=0.01 for the time-dependent model). For CCR2-64I, the hazard ratio for death was 0.69 (95% CI 0.39-1.21) in the first 6 years of life and 2.56 (95% CI 1.26-5.20) in subsequent years (P<0.01 for the time-dependent model). CCR5-delta32 and CCR2-64I offered no clear protection after clinical AIDS had developed. The CCR5-delta32 and CCR2-64I alleles are associated with a decreased risk of death among perinatally infected children, but only for the first years of life.

Medicinal chemistry of small molecule CCR5 antagonists for blocking HIV-1 entry: a review of structural evolution.

PubMed

Tian, Ye; Zhang, Dujuan; Zhan, Peng; Liu, Xinyong

2014-01-01

CCR5, a member of G protein-coupled receptors superfamily, plays an important role in the HIV-1 entry process. Antagonism of this receptor finally leads to the inhibition of R5 strains of HIV entry into the human cells. The identification of CCR5 antagonists as antiviral agents will provide more option for HAART. Now, more than a decade after the first small molecule CCR5 inhibitor was discovered, great achievements have been made. In this article, we will give a brief introduction of several series of small molecule CCR5 antagonists, focused on their appealing structure evolution, essential SAR information and thereof the enlightenment of strategies on CCR5 inhibitors design.

Loss of PPAR gamma in immune cells impairs the ability of abscisic acid to improve insulin sensitivity by suppressing monocyte chemoattractant protein-1 expression and macrophage infiltration into white adipose tissue.

PubMed

Guri, Amir J; Hontecillas, Raquel; Ferrer, Gerardo; Casagran, Oriol; Wankhade, Umesh; Noble, Alexis M; Eizirik, Decio L; Ortis, Fernanda; Cnop, Miriam; Liu, Dongmin; Si, Hongwei; Bassaganya-Riera, Josep

2008-04-01

Abscisic acid (ABA) is a natural phytohormone and peroxisome proliferator-activated receptor gamma (PPARgamma) agonist that significantly improves insulin sensitivity in db/db mice. Although it has become clear that obesity is associated with macrophage infiltration into white adipose tissue (WAT), the phenotype of adipose tissue macrophages (ATMs) and the mechanisms by which insulin-sensitizing compounds modulate their infiltration remain unknown. We used a loss-of-function approach to investigate whether ABA ameliorates insulin resistance through a mechanism dependent on immune cell PPARgamma. We characterized two phenotypically distinct ATM subsets in db/db mice based on their surface expression of F4/80. F4/80(hi) ATMs were more abundant and expressed greater concentrations of chemokine receptor (CCR) 2 and CCR5 when compared to F4/80(lo) ATMs. ABA significantly decreased CCR2(+) F4/80(hi) infiltration into WAT and suppressed monocyte chemoattractant protein-1 (MCP-1) expression in WAT and plasma. Furthermore, the deficiency of PPARgamma in immune cells, including macrophages, impaired the ability of ABA to suppress the infiltration of F4/80(hi) ATMs into WAT, to repress WAT MCP-1 expression and to improve glucose tolerance. We provide molecular evidence in vivo demonstrating that ABA improves insulin sensitivity and obesity-related inflammation by inhibiting MCP-1 expression and F4/80(hi) ATM infiltration through a PPARgamma-dependent mechanism.

HIV envelope-mediated, CCR5/α4β7-dependent killing of CD4-negative γδ T cells which are lost during progression to AIDS.

PubMed

Li, Haishan; Pauza, C David

2011-11-24

HIV infects and replicates in CD4+ T cells but effects on host immunity and disease also involve depletion, hyper-activation, and modification of CD4-negative cell populations. In particular, the depletion of CD4-negative γδ T cells is common to all HIV+ individuals. We found that soluble or cell-associated envelope glycoproteins from CCR5-tropic strains of HIV could bind, activates the p38-caspase pathway, and induce the death of γδ cells. Envelope binding requires integrin α4β7 and chemokine receptor CCR5 which are at high levels and form a complex on the γδ T cell membrane. This receptor complex facilitated V3 loop binding to CCR5 in the absence of CD4-induced conformational changes. Cell death was increased by antigen stimulation after exposure to envelope glycoprotein. Direct signaling by envelope glycoprotein killed CD4-negative γδ T cells and reproduced a defect observed in all patients with HIV disease.

The CC chemokine receptor 5 regulates olfactory and social recognition in mice.

PubMed

Kalkonde, Y V; Shelton, R; Villarreal, M; Sigala, J; Mishra, P K; Ahuja, S S; Barea-Rodriguez, E; Moretti, P; Ahuja, S K

2011-12-01

Chemokines are chemotactic cytokines that regulate cell migration and are thought to play an important role in a broad range of inflammatory diseases. The availability of chemokine receptor blockers makes them an important therapeutic target. In vitro, chemokines are shown to modulate neurotransmission. However, it is not very clear if chemokines play a role in behavior and cognition. Here we evaluated the role of CC chemokine receptor 5 (CCR5) in various behavioral tasks in mice using Wt (Ccr5⁺/⁺) and Ccr5-null (Ccr5⁻/⁻)mice. Ccr5⁻/⁻ mice showed enhanced social recognition. Administration of CC chemokine ligand 3 (CCL3), one of the CCR5-ligands, impaired social recognition. Since the social recognition task is dependent on the sense of olfaction, we tested olfactory recognition for social and non-social scents in these mice. Ccr5⁻/⁻ mice had enhanced olfactory recognition for both these scents indicating that enhanced performance in social recognition task could be due to enhanced olfactory recognition in these mice. Spatial memory and aversive memory were comparable in Wt and Ccr5⁻/⁻ mice. Collectively, these results suggest that chemokines/chemokine receptors might play an important role in olfactory recognition tasks in mice and to our knowledge represents the first direct demonstration of an in vivo role of CCR5 in modulating social behavior in mice. These studies are important as CCR5 blockers are undergoing clinical trials and can potentially modulate behavior. Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.

Murine lung eosinophil activation and chemokine production in allergic airway inflammation

PubMed Central

Rose, C Edward; Lannigan, Joanne A; Kim, Paul; Lee, James J; Fu, Shu Man; Sung, Sun-sang J

2010-01-01

Eosinophils play important roles in asthma and lung infections. Murine models are widely used for assessing the functional significance and mechanistic basis for eosinophil involvements in these diseases. However, little is known about tissue eosinophils in homeostasis. In addition, little data on eosinophil chemokine production during allergic airway inflammation are available. In this study, the properties and functions of homeostatic and activated eosinophils were compared. Eosinophils from normal tissues expressed costimulation and adhesion molecules B7-1, B7-2 and ICAM-1 for Ag presentation but little major histocompatibility complex (MHC) class II, and were found to be poor stimulators of T-cell proliferation. However, these eosinophils expressed high levels of chemokine mRNA including C10, macrophage inflammatory protein (MIP)-1α, MIP-1γ, MIP-2, eotaxin and monocyte chemoattractant protein-5 (MCP-5), and produced chemokine proteins. Eosinophil intracellular chemokines decreased rapidly with concomitant surface marker downregulation upon in vitro culturing consistent with piecemeal degranulation. Lung eosinophils from mice with induced allergic airway inflammation exhibited increased chemokines mRNA expression and chemokines protein production and upregulated MHC class II and CD11c expression. They were also found to be the predominant producers of the CCR1 ligands CCL6/C10 and CCL9/MIP-1γ in inflamed lungs. Eosinophil production of C10 and MIP-1γ correlated with the marked influx of CD11bhigh lung dendritic cells during allergic airway inflammation and the high expression of CCR1 on these dendritic cells (DCs). The study provided baseline information on tissue eosinophils, documented the upregulation of activation markers and chemokine production in activated eosinophils, and indicated that eosinophils were a key chemokine-producing cell type in allergic lung inflammation. PMID:20622891

Characterization of developmental and stress mediated expression of cinnamoyl-CoA reductase (CCR) in kenaf (Hibiscus cannabinus L.)

USDA-ARS?s Scientific Manuscript database

Cinnamoyl-CoA reductase (CCR) is an important enzyme for lignin biosynthesis as it catalyzes the first specific committed step in monolignol biosynthesis. We have cloned a full length coding sequence of CCR from kenaf (Hibiscus cannabinus L.), which contains a 1,020-bp open reading frame (ORF), enco...

Chemokine (C-C Motif) Receptor 2 Mediates Dendritic Cell Recruitment to the Human Colon but Is Not Responsible for Differences Observed in Dendritic Cell Subsets, Phenotype, and Function Between the Proximal and Distal Colon.

PubMed

Bernardo, David; Durant, Lydia; Mann, Elizabeth R; Bassity, Elizabeth; Montalvillo, Enrique; Man, Ripple; Vora, Rakesh; Reddi, Durga; Bayiroglu, Fahri; Fernández-Salazar, Luis; English, Nick R; Peake, Simon T C; Landy, Jon; Lee, Gui H; Malietzis, George; Siaw, Yi Harn; Murugananthan, Aravinth U; Hendy, Phil; Sánchez-Recio, Eva; Phillips, Robin K S; Garrote, Jose A; Scott, Paul; Parkhill, Julian; Paulsen, Malte; Hart, Ailsa L; Al-Hassi, Hafid O; Arranz, Eduardo; Walker, Alan W; Carding, Simon R; Knight, Stella C

2016-01-01

Most knowledge about gastrointestinal (GI)-tract dendritic cells (DC) relies on murine studies where CD103 + DC specialize in generating immune tolerance with the functionality of CD11b +/- subsets being unclear. Information about human GI-DC is scarce, especially regarding regional specifications. Here, we characterized human DC properties throughout the human colon. Paired proximal (right/ascending) and distal (left/descending) human colonic biopsies from 95 healthy subjects were taken; DC were assessed by flow cytometry and microbiota composition assessed by 16S rRNA gene sequencing. Colonic DC identified were myeloid (mDC, CD11c + CD123 - ) and further divided based on CD103 and SIRPα (human analog of murine CD11b) expression. CD103 - SIRPα + DC were the major population and with CD103 + SIRPα + DC were CD1c + ILT3 + CCR2 + (although CCR2 was not expressed on all CD103 + SIRPα + DC). CD103 + SIRPα - DC constituted a minor subset that were CD141 + ILT3 - CCR2 - . Proximal colon samples had higher total DC counts and fewer CD103 + SIRPα + cells. Proximal colon DC were more mature than distal DC with higher stimulatory capacity for CD4 + CD45RA + T-cells. However, DC and DC-invoked T-cell expression of mucosal homing markers (β7, CCR9) was lower for proximal DC. CCR2 was expressed on circulating CD1c + , but not CD141 + mDC, and mediated DC recruitment by colonic culture supernatants in transwell assays. Proximal colon DC produced higher levels of cytokines. Mucosal microbiota profiling showed a lower microbiota load in the proximal colon, but with no differences in microbiota composition between compartments. Proximal colonic DC subsets differ from those in distal colon and are more mature. Targeted immunotherapy using DC in T-cell mediated GI tract inflammation may therefore need to reflect this immune compartmentalization.

The possible role of CCR5Δ32 mutation in Crimean-Congo hemorrhagic fever infection.

PubMed

Rustemoglu, Aydin; Ekinci, Duygu; Nursal, Ayşe F; Barut, Sener; Duygu, Fazilet; Günal, Özgür

2017-10-01

Crimean-Congo hemorrhagic fever infection (CCHF) is a viral zoonosis. The pathogenesis of this disease has not been established so far, however, cytokines account for its progression and outcome. The aim of the present study is to investigate the association between chemokine receptor 5 (CCR5) gene Δ32 mutation and pathogenity, severity, and mortality of CCHF. This case-control study included 133 CCHF patients diagnosed by detection of CCHV RNA positivity and 97 healthy control subjects. CCR5 gene Δ32 mutation analyzed by polymerase chain reaction (PCR) method. The results were compared by using SPSS 16.0 and WINPEPI software's. The genotype distribution and allele frequency of the CCR5Δ32 were statistically different between the patients and the control group (P = 0.017; OR: 4.98 95% CI = 1.65-14.99 and P = 0.019; OR:4.76 95%CI = 1.30-17.50, respectively). CCR5/CCR5 (W/W) genotype and W allele of CCR5 gene were more common in patient group than in controls. There was no significant difference in severe and mild cases with regard to genotype distribution and allele distribution of CCR5Δ32 mutation (P >0.05). These results suggest that the CCR5 gene and its product might play a role in the pathogenesis of CCHF disease. Future studies will help us to uncover the exact role of CCR5 in the pathogenesis and prognosis of CCHF and to treat the disease. © 2017 Wiley Periodicals, Inc.

Chemokine receptor antagonists: part 2.

PubMed

Pease, James E; Horuk, Richard

2009-02-01

The first part of this two-part review discussed approaches to generating antagonists for some of the CC chemokine receptors, including CCR1, CCR2, CCR3, and CCR4. This second part of the series concludes the review by describing antagonists for CCR5, CCR8, CCR9, CXCR3, CXCR4, and promiscuous antagonists. Chemokine receptor antagonists have found mixed success as therapeutics. Although one antagonist--maraviroc, a CCR5 inhibitor to treat AIDS--has been registered as an approved drug, this is the only success so far. There have been many failures in the clinic and we discuss the idea of promiscuous receptor antagonists as an alternative approach.

Chemokine (C-C motif) receptor 5-using envelopes predominate in dual/mixed-tropic HIV from the plasma of drug-naive individuals.

PubMed

Irlbeck, David M; Amrine-Madsen, Heather; Kitrinos, Kathryn M; Labranche, Celia C; Demarest, James F

2008-07-31

HIV-1 utilizes CD4 and either chemokine (C-C motif) receptor 5 (CCR5) or chemokine (C-X-C motif) receptor 4 (CXCR4) to gain entry into host cells. Small molecule CCR5 antagonists are currently being developed for the treatment of HIV-1 infection. Because HIV-1 may also use CXCR4 for entry, the use of CCR5 entry inhibitors is controversial for patients harboring CCR5-using and CXCR4-using (dual/mixed-tropic) viruses. The goal of the present study was to determine the proportion of CCR5-tropic and CXCR4-tropic viruses in dual/mixed-tropic virus isolates from drug-naïve patients and the phenotypic and genotypic relationships of viruses that use CCR5 or CXCR4 or both. Fourteen antiretroviral-naive HIV-1-infected patients were identified as having population coreceptor tropism readout of dual/mixed-tropic viruses. Intrapatient comparisons of coreceptor tropism and genotype of env clones were conducted on plasma virus from each patient. Population HIV-1 envelope tropism and susceptibility to the CCR5 entry inhibitor, aplaviroc, were performed using the Monogram Biosciences Trofile Assay. Twelve env clones from each patient were analyzed for coreceptor tropism, aplaviroc sensitivity, genotype, and intrapatient phylogenetic relationships. Viral populations from antiretroviral-naive patients with dual/mixed-tropic virus are composed primarily of CCR5-tropic env clones mixed with those that use both coreceptors (R5X4-tropic) and, occasionally, CXCR4-tropic env clones. Interestingly, the efficiency of CXCR4 use by R5X4-tropic env clones varied with their genetic relationships to CCR5-tropic env clones from the same patient. These data show that the majority of viruses in these dual/mixed-tropic populations use CCR5 and suggest that antiretroviral-naive patients may benefit from combination therapy that includes CCR5 entry inhibitors.

Structure of CC Chemokine Receptor 5 with a Potent Chemokine Antagonist Reveals Mechanisms of Chemokine Recognition and Molecular Mimicry by HIV

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zheng, Yi; Han, Gye Won; Abagyan, Ruben

CCR5 is the primary chemokine receptor utilized by HIV to infect leukocytes, whereas CCR5 ligands inhibit infection by blocking CCR5 engagement with HIV gp120. To guide the design of improved therapeutics, we solved the structure of CCR5 in complex with chemokine antagonist [5P7]CCL5. Several structural features appeared to contribute to the anti-HIV potency of [5P7]CCL5, including the distinct chemokine orientation relative to the receptor, the near-complete occupancy of the receptor binding pocket, the dense network of intermolecular hydrogen bonds, and the similarity of binding determinants with the FDA-approved HIV inhibitor Maraviroc. Molecular modeling indicated that HIV gp120 mimicked the chemokinemore » interaction with CCR5, providing an explanation for the ability of CCR5 to recognize diverse ligands and gp120 variants. Our findings reveal that structural plasticity facilitates receptor-chemokine specificity and enables exploitation by HIV, and provide insight into the design of small molecule and protein inhibitors for HIV and other CCR5-mediated diseases.« less

Exceptional complex chromosomal rearrangements in three generations.

PubMed

Kartapradja, Hannie; Marzuki, Nanis Sacharina; Pertile, Mark D; Francis, David; Suciati, Lita Putri; Anggaratri, Helena Woro; Ambarwati, Debby Dwi; Idris, Firman Prathama; Lesmana, Harry; Trimarsanto, Hidayat; Paramayuda, Chrysantine; Harahap, Alida Roswita

2015-01-01

We report an exceptional complex chromosomal rearrangement (CCR) found in three individuals in a family that involves 4 chromosomes with 5 breakpoints. The CCR was ascertained in a phenotypically abnormal newborn with additional chromosomal material on the short arm of chromosome 4. Maternal karyotyping indicated that the mother carried an apparently balanced CCR involving chromosomes 4, 6, 11, and 18. Maternal transmission of the derivative chromosome 4 resulted in partial trisomy for chromosomes 6q and 18q and a partial monosomy of chromosome 4p in the proband. Further family studies found that the maternal grandmother carried the same apparently balanced CCR as the proband's mother, which was confirmed using the whole chromosome painting (WCP) FISH. High resolution whole genome microarray analysis of DNA from the proband's mother found no evidence for copy number imbalance in the vicinity of the CCR translocation breakpoints, or elsewhere in the genome, providing evidence that the mother's and grandmother's CCRs were balanced at a molecular level. This structural rearrangement can be categorized as an exceptional CCR due to its complexity and is a rare example of an exceptional CCR being transmitted in balanced and/or unbalanced form across three generations.

CCR5 receptor antagonists in preclinical to phase II clinical development for treatment of HIV

PubMed Central

Kim, Michelle B.; Giesler, Kyle E.; Tahirovic, Yesim A.; Truax, Valarie M.; Liotta, Dennis C.; Wilson, Lawrence J.

2018-01-01

Introduction The chemokine receptor CCR5 has garnered significant attention in recent years as a target to treat HIV infection largely due to the approval and success of the drug Maraviroc. The side effects and inefficiencies with other first generation agents led to failed clinical trials, prompting the development of newer CCR5 antagonists. Areas covered This review aims to survey the current status of ‘next generation’ CCR5 antagonists in the preclinical pipeline with an emphasis on emerging agents for the treatment of HIV infection. These efforts have culminated in the identification of advanced second-generation agents to reach the clinic and the dual CCR5/CCR2 antagonist Cenicriviroc as the most advanced currently in phase II clinical studies. Expert opinion The clinical success of CCR5 inhibitors for treatment of HIV infection has rested largely on studies of Maraviroc and a second-generation dual CCR5/CCR2 antagonist Cenicriviroc. Although research efforts identified several promising preclinical candidates, these were dropped during early clinical studies. Despite patient access to Maraviroc, there is insufficient enthusiasm surrounding its use as front-line therapy for treatment of HIV. The non-HIV infection related development activities for Maraviroc and Cenicriviroc may help drive future interests. PMID:27791451

mTOR signaling promotes foam cell formation and inhibits foam cell egress through suppressing the SIRT1 signaling pathway.

PubMed

Zheng, Haixiang; Fu, Yucai; Huang, Yusheng; Zheng, Xinde; Yu, Wei; Wang, Wei

2017-09-01

Atherosclerosis (AS) is a chronic immuno‑inflammatory disease accompanied by dyslipidemia. The authors previously demonstrated that sirtuin 1 (SIRT1) may prevent atherogenesis through influencing the liver X receptor/C‑C chemokine receptor type 7/nuclear factor‑κB (LXR‑CCR7/NF‑κB) signaling pathway. Previous studies have suggested a role for mammalian target of rapamycin (mTOR) signaling in the pathogenesis of cardiovascular diseases. The present study investigated the potential association between mTOR signaling and SIRT1‑LXR‑CCR7/NF‑κB signaling (SIRT1 signaling) in AS pathogenesis. To induce foam cell formation, U937 cells were differentiated into macrophages by exposure to phorbol 12‑myristate 13‑acetate (PMA) for 24 h, followed by treatment with palmitate and oxidized low density lipoprotein for a further 24 h. Oil red O staining revealed a large accumulation of lipid droplets present in foam cells. Western blot analysis demonstrated increased protein levels of phosphorylated (p)‑mTOR and its downstream factor p‑ribosomal protein S6 kinase (p70S6K). Reverse transcription‑quantitative polymerase chain reaction and western blot analyses additionally revealed decreased expression of SIRT1, LXRα and CCR7 and increased expression of NF‑κB and its downstream factor tumor necrosis factor‑α (TNF‑α) in an atherogenetic condition induced by lysophosphatidic acid (LPA). In addition, abundant lipid droplets accumulated in U937‑LPA‑treated foam cells. Rapamycin, an mTOR inhibitor, suppressed the expression and activity of mTOR and p70S6K, however enhanced expression of SIRT1, LXRα, and CCR7. Conversely, rapamycin deceased TNF‑α and NF‑κB activity, the latter of which was further confirmed by immunofluorescence analysis demonstrating increased levels of NF‑κB present in the cytoplasm compared with the nucleus. The findings of the present study suggest that mTOR signaling promotes foam cell formation and inhibits foam cell egress via suppression of SIRT1 signaling.

CCL2 binding is CCR2 independent in primary adult human astrocytes.

PubMed

Fouillet, A; Mawson, J; Suliman, O; Sharrack, B; Romero, I A; Woodroofe, M N

2012-02-09

Chemokines are low relative molecular mass proteins, which have chemoattractant actions on many cell types. The chemokine, CCL2, has been shown to play a major role in the recruitment of monocytes in central nervous system (CNS) lesions in multiple sclerosis (MS). Since resident astrocytes constitute a major source of chemokine synthesis including CCL2, we were interested to assess the regulation of CCL2 by astrocytes. We showed that CCL2 bound to the cell surface of astrocytes and binding was not modulated by inflammatory conditions. However, CCR2 protein was not detected nor was activation of the classical CCR2 downstream signaling pathways. Recent studies have shown that non-signaling decoy chemokine receptors bind and modulate the expression of chemokines at site of inflammation. Here, we show that the D6 chemokine decoy receptor is constitutively expressed by primary human adult astrocytes at both mRNA and protein level. In addition, CCL3, which binds to D6, but not CCL19, which does not bind to D6, displaced CCL2 binding to astrocytes; indicating that CCL2 may bind to this cell type via the D6 receptor. Our results suggest that CCL2 binding to primary adult human astrocytes is CCR2-independent and is likely to be mediated via the D6 decoy chemokine receptor. Therefore we propose that astrocytes are implicated in both the establishment of chemokine gradients for the migration of leukocytes into and within the CNS and in the regulation of CCL2 levels at inflammatory sites in the CNS. Copyright © 2011 Elsevier B.V. All rights reserved.

The effect of pharmacological PI3Kγ inhibitor on eotaxin-induced human eosinophil functions.

PubMed

Saito, Yukiko; Takeda, Masahide; Nishikawa, Junko; Konno, Yasunori; Tamaki, Mami; Itoga, Masamichi; Kobayashi, Yoshiki; Moritoki, Yuki; Ito, Wataru; Chihara, Junichi; Ueki, Shigeharu

2014-04-01

Asthma is characterized by chronic inflammation caused by activation of immune cells including Th2 lymphocytes and eosinophils. Phosphoinositide 3-kinase (PI3K) γ deficient asthmatic mice did not develop lung eosinophilia, although the detailed mechanisms are not well known. A CC chemokine eotaxin (CCL11) plays a prominent role in developing eosinophilic inflammation through CCR3. In this study, we tested the roles of PI3Kγ in eotaxin-induced eosinophil functions using a pharmacological inhibitor. Human peripheral blood eosinophils were isolated by CD16-negative selection method. The effect of AS605240, synthetic PI3Kγ inhibitor on eotaxin-induced adhesion, chemotaxis, and degranulation were studied using intracellular adhesion molecule-1 (ICAM-1)-coated plates, Boyden chamber system, ELISA for eosinophil-derived neurotoxin (EDN) levels in the culture supernatant, respectively. CCR3 expression levels and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation were assessed by flowcytometry. Involvement of PI3Kγ in spontaneous apoptosis was studied using flowcytometry. Although AS605240 did not affect the eosinophil spontaneous apoptosis, eotaxin-induced chemotaxis, adhesion to ICAM-1 coated plate, and EDN release were inhibited by AS605240. AS605240 also inhibited the eotaxin-induced ERK1/2 phosphorylation without down-regulation of surface CCR3 expression. These results indicate that PI3Kγ inhibitor attenuates eotaxin-induced eosinophil functions by suppressing the downstream signaling of CCR3 without significant cytotoxicity. PI3Kγ plays an important role in the development of eosinophilic inflammation and blockade of PI3Kγ might be a therapeutic strategy for treatment of eosinophil-related diseases including asthma. Crown Copyright © 2013. Published by Elsevier Ltd. All rights reserved.

CCR5 chemokine receptor gene polymorphisms in ocular toxoplasmosis.

PubMed

de Faria Junior, Geraldo M; Ayo, Christiane M; de Oliveira, Amanda P; Lopes, Alessandro G; Frederico, Fábio B; Silveira-Carvalho, Aparecida P; Previato, Mariana; Barbosa, Amanda P; Murata, Fernando H A; de Almeida Junior, Gildásio Castello; Siqueira, Rubens Camargo; de Mattos, Luiz C; Brandão de Mattos, Cinara C

2018-02-01

CC chemokine receptor type 5 (CCR5) is a chemokine receptor that influences the immune response to infectious and parasitic diseases. This study aimed to determine whether the CCR5Δ32 and CCR5 59029 A/G polymorphisms are associated with the development of ocular toxoplasmosis in humans. Patients with positive serology for Toxoplasma gondii were analyzed and grouped as 'with ocular toxoplasmosis' (G1: n=160) or 'without ocular toxoplasmosis' (G2: n=160). A control group (G3) consisted of 160 individuals with negative serology. The characterization of the CCR5Δ32 and CCR5 59029 A/G polymorphisms was by PCR and by PCR-RFLP, respectively. The difference between the groups with respect to the mean age (G1: mean age: 47.3, SD±19.3, median: 46 [range: 18-95]; G2: mean age: 61.3, SD±13.7, median: 61 [range: 21-87]; G3: mean age: 38.8, SD±17.9, median: 34 [range: 18-80]) was statistically significant (G1 vs.G2: p-value <0.0001; t=7.21; DF=318; G1 vs.G3: p-value <0.0001; t=4.32; DF=318; G2 vs. G3: p-value <0.0001; t=9.62; DF=318). The Nagelkerke r 2 value was 0.040. There were statistically significant differences for the CCR5/CCR5 (p-value=0.008; OR=0.261), AA (p-value=0.007; OR=2.974) and AG genotypes (p-value=0.018; OR=2.447) between G1 and G2. Individuals with the CCR5/CCR5 genotype and simultaneously the CCR5-59029 AA or AG genotypes have a greater risk of developing ocular toxoplasmosis (4% greater), which may be associated with a strong and persistent inflammatory response in ocular tissue. Copyright © 2017 Elsevier B.V. All rights reserved.

In vivo evolution of HIV-1 co-receptor usage and sensitivity to chemokine-mediated suppression.

PubMed

Scarlatti, G; Tresoldi, E; Björndal, A; Fredriksson, R; Colognesi, C; Deng, H K; Malnati, M S; Plebani, A; Siccardi, A G; Littman, D R; Fenyö, E M; Lusso, P

1997-11-01

Following the identification of the C-C chemokines RANTES, MIP-1alpha and MIP-1beta as major human immunodeficiency virus (HIV)-suppressive factors produced by CD8+ T cells, several chemokine receptors were found to serve as membrane co-receptors for primate immunodeficiency lentiretroviruses. The two most widely used co-receptors thus far recognized, CCR5 and CXCR4, are expressed by both activated T lymphocytes and mononuclear phagocytes. CCR5, a specific RANTES, MIP-1alpha and MIP-1 receptor, is used preferentially by non-MT2-tropic HIV-1 and HIV-2 strains and by simian immunodeficiency virus (SIV), whereas CXCR4, a receptor for the C-X-C chemokine SDF-1, is used by MT2-tropic HIV-1 and HIV-2, but not by SIV. Other receptors with a more restricted cellular distribution, such as CCR2b, CCR3 and STRL33, can also function as co-receptors for selected viral isolates. The third variable region (V3) of the gp120 envelope glycoprotein of HIV-1 has been fingered as a critical determinant of the co-receptor choice. Here, we document a consistent pattern of evolution of viral co-receptor usage and sensitivity to chemokine-mediated suppression in a longitudinal follow-up of children with progressive HIV-1 infection. Viral isolates obtained during the asymptomatic stages generally used only CCR5 as a co-receptor and were inhibited by RANTES, MIP-1alpha and MIP-1beta, but not by SDF-1. By contrast, the majority of the isolates derived after the progression of the disease were resistant to C-C chemokines, having acquired the ability to use CXCR4 and, in some cases, CCR3, while gradually losing CCR5 usage. Surprisingly, most of these isolates were also insensitive to SDF-1, even when used in combination with RANTES. An early acquisition of CXCR4 usage predicted a poor prognosis. In children who progressed to AIDS without a shift to CXCR4 usage, all the sequential isolates were CCR5-dependent but showed a reduced sensitivity to C-C chemokines. Discrete changes in the V3 domain of gp120 were associated with the loss of sensitivity to C-C chemokines and the shift in co-receptor usage. These results suggest an adaptive evolution of HIV-1 in vivo, leading to escape from the control of the antiviral C-C chemokines.

Dynamic Modulation of Expression of Lentiviral Restriction Factors in Primary CD4+ T Cells following Simian Immunodeficiency Virus Infection.

PubMed

Rahmberg, Andrew R; Rajakumar, Premeela A; Billingsley, James M; Johnson, R Paul

2017-04-01

Although multiple restriction factors have been shown to inhibit HIV/SIV replication, little is known about their expression in vivo Expression of 45 confirmed and putative HIV/SIV restriction factors was analyzed in CD4 + T cells from peripheral blood and the jejunum in rhesus macaques, revealing distinct expression patterns in naive and memory subsets. In both peripheral blood and the jejunum, memory CD4 + T cells expressed higher levels of multiple restriction factors compared to naive cells. However, relative to their expression in peripheral blood CD4 + T cells, jejunal CCR5 + CD4 + T cells exhibited significantly lower expression of multiple restriction factors, including APOBEC3G , MX2 , and TRIM25 , which may contribute to the exquisite susceptibility of these cells to SIV infection. In vitro stimulation with anti-CD3/CD28 antibodies or type I interferon resulted in upregulation of distinct subsets of multiple restriction factors. After infection of rhesus macaques with SIVmac239, the expression of most confirmed and putative restriction factors substantially increased in all CD4 + T cell memory subsets at the peak of acute infection. Jejunal CCR5 + CD4 + T cells exhibited the highest levels of SIV RNA, corresponding to the lower restriction factor expression in this subset relative to peripheral blood prior to infection. These results illustrate the dynamic modulation of confirmed and putative restriction factor expression by memory differentiation, stimulation, tissue microenvironment and SIV infection and suggest that differential expression of restriction factors may play a key role in modulating the susceptibility of different populations of CD4 + T cells to lentiviral infection. IMPORTANCE Restriction factors are genes that have evolved to provide intrinsic defense against viruses. HIV and simian immunodeficiency virus (SIV) target CD4 + T cells. The baseline level of expression in vivo and degree to which expression of restriction factors is modulated by conditions such as CD4 + T cell differentiation, stimulation, tissue location, or SIV infection are currently poorly understood. We measured the expression of 45 confirmed and putative restriction factors in primary CD4 + T cells from rhesus macaques under various conditions, finding dynamic changes in each state. Most dramatically, in acute SIV infection, the expression of almost all target genes analyzed increased. These are the first measurements of many of these confirmed and putative restriction factors in primary cells or during the early events after SIV infection and suggest that the level of expression of restriction factors may contribute to the differential susceptibility of CD4 + T cells to SIV infection. Copyright © 2017 American Society for Microbiology.

HIV coreceptor phenotyping in the clinical setting.

PubMed

Low, Andrew J; Swenson, Luke C; Harrigan, P Richard

2008-01-01

The introduction of CCR5 antagonists increases the options available for constructing antiretroviral regimens. However, this option is coupled with the caveat that patients should be tested for HIV coreceptor tropism prior to initiating CCR5 antagonist-based therapy. Failure to screen for CXCR4 usage increases the risk of using an ineffective drug, thus reducing the likelihood of viral suppression and increasing their risk for developing antiretroviral resistance. This review discusses current and future methods of determining HIV tropism, with a focus on their utility in the clinical setting for screening purposes. Some of these methods include recombinant phenotypic tests, such as the Monogram Trofile assay, as well as genotype-based predictors, heteroduplex tracking assays, and flow cytometry based methods. Currently, the best evidence supports the use of phenotypic methods, although other methods of screening for HIV coreceptor usage prior to the administration of CCR5 antagonists may reduce costs and increase turnaround time over phenotypic methods. The presence of low levels of X4 virus is a challenge to all assay methods, resulting in reduced sensitivity in clinical, patient-derived samples when compared to clonally derived samples. Gaining a better understanding of the output of these assays and correlating them with clinical progression and therapy response will provide some indication on how both genotype-based, and phenotypic assays for determining HIV coreceptor usage can be improved. In addition, leveraging new technologies capable of detecting low-level minority species may provide the most significant advances in ensuring that individuals with low levels of dual/mixed tropic virus are not inadvertently prescribed CCR5 antagonists.

Low-Dose Radiation Promotes Dendritic Cell Migration and IL-12 Production via the ATM/NF-KappaB Pathway.

PubMed

Yu, Nan; Wang, Sinian; Song, Xiujun; Gao, Ling; Li, Wei; Yu, Huijie; Zhou, Chuanchuan; Wang, Zhenxia; Li, Fengsheng; Jiang, Qisheng

2018-04-01

For dendritic cells (DCs) to initiate an immune response, their ability to migrate and to produce interleukin-12 (IL-12) is crucial. It has been previously shown that low-dose radiation (LDR) promoted IL-12 production by DCs, resulting in increased DC activity that contributed to LDR hormesis in the immune system. However, the molecular mechanism of LDR-induced IL-12 production, as well as the effect of LDR on DC migration capacity require further elucidation. Using the JAWSII immortalized mouse dendritic cell line, we showed that in vitro X-ray irradiation (0.2 Gy) of DCs significantly increased DC migration and IL-12 production, and upregulated CCR7. The neutralizing antibody against CCR7 has been shown to abolish LDR-enhanced DC migration, demonstrating that CCR7 mediates LDR-promoting DC migration. We identified nuclear factor kappaB (NF-κB) as the central signaling pathway that mediated LDR-enhanced expression of IL-12 and CCR7 based on findings that 0.2 Gy X-ray irradiation activated NF-κB, showing increased nuclear p65 translocation and NF-κB DNA-binding activity, while an NF-κB inhibitor blocked LDR-enhanced expression of IL-12 and CCR7, as well as DC migration. Finally, we demonstrated that 0.2 Gy X-ray irradiation promoted ATM phosphorylation and reactive oxygen species generation; however, only the ATM inhibitor abolished the LDR-induced NF-κB-mediated expression of IL-12 and CCR7. Altogether, our data show that exposure to LDR resulted in a hormetic effect on DCs regarding CCR7-mediated migration and IL-12 production by activating the ATM/NF-κB pathway.

EXPRESSION, PURIFICATION AND IN VITRO FUNCTIONAL RECONSTITUTION OF THE CHEMOKINE RECEPTOR CCR1

PubMed Central

Allen, Samantha J.; Ribeiro, Sofia; Horuk, Richard; Handel, Tracy M.

2009-01-01

Chemokine receptors are a specific class of G protein-coupled receptors (GPCRs) that control cell migration associated with routine immune surveillance, inflammation and development. In addition to their roles in normal physiology, these receptors and their ligands are involved in a large number of inflammatory diseases, cancer and AIDS, making them prime therapeutic targets in the pharmaceutical industry. Like other GPCRs, a significant obstacle in determining structures and characterizing mechanisms of activation has been the difficulty in obtaining high levels of pure, functional receptor. Here we describe a systematic effort to express the chemokine receptor CCR1 in mammalian cells, and to purify and reconstitute it in functional form. The highest expression levels were obtained using an inducible HEK293 system. The receptor was purified using a combination of N- (StrepII or Hemagglutinin) and C-terminal (His8) affinity tags. Function was assessed by ligand binding using a novel fluorescence polarization assay with fluorescein-labeled chemokine. A strict dependence of function on the detergent composition was observed, as solubilization of CCR1 in n-dodecyl-β-D-maltopyranoside/cholesteryl hemisuccinate yielded functional receptor with a Kd of 21 nM for the chemokine CCL14, whereas it was non-functional in phosphocholine detergents. Differences in function were observed despite the fact that both these detergent types maintained the receptor in a state characterized by monomers and small oligomers, but not large aggregates. While optimization is still warranted, yields of ~ 0.1–0.2mgs of pure functional receptor per 109 cells will permit biophysical studies of this medically important receptor. PMID:19275940

Isoleucine/leucine2 is essential for chemoattractant activity of beta-defensin Defb14 through chemokine receptor 6.

PubMed

Tyrrell, Christine; De Cecco, Martin; Reynolds, Natalie L; Kilanowski, Fiona; Campopiano, Dominic; Barran, Perdita; Macmillan, Derek; Dorin, Julia R

2010-03-01

Beta-defensins are both antimicrobial and able to chemoattract various immune cells including immature dendritic cells and CD4 T cells through CCR6. They are short, cationic peptides with a highly conserved six-cysteine motif. It has been shown that only the fifth cysteine is critical for chemoattraction of cells expressing CCR6. In order to identify other residues essential for functional interaction with CCR6 we used a library of peptide deletion derivatives based on Defb14. Loss of the initial two amino acids from the Defb14-1C(V) derivative destroys its ability to chemoattract cells expressing CCR6. As the second amino acid is an evolutionarily conserved leucine, we make full-length Defb14-1C(V) peptides with substitution of the leucine(2) for glycine (L2G), lysine (L2K) or isoleucine (L2I). Defb14-1C(V) L2G and L2K and are unable to chemoattract CCR6 expressing cells but the semi-conservative change L2I has activity. By circular dichroism spectroscopy we can see no evidence for a significant change in secondary structure as a consequence of these substitutions and so cannot attribute loss of chemotactic activity with disruption of the N-terminal helix. We conclude that isoleucine/leucine in the N-terminal alpha-helix region of this beta-defensin is essential for CCR6-mediated chemotaxis. Copyright 2009 Elsevier Ltd. All rights reserved.

Oral CCR5 inhibitors: will they make it through?

PubMed

Biswas, Priscilla; Nozza, Silvia; Scarlatti, Gabriella; Lazzarin, Adriano; Tambussi, Giuseppe

2006-05-01

The therapeutic armamentarium against HIV has recently gained a drug belonging to a novel class of antiretrovirals, the entry inhibitors. The last decade has driven an in-depth knowledge of the HIV entry process, unravelling the multiple engagements of the HIV envelope proteins with the cellular receptorial complex that is composed of a primary receptor (CD4) and a co-receptor (CCR5 or CXCR4). The vast majority of HIV-infected subjects exhibit biological viral variants that use CCR5 as a co-receptor. Individuals with a mutated CCR5 gene, both homo- and heterozygotes, appear to be healthy. For these and other reasons, CCR5 represents an appealing target for treatment intervention, although certain challenges can not be ignored. Promising small-molecule, orally bioavailable CCR5 antagonists are under development for the treatment of HIV-1 infection.

HIV blocking antibodies following immunisation with chimaeric peptides coding a short N-terminal sequence of the CCR5 receptor.

PubMed

Chain, Benjamin M; Noursadeghi, Mahdad; Gardener, Michelle; Tsang, Jhen; Wright, Edward

2008-10-23

The chemokine receptor CCR5 is required for cellular entry by many strains of HIV, and provides a potential target for molecules, including antibodies, designed to block HIV transmission. This study investigates a novel approach to stimulate antibodies to CCR5. Rabbits were immunised with chimaeric peptides which encode a short fragment of the N-terminal sequence of CCR5, as well as an unrelated T cell epitope from Tetanus toxoid. Immunisation with these chimaeric peptides generates a strong antibody response which is highly focused on the N-terminal CCR5 sequence. The antibody to the chimaeric peptide containing an N-terminal methionine also recognises the full length CCR5 receptor on the cell surface, albeit at higher concentrations. Further comparison of binding to intact CCR5 with binding to CCR5 peptide suggest that the receptor specific antibody generated represents a very small fragment of the total anti-peptide antibody. These findings are consistent with the hypothesis that the N-terminal peptide in the context of the intact receptor has a different structure to that of the synthetic peptide. Finally, the antibody was able to block HIV infection of macrophages in vitro. Thus results of this study suggest that N-terminal fragments of CCR5 may provide potential immunogens with which to generate blocking antibodies to this receptor, while avoiding the dangers of including T cell auto-epitopes.

HIV blocking antibodies following immunisation with chimaeric peptides coding a short N-terminal sequence of the CCR5 receptor

PubMed Central

Chain, Benjamin M.; Noursadeghi, Mahdad; Gardener, Michelle; Tsang, Jhen; Wright, Edward

2008-01-01

The chemokine receptor CCR5 is required for cellular entry by many strains of HIV, and provides a potential target for molecules, including antibodies, designed to block HIV transmission. This study investigates a novel approach to stimulate antibodies to CCR5. Rabbits were immunised with chimaeric peptides which encode a short fragment of the N-terminal sequence of CCR5, as well as an unrelated T cell epitope from Tetanus toxoid. Immunisation with these chimaeric peptides generates a strong antibody response which is highly focused on the N-terminal CCR5 sequence. The antibody to the chimaeric peptide containing an N-terminal methionine also recognises the full length CCR5 receptor on the cell surface, albeit at higher concentrations. Further comparison of binding to intact CCR5 with binding to CCR5 peptide suggest that the receptor specific antibody generated represents a very small fragment of the total anti-peptide antibody. These findings are consistent with the hypothesis that the N-terminal peptide in the context of the intact receptor has a different structure to that of the synthetic peptide. Finally, the antibody was able to block HIV infection of macrophages in vitro. Thus results of this study suggest that N-terminal fragments of CCR5 may provide potential immunogens with which to generate blocking antibodies to this receptor, while avoiding the dangers of including T cell auto-epitopes. PMID:18765264

Short communication: identification of the conformational requirement for the specificities of coreceptors for human and simian immunodeficiency viruses.

PubMed

Shimizu, Nobuaki; Tanaka, Atsushi; Jinno-Oue, Atsushi; Mori, Takahisa; Ohtsuki, Takahiro; Hoshino, Hiroo

2010-03-01

More than 10 G protein-coupled receptors (GPCRs) work as coreceptors for human and simian immunodeficiency viruses (HIVs/SIVs); however, structural features critical for coreceptor activity have not been identified. Our objective was to elucidate the structural requirement of coreceptor activities. Amino-terminal regions (NTRs), extracellular loops (ECLs), and the undecapeptidyl arch (UPA) in the second ECL have been shown to be important for coreceptor function. We made chimeric coreceptors for these regions between CCR5 and GPR1, which is genetically distant from CCR5, and analyzed their activities. The coreceptor activity and specificity of CCR5 were maintained when its NTR or UPA was replaced with GPR1. In contrast, the GPR1 chimera with CCR5 NTR was used by HIV-1 strains that can use only CCR5, but not both CCR5 and CXCR4, or GPR1. GPR1 chimera with CCR5 UPA almost lost activity. All ECL chimeras could hardly maintain activity. Thus, CCR5 is more flexibly acceptable to heterologous NTR and UPA than GPR1, suggesting the existence of conformational differences made by the integration of multiple extracellular regions. This conformation may specifically interact with HIV-1 in a strain-dependent manner. Identification of a factor that is critical to make this conformation will contribute to understanding the mechanism of coreceptor function of GPCRs. For this, the coreceptor activity of GPR1, which is genetically distant from CCR5, will be a useful tool.

CCR4 frameshift mutation identifies a distinct group of adult T cell leukaemia/lymphoma with poor prognosis.

PubMed

Yoshida, Noriaki; Miyoshi, Hiroaki; Kato, Takeharu; Sakata-Yanagimoto, Mamiko; Niino, Daisuke; Taniguchi, Hiroaki; Moriuchi, Yukiyoshi; Miyahara, Masaharu; Kurita, Daisuke; Sasaki, Yuya; Shimono, Joji; Kawamoto, Keisuke; Utsunomiya, Atae; Imaizumi, Yoshitaka; Seto, Masao; Ohshima, Koichi

2016-04-01

Adult T cell leukaemia/lymphoma (ATLL) is an intractable T cell neoplasm caused by human T cell leukaemia virus type 1. Next-generation sequencing-based comprehensive mutation studies have revealed recurrent somatic CCR4 mutations in ATLL, although clinicopathological findings associated with CCR4 mutations remain to be delineated. In the current study, 184 cases of peripheral T cell lymphoma, including 113 cases of ATLL, were subjected to CCR4 mutation analysis. This sequence analysis identified mutations in 27% (30/113) of cases of ATLL and 9% (4/44) of cases of peripheral T cell lymphoma not otherwise specified. Identified mutations included nonsense (NS) and frameshift (FS) mutations. No significant differences in clinicopathological findings were observed between ATLL cases stratified by presence of CCR4 mutation. All ATLL cases with CCR4 mutations exhibited cell-surface CCR4 positivity. Semi-quantitative CCR4 protein analysis of immunohistochemical sections revealed higher CCR4 expression in cases with NS mutations of CCR4 than in cases with wild-type (WT) CCR4. Furthermore, among ATLL cases, FS mutation was significantly associated with a poor prognosis, compared with NS mutation and WT CCR4. These results suggest that CCR4 mutation is an important determinant of the clinical course in ATLL cases, and that NS and FS mutations of CCR4 behave differently with respect to ATLL pathophysiology. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Role of the frequency of blood CD4{sup +} CXCR5{sup +} CCR6{sup +} T cells in autoimmunity in patients with Sjoegren's syndrome

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Xue-yi; Wu, Zhen-biao; Ding, Jin

2012-06-01

Highlights: Black-Right-Pointing-Pointer The frequency of CD4{sup +} CXCR5{sup +} CCR6{sup +} T cells increased in pSS patients and positively correlated with autoantibodies in the blood. Black-Right-Pointing-Pointer CD4{sup +} CXCR5{sup +} CCR6{sup +} T cells in blood invariably coexpressed PD-1, ICOS, CD40L, Bcl-6 and secreted IL-21 after stimulated by PHA. Black-Right-Pointing-Pointer CD4{sup +} CXCR5{sup +} CCR6{sup +} Tfh cells in blood may be suitable biomarkers for the evaluation of the active immune stage of pSS patients. -- Abstract: The blood CD4{sup +} CXCR5{sup +} T cells, known as 'circulating' Tfh, have been shown to efficiently induce naieve B cells to producemore » immunoglobulin. They play an important role in certain autoimmune diseases. In the present study, we show for the first time that the frequency of CD4{sup +} CXCR5{sup +} T cells is increased in pSS patients and positively correlated with autoantibodies in the blood. The concentration of Th17-like subsets (CD4{sup +} CXCR5{sup +} CCR6{sup +}) in pSS patients was found to be significantly higher than in healthy controls. Functional assays showed that activated Th17-like subtypes in the blood display the key features of Tfh cells, including invariably coexpressed PD-1, ICOS, CD40L and IL-21. Th17 subsets were found to highly express Bcl-6 protein and Th1 and Th2 were not. Bcl-6 is believed to be a master transforming factor for Tfh cell differentiation and facilitate B cell proliferation and somatic hypermutation within the germinal center. These data indicate that Th17 subsets of CD4{sup +} CXCR5{sup +} T cells in the blood may participate in the antibody-related immune responses and that high frequency of CD4{sup +} CXCR5{sup +} CCR6{sup +} Tfh cells in blood may be suitable biomarkers for the evaluation of the active immune stage of pSS patients. It might provide insights into the pathogenesis and perhaps help researchers identify novel therapeutic targets for pSS.« less

Molecular and Bioenergetic Differences between Cells with African versus European Inherited Mitochondrial DNA Haplogroups: Implications for Population Susceptibility to Diseases

PubMed Central

Kenney, M. Cristina; Chwa, Marilyn; Atilano, Shari R.; Falatoonzadeh, Payam; Ramirez, Claudio; Malik, Deepika; Tarek, Mohamed; Cáceres del Carpio, Javier; Nesburn, Anthony B.; Boyer, David S.; Kuppermann, Baruch D.; Vawter, Marquis P.; Jazwinski, S. Michal; Miceli, Michael V.; Wallace, Douglas C.; Udar, Nitin

2015-01-01

The geographic origins of populations can be identified by their maternally inherited mitochondrial DNA (mtDNA) haplogroups. This study compared human cybrids (cytoplasmic hybrids), which are cell lines with identical nuclei but mitochondria from different individuals with mtDNA from either the H haplogroup or L haplogroup backgrounds. The most common European haplogroup is H while individuals of maternal African origin are of the L haplogroup. Despite lower mtDNA copy numbers, L cybrids had higher expression levels for nine mtDNA-encoded respiratory complex genes, decreased ATP turnover rates and lower levels of ROS production, parameters which are consistent with more efficient oxidative phosphorylation. Surprisingly, GeneChip arrays showed that the L and H cybrids had major differences in expression of genes of the canonical complement system (5 genes), dermatan/chondroitin sulfate biosynthesis (5 genes) and CCR3 signaling (9 genes). Quantitative nuclear gene expression studies confirmed that L cybrids had (a) lower expression levels of complement pathway and innate immunity genes and (b) increased levels of inflammation-related signaling genes, which are critical in human diseases. Our data support the hypothesis that mtDNA haplogroups representing populations from different geographic origins may play a role in differential susceptibilities to diseases. PMID:24200652

Leukotactin-1/CCL15 induces cell migration and differentiation of human eosinophilic leukemia EoL-1 cells through PKCdelta activation.

PubMed

Lee, Ji-Sook; Kim, In Sik

2010-06-01

Leukotactin-1 (Lkn-1)/CCL15 is a CC chemokine that binds to the CCR1 and CCR3. Lkn-1 functions as an essential factor in the migration of monocytes, lymphocytes, and neutrophils. Although eosinophils express both receptors, the role of Lkn-1 in immature eosinophils remains to be elucidated. In this present study, we investigated the contribution of the CCR1-binding chemokines to chemotactic activity and in the differentiation in the human eosinophilic leukemia cell line EoL-1. Lkn-1 induced the stronger migration of EoL-1 cells than other CCR1-binding chemokines such as RANTES/CCL5, MIP-1alpha/CCL3 and HCC-4/CCL16. Lkn-1-induced chemotaxis was inhibited by pertussis toxin, an inhibitor of G(i)/G(o) protein; U73122, an inhibitor of phospholipase C and rottlerin, an inhibitor of protein kinase C delta (PKCdelta). Lkn-1 increased PKCdelta activity, which was partially blocked by the pertussis toxin and U73122. Lkn-1 enhanced the butyric acid-induced differentiation via PKCdelta after binding to the increased CCR1 because Lkn-1 caused EoL-1 cells to change morphologically into mature eosinophil-like cells. Likewise, Lkn-1 increased the expression of both eosinophil peroxidase (EPO) and the major basic protein (MBP). PKCdelta activation due to Lkn-1 is involved in migration, as well as the butyric acid-induced differentiation. This finding contributes to an understanding of CC chemokines in eosinophil biology and to the development of novel therapies for the treatment of eosinophilic disorders. This study suggests the pivotal roles of Lkn-1 in the regulation of the movement and development of eosinophils.

Oxidized Lipids and Lysophosphatidylcholine Induce the Chemotaxis, Up-Regulate the Expression of CCR9 and CXCR4 and Abrogate the Release of IL-6 in Human Monocytes

PubMed Central

Rolin, Johannes; Vego, Heidi; Maghazachi, Azzam A.

2014-01-01

Lipids through regulation of chronic inflammation play key roles in the development of various diseases. Here, we report that a mixed population of human primary monocytes migrated towards LPC, as well as oxidized linoleic acid isoforms 9-S-HODE, 9-R-HODE and 13-R-HODE. Incubation with 9-R-HODE, 13-R-HODE and LPC resulted in increased expression of CXCR4, the receptor for SDF-1α/CXCL12, correlated with increased monocyte migration towards SDF-1α/CXCL12. Further, we report increased expression of CCR9, the receptor for TECK/CCL25, after stimulation with these lipids. Upon examining the migratory response towards TECK/CCL25, it was observed that an increase in CCR9 expression upon pre-treatment with 9-S-HODE, 9-R-HODE, 13-R-HODE and LPC resulted in increased migration of monocytes expressing CCR9. Only LPC but not any other lipid examined increased the influx of intracellular Ca2+ in monocytes. Finally, 9-S-HODE, 9-R-HODE, 13-R-HODE, or LPC inhibited the release of IL-6 from monocytes suggesting that these lipids may play important role in controlling inflammatory responses. PMID:25251539

Fluctuations in Blood Marginal Zone B-Cell Frequencies May Reflect Migratory Patterns Associated with HIV-1 Disease Progression Status

PubMed Central

Poudrier, Johanne; Roger, Michel

2016-01-01

We have previously shown that overexpression of BLyS/BAFF was associated with increased relative frequencies of innate “precursor” marginal zone (MZ)-like B-cells in the blood of HIV-1-infected rapid and classic progressors. However, along with relatively normal BLyS/BAFF expression levels, these cells remain unaltered in elite-controllers (EC), rather, percentages of more mature MZ-like B-cells are decreased in the blood of these individuals. Fluctuations in frequencies of blood MZ-like B-cell populations may reflect migratory patterns associated with disease progression status, suggesting an important role for these cells in HIV-1 pathogenesis. We have therefore longitudinally measured plasma levels of B-tropic chemokines by ELISA-based technology as well as their ligands by flow-cytometry on blood B-cell populations of HIV-1-infected individuals with different rates of disease progression and uninfected controls. Migration potential of B-cell populations from these individuals were determined by chemotaxis assays. We found important modulations of CXCL13-CXCR5, CXCL12-CXCR4/CXCR7, CCL20-CCR6 and CCL25-CCR9 chemokine-axes and increased cell migration patterns in HIV progressors. Interestingly, frequencies of CCR6 expressing cells were significantly elevated within the precursor MZ-like population, consistent with increased migration in response to CCL20. Although we found little modulation of chemokine-axes in EC, cell migration was greater than that observed for uninfected controls, especially for MZ-like B-cells. Overall the immune response against HIV-1 may involve recruitment of MZ-like B-cells to peripheral sites. Moreover, our findings suggest that “regulated” attraction of these cells in a preserved BLyS/BAFF non-inflammatory environment, such as encountered in EC could be beneficial to the battle and even control of HIV. PMID:27203285

Follicular helper T cells in peripheral blood of patients with rheumatoid arthritis.

PubMed

Costantino, Alicia Beatriz; Acosta, Cristina Del Valle; Onetti, Laura; Mussano, Eduardo; Cadile, Ignacio Isaac; Ferrero, Paola Virginia

Rheumatoid arthritis (RA) is a chronic autoimmune disease that is characterized by the presence of different autoantibodies such as rheumatoid factor (RF) and anti-citrullinated protein antibodies. CD4T cells expressing CXCR5, referred as follicular helper T cells (Tfh), collaborate with B cells to produce antibodies. Differential expression of CXCR3 and CCR6 within CD4 + CXCR5 + T cells defines three mayor subsets: CXCR3 + CCR6 - (Tfh1), CXCR3 - CCR6 - (Tfh2) and CXCR3 - CCR6 + (Tfh17). The aim of the study was to assess whether there is an association between the percentage of these cells and RA and whether there is a correlation with disease activity. Twenty-four RA patients, 22 healthy controls (HC) and 16 undifferentiated arthritis (UA) patients were included. Percentage of CD4 + CXCR5 + T cells and their subsets were analyzed by flow cytometry. No differences were found in the percentages of CD4 + CXCR5 + T cells in the comparison of RA vs HC or RA vs UA patients. Tfh1, Tfh2 and Tfh17 subsets showed no differences either. There was no correlation between CD4 + CXCR5 + T cells, Tfh1, Tfh2 and Tfh17, and Disease Activity Score in twenty-eight joints (DAS28) or erythrocyte sedimentation rate. Surprisingly, there was a positive correlation between Tfh17 cells and C-reactive protein. Finally, there was no correlation between CD4 + CXCR5 + T cells, or their subsets, and anti-mutated citrullinated vimentin, or between the cells and RF. There were no differences between the percentages of CD4 + CXCR5 + T cells and their subsets in peripheral blood of RA patients and the percentages of cells in the control groups. This finding does not rule out a pathogenic role of these cells in the development and activity of RA. Copyright © 2016 Elsevier España, S.L.U. and Sociedad Española de Reumatología y Colegio Mexicano de Reumatología. All rights reserved.

Co-Introduced Functional CCR2 Potentiates In Vivo Anti-Lung Cancer Functionality Mediated by T Cells Double Gene-Modified to Express WT1-Specific T-Cell Receptor

PubMed Central

Asai, Hiroaki; Fujiwara, Hiroshi; An, Jun; Ochi, Toshiki; Miyazaki, Yukihiro; Nagai, Kozo; Okamoto, Sachiko; Mineno, Junichi; Kuzushima, Kiyotaka; Shiku, Hiroshi; Inoue, Hirofumi; Yasukawa, Masaki

2013-01-01

Background and Purpose Although gene-modification of T cells to express tumor-related antigen-specific T-cell receptor (TCR) or chimeric antigen receptor (CAR) has clinically proved promise, there still remains room to improve the clinical efficacy of re-directed T-cell based antitumor adoptive therapy. In order to achieve more objective clinical responses using ex vivo-expanded tumor-responsive T cells, the infused T cells need to show adequate localized infiltration into the tumor. Methodology/Principal Findings Human lung cancer cells variously express a tumor antigen, Wilms' Tumor gene product 1 (WT1), and an inflammatory chemokine, CCL2. However, CCR2, the relevant receptor for CCL2, is rarely expressed on activated T-lymphocytes. A HLA-A2402+ human lung cancer cell line, LK79, which expresses high amounts of both CCL2 and WT1 mRNA, was employed as a target. Normal CD8+ T cells were retrovirally gene-modified to express both CCR2 and HLA-A*2402-restricted and WT1235–243 nonapeptide-specific TCR as an effector. Anti-tumor functionality mediated by these effector cells against LK79 cells was assessed both in vitro and in vivo. Finally the impact of CCL2 on WT1 epitope-responsive TCR signaling mediated by the effector cells was studied. Introduced CCR2 was functionally validated using gene-modified Jurkat cells and human CD3+ T cells both in vitro and in vivo. Double gene-modified CD3+ T cells successfully demonstrated both CCL2-tropic tumor trafficking and cytocidal reactivity against LK79 cells in vitro and in vivo. CCL2 augmented the WT1 epitope-responsive TCR signaling shown by relevant luciferase production in double gene-modified Jurkat/MA cells to express luciferase and WT1-specific TCR, and CCL2 also dose-dependently augmented WT1 epitope-responsive IFN-γ production and CD107a expression mediated by these double gene-modifiedCD3+ T cells. Conclusion/Significance Introduction of the CCL2/CCR2 axis successfully potentiated in vivo anti-lung cancer reactivity mediated by CD8+ T cells double gene-modified to express WT1-specific TCR and CCR2 not only via CCL2-tropic tumor trafficking, but also CCL2-enhanced WT1-responsiveness. PMID:23441216

Lignin biosynthesis in wheat (Triticum aestivum L.): its response to waterlogging and association with hormonal levels.

PubMed

Nguyen, Tran-Nguyen; Son, SeungHyun; Jordan, Mark C; Levin, David B; Ayele, Belay T

2016-01-25

Lignin is an important structural component of plant cell wall that confers mechanical strength and tolerance against biotic and abiotic stressors; however it affects the use of biomass such as wheat straw for some industrial applications such as biofuel production. Genetic alteration of lignin quantity and quality has been considered as a viable option to overcome this problem. However, the molecular mechanisms underlying lignin formation in wheat biomass has not been studied. Combining molecular and biochemical approaches, the present study investigated the transcriptional regulation of lignin biosynthesis in two wheat cultivars with varying lodging characteristics and also in response to waterlogging. It also examined the association of lignin level in tissues with that of plant hormones implicated in the control of lignin biosynthesis. Analysis of lignin biosynthesis in the two wheat cultivars revealed a close association of lodging resistance with internode lignin content and expression of 4-coumarate:CoA ligase1 (4CL1), p-coumarate 3-hydroxylase1 (C3H1), cinnamoyl-CoA reductase2 (CCR2), ferulate 5-hydroxylase2 (F5H2) and caffeic acid O-methyltransferase2 (COMT2), which are among the genes highly expressed in wheat tissues, implying the importance of these genes in mediating lignin deposition in wheat stem. Waterlogging of wheat plants reduced internode lignin content, and this effect is accompanied by transcriptional repression of three of the genes characterized as highly expressed in wheat internode including phenylalanine ammonia-lyase6 (PAL6), CCR2 and F5H2, and decreased activity of PAL. Expression of the other genes was, however, induced by waterlogging, suggesting their role in the synthesis of other phenylpropanoid-derived molecules with roles in stress responses. Moreover, difference in internode lignin content between cultivars or change in its level due to waterlogging is associated with the level of cytokinin. Lodging resistance, tolerance against biotic and abiotic stresses and feedstock quality of wheat biomass are closely associated with its lignin content. Therefore, the findings of this study provide important insights into the molecular mechanisms underlying lignin formation in wheat, an important step towards the development of molecular tools that can facilitate the breeding of wheat cultivars for optimized lignin content and enhanced feedstock quality without affecting other lignin-related agronomic benefits.

Rapid and Simple Phenotypic Assay for Drug Susceptibility of Human Immunodeficiency Virus Type 1 Using CCR5-Expressing HeLa/CD4+ Cell Clone 1-10 (MAGIC-5)

PubMed Central

Hachiya, Atsuko; Aizawa-Matsuoka, Saori; Tanaka, Mari; Takahashi, Yukiko; Ida, Setsuko; Gatanaga, Hiroyuki; Hirabayashi, Yoshihiro; Kojima, Asato; Tatsumi, Masashi; Oka, Shinichi

2001-01-01

We describe a rapid and simple novel phenotypic assay for drug susceptibility of human immunodeficiency virus type-1 (HIV-1) using a CCR5-expressing HeLa/CD4+ cell clone 1-10 (MAGIC-5). MAGIC-5 cells produced large amounts of HIV-1 in culture supernatants, which enabled us to perform the phenotypic resistance assay. Determination of HIV-1 susceptibility to various protease inhibitors (PI) and nucleoside reverse transcriptase inhibitors was completed within 15 days in T-cell-tropic (X4) and macrophage-tropic (R5) viruses using fresh plasma samples containing at least 104 copies/ml. The nucleotide sequence of the envelope V3 region of HIV-1 in plasma was almost identical to that of the virus isolated by MAGIC-5 cells, suggesting a lack of selection bias in our assay. The assay variability was confined to within five-fold in all drugs examined. Accordingly, we used a 10-fold increase in the 50% inhibitory concentration as the cutoff value for viral resistance in the present assay. HIV-1 resistant to lamivudine, which was not detected by conventional genotypic assays, was isolated. In HIV-1 with PI-associated primary amino acid substitutions, our assay showed that drug resistance profiles correlated well with previously reported genotypic-assay data. Furthermore, our assay provided comprehensive results regarding PI resistance in the presence of multiple mutations. The novel assay successfully quantified the level of resistance of clinical HIV-1 isolates to a battery of anti-HIV drugs, indicating its clinical usefulness, particularly in patients who failed to respond to antiretroviral chemotherapy. PMID:11158746

CC chemokine ligand 2 and CXC chemokine ligand 8 as neutrophil chemoattractant factors in canine idiopathic polyarthritis.

PubMed

Murakami, Kohei; Maeda, Shingo; Yonezawa, Tomohiro; Matsuki, Naoaki

2016-12-01

Canine idiopathic polyarthritis (IPA) is characterized by increased numbers of polymorphonuclear leukocytes (PMNs) in the synovial fluid (SF). In humans, CC chemokine ligand 2 (CCL2) and CXC chemokine ligand 8 (CXCL8) recruit monocytes and neutrophils, respectively, and are involved in various inflammatory disorders. The aim of this study was to assess the roles of these chemokines in driving PMNs infiltration into the joints of dogs with IPA. SF samples were collected from dogs with IPA (n=19) and healthy controls (n=8), and the concentrations of SF CCL2 and CXCL8 were determined by ELISA. Dogs with IPA had significantly higher concentrations of CCL2 (3316±2452pg/ml, mean±SD) and CXCL8 (3668±3879pg/ml) compared with the healthy controls (235±45pg/ml and <15.6pg/ml, respectively). Then, an in vitro chemotaxis assay was performed using a modified Boyden chamber (pore size: 3μm). SF from IPA dogs had a chemoattractant activity for PMNs that purified from the peripheral blood of a healthy dog. We subsequently found that combination treatment with MK-0812 (an antagonist of CCL2 receptor) and repertaxin (an antagonist of CXCL8 receptors) significantly inhibited the migration of PMNs to SF from IPA dogs. Thus, expression of the CCL2 receptor (chemokine (CC motif) receptor 2 (CCR2)) was examined using polymerase chain reaction and immunocytochemistry. Canine peripheral blood PMNs exhibited significantly higher CCR2 mRNA expression levels than those in monocytes. In addition, we observed strong CCR2 expression on PMNs obtained from healthy controls and IPA dogs, although mononuclear cells did not express CCR2. Taken together, the data suggest that CCL2 acts as a canine PMNs chemotactic factor as well as CXCL8 and both CCL2 and CXCL8 facilitate the infiltration of PMNs into the joints of dogs with IPA. Copyright © 2016 Elsevier B.V. All rights reserved.

Chemokine CCL2–CCR2 Signaling Induces Neuronal Cell Death via STAT3 Activation and IL-1β Production after Status Epilepticus

PubMed Central

Tian, Dai-Shi; Feng, Li-Jie; Liu, Jun-Li

2017-01-01

Elevated levels of chemokine C-C motif ligand 2 (CCL2) and its receptor CCR2 have been reported in patients with temporal lobe epilepsy and in experimental seizures. However, the functional significance and molecular mechanism underlying CCL2–CCR2 signaling in epileptic brain remains largely unknown. In this study, we found that the upregulated CCL2 was mainly expressed in hippocampal neurons and activated microglia from mice 1 d after kainic acid (KA)-induced seizures. Taking advantage of CX3CR1GFP/+:CCR2RFP/+ double-transgenic mice, we demonstrated that CCL2–CCR2 signaling has a role in resident microglial activation and blood-derived monocyte infiltration. Moreover, seizure-induced degeneration of neurons in the hippocampal CA3 region was attenuated in mice lacking CCL2 or CCR2. We further showed that CCR2 activation induced STAT3 (signal transducer and activator of transcription 3) phosphorylation and IL-1β production, which are critical for promoting neuronal cell death after status epilepticus. Consistently, pharmacological inhibition of STAT3 by WP1066 reduced seizure-induced IL-1β production and subsequent neuronal death. Two weeks after KA-induced seizures, CCR2 deficiency not only reduced neuronal loss, but also attenuated seizure-induced behavioral impairments, including anxiety, memory decline, and recurrent seizure severity. Together, we demonstrated that CCL2–CCR2 signaling contributes to neurodegeneration via STAT3 activation and IL-1β production after status epilepticus, providing potential therapeutic targets for the treatment of epilepsy. SIGNIFICANCE STATEMENT Epilepsy is a global concern and epileptic seizures occur in many neurological conditions. Neuroinflammation associated with microglial activation and monocyte infiltration are characteristic of epileptic brains. However, molecular mechanisms underlying neuroinflammation in neuronal death following epilepsy remain to be elucidated. Here we demonstrate that CCL2–CCR2 signaling is required for monocyte infiltration, which in turn contributes to kainic acid (KA)-induced neuronal cell death. The downstream of CCR2 activation involves STAT3 (signal transducer and activator of transcription 3) phosphorylation and IL-1β production. Two weeks after KA-induced seizures, CCR2 deficiency not only reduced neuronal loss, but also attenuated seizure-induced behavioral impairments, including anxiety, memory decline, and recurrent seizure severity. The current study provides a novel insight on the function and mechanisms of CCL2–CCR2 signaling in KA-induced neurodegeneration and behavioral deficits. PMID:28716963

Polymorphisms of CCL3L1/CCR5 genes and recurrence of hepatitis B in liver transplant recipients.

PubMed

Li, Hong; Xie, Hai-Yang; Zhou, Lin; Wang, Wei-Lin; Liang, Ting-Bo; Zhang, Min; Zheng, Shu-Sen

2011-12-01

The genetic diversity of chemokines and chemokine receptors has been associated with the outcome of hepatitis B virus infection. The aim of this study was to evaluate whether the copy number variation in the CCL3L1 gene and the polymorphisms of CCR5Δ32 and CCR5-2459A→G (rs1799987) are associated with recurrent hepatitis B in liver transplantation for hepatitis B virus infection-related end-stage liver disease. A total of 185 transplant recipients were enrolled in this study. The genomic DNA was extracted from whole blood, the copy number of the CCL3L1 gene was determined by a quantitative real-time PCR based assay, CCR5Δ32 was detected by a sizing PCR method, and a single-nucleotide polymorphism in CCR5-2459 was detected by restriction fragment length polymorphism PCR. No CCR5Δ32 mutation was detected in any of the individuals from China. Neither copy number variation nor polymorphism in CCR5-2459 was associated with post-transplant re-infection with hepatitis B virus. However, patients with fewer copies (<4) of the CCL3L1 gene compared with the population median in combination with the CCR5G allele had a significantly higher risk for recurrent hepatitis B (odds ratio=1.93, 95% CI: 1.00-3.69; P=0.047). Patients possessing the compound decreased functional genotype of both CCL3L1 and CCR5 genes might be more likely to have recurrence of hepatitis B after transplantation.

Rapid and transient upregulation of CCL11 (eotaxin-1) in mouse ovary during terminal stages of follicular development.

PubMed

Kuwabara, Yoshimitsu; Katayama, Akira; Igarashi, Tsutomu; Tomiyama, Ryoko; Piao, Hua; Kaneko, Reika; Abe, Takashi; Mine, Katsuya; Akira, Shigeo; Orimo, Hideo; Takeshita, Toshiyuki

2012-05-01

This study aimed to investigate the regulation of expression, localization and physiological role of the CCL11/CCR3 axis in mouse ovary during the periovulatory period. CCL11/CCR3 expression in the mouse ovary after treatment with pregnant mare serum gonadotropin (PMSG) followed by human chorionic gonadotropin (hCG) 48 hr later was assessed in vivo and in 3-dimensional cultures in vitro. Real-time RT-PCR analyses revealed transient CCL11 mRNA upregulation 6 hr after hCG treatment. Immunohistochemical staining of serial ovarian sections demonstrated overlapping expression of CCL11, CCR3 and CD31 endothelial cell marker in the theca-interstitial layer at 10 hr after hCG treatment. In vitro 3-dimensional cultures of periovulatory ovarian tissues demonstrated that treatment with anti-CCL11 neutralizing antibody significantly decreased CD31 transcript. Gonadotropin surge leads to transient CCL11/CCR3 axis upregulation in the ovarian theca-interstitial layer, suggesting that it is involved in periovulatory physiological processes by affecting follicular vessels. © 2012 John Wiley & Sons A/S.

Analysis of the Spanish CCR5-∆32 inventory of cord blood units: lower cell counts in homozygous donors.

PubMed

Enrich, Emma; Vidal, Francisco; Sánchez-Gordo, Francisco; Gómez-Zumaquero, Juan M; Balas, Antonio; Rudilla, Francesc; Barea, Luisa; Castro, Ana; Larrea, Luis; Perez-Vaquero, Miguel A; Prat, Isidro; Querol, Sergio; Garrido, Gregorio; Matesanz, Rafael; Carreras, Enric; Duarte, Rafael F

2018-02-06

The possibility to use CCR5-∆32 umbilical cord blood to cure HIV infection in patients in need of a hematopoietic transplant has been suggested. The less stringent HLA compatibility needed in this type of transplant facilitates the search of a suitable donor having the CCR5-∆32 mutation. To achieve an inventory of CCR5-∆32 cord blood units, the 20,236 best cell quality units of the Spanish Registry were genotyped. Furthermore, their CD34 + and total nucleated cells counts, blood type, gender, HLA and donor's geographical and ancestral origin were analyzed. The results showed 130 (0.64%) units homozygous for the deletion, 2,646 (13.08%) heterozygous and 17,460 (86.28%) did not present the mutation. Interestingly, a significant lower amount of CD34 + cells was found in the CCR5-∆32 homozygous units. In addition, a significant association was found among donor's ancestral origin and the mutation, with a higher percentage of CCR5-∆32 units with a European ancestry. In summary, identification of a relatively high number of CCR5-∆32 units is feasible and will facilitate the development of clinical trials for HIV cure in patients requiring hematopoietic transplantation. Further studies are required to understand the significance of lower cell counts within the CCR5-∆32 homozygous group and its clinical impact.

Wu-Tou Decoction in Rheumatoid Arthritis: Integrating Network Pharmacology and In Vivo Pharmacological Evaluation

PubMed Central

Guo, Qingqing; Zheng, Kang; Fan, Danping; Zhao, Yukun; Li, Li; Bian, Yanqin; Qiu, Xuemei; Liu, Xue; Zhang, Ge; Ma, Chaoying; He, Xiaojuan; Lu, Aiping

2017-01-01

Purpose: This study aimed to explore underlying action mechanism of Wu-Tou decoction (WTD) in rheumatoid arthritis (RA) through network pharmacology prediction and experimental verification. Methods: Chemical compounds and human target proteins of WTD as well as RA-related human genes were obtained from TCM Database @ Taiwan, PubChem and GenBank, respectively. Subsequently, molecular networks and canonical pathways presumably involved in the treatment of WTD on RA were generated by ingenuity pathway analysis (IPA) software. Furthermore, experimental validation was carried out with MIP-1β-induced U937 cell model and collagen induced arthritis (CIA) rat model. Results: CCR5 signaling pathway in macrophages was shown to be the top one shared signaling pathway associated with both cell immune response and cytokine signaling. In addition, protein kinase C (PKC) δ and p38 in this pathway were treated as target proteins of WTD in RA. In vitro experiments indicated that WTD inhibited MIP-1β-induced production of TNF-α, MIP-1α, and RANTES as well as phosphorylation of CCR5, PKC δ, and p38 in U937 cells. WTD treatment maintained the inhibitory effects on production of TNF-α and RANTES in MIP-1β-induced U937 cells after CCR5 knockdown. In vivo experiments demonstrated that WTD ameliorated symptoms in CIA rats, decreased the levels of IL-1β, IL-2, IL-6, TNF-α, MIP-1α, MIP-2, RANTES, and IP-10 in serum of CIA rats, as well as mRNA levels of MIP-1α, MIP-2, RANTES, and IP-10 in ankle joints of CIA rats. Furthermore, WTD also lowered the phosphorylation levels of CCR5, PKC δ and p38 in both ankle joints and macrophages in ankle joints from CIA rats. Conclusion: It was demonstrated in this research that WTD played a role in inhibiting inflammatory response in RA which was closely connected with the modulation effect of WTD on CCR5 signaling pathway in macrophages. PMID:28515692

CCR7 and CXCR4 Expression in Primary Head and Neck Squamous Cell Carcinomas and Nodal Metastases – a Clinical and Immunohistochemical Study

PubMed Central

Al-Jokhadar, Maya; Al-Mandily, Ahmad; Zaid, Khaled; Maalouf, Elie Azar

2017-01-01

Background: Squamous cell carcinomas (SCCs) are common head and neck malignancies demonstrating lymph node LN involvement. Recently chemokine receptor overxpression has been reported in many cancers. Of particular interest, CCR7 appears to be a strong mediator of LN metastases, while CXCR4 may mediate distant metastases. Any relations between their expression in primary HNSCCs and metastatic lymph nodes need to be clarified. Aims: To investigate CCR7 andCXCR4 expression in primary HNSCCs of all tumor sizes, clinical stages and histological grades, as well as involved lymph nodes, then make comparisons, also with control normal oral epithelium. Materials and Methods: The sample consisted of 60 formalin-fixed, paraffin-embedded specimens of primary HNSCCs, 77 others of metastasi-positive lymph nodes, and 10 of control normal oral epithelial tissues. Sections were conventionally stained with H&E and immunohistochemically with monoclonal anti-CCR7 and monoclonal anti-CXCR4 antibodies. Positive cells were counted under microscopic assessment in four fields (X40) per case. Results: There was no variation among primary HNSCC tumors staining positive for CCR7 and CXCR4 with tumor size of for CCR7 with lymph node involvement. However, a difference was noted between primary HNSCC tumors stained by CXCR4 with a single as compared to more numerous node involvement. CXCR4 appear to vary with the clinical stagebut no links were noted with histological grades. Staining for primary HNSCC tumors and metastatic lymph nodes correlated. PMID:28547946

CCR7 Maintains Nonresolving Lymph Node and Adipose Inflammation in Obesity

PubMed Central

Hellmann, Jason; Sansbury, Brian E.; Holden, Candice R.; Tang, Yunan; Wong, Blenda; Wysoczynski, Marcin; Rodriguez, Jorge; Bhatnagar, Aruni; Hill, Bradford G.

2016-01-01

Accumulation of immune cells in adipose tissue promotes insulin resistance in obesity. Although innate and adaptive immune cells contribute to adipose inflammation, the processes that sustain these interactions are incompletely understood. Here we show that obesity promotes the accumulation of CD11c+ adipose tissue immune cells that express C-C chemokine receptor 7 (CCR7) in mice and humans, and that CCR7 contributes to chronic inflammation and insulin resistance. We identified that CCR7+ macrophages and dendritic cells accumulate in adipose tissue in close proximity to lymph nodes (LNs) (i.e., perinodal) and visceral adipose. Consistent with the role of CCR7 in regulating the migration of immune cells to LNs, obesity promoted the accumulation of CD11c+ cells in LNs, which was prevented by global or hematopoietic deficiency of Ccr7. Obese Ccr7−/− mice had reduced accumulation of CD8+ T cells, B cells, and macrophages in adipose tissue, which was associated with reduced inflammatory signaling. This reduction in maladaptive inflammation translated to increased insulin signaling and improved glucose tolerance in obesity. Therapeutic administration of an anti-CCR7 antibody phenocopied the effects of genetic Ccr7 deficiency in mice with established obesity. These results suggest that CCR7 plays a causal role in maintaining innate and adaptive immunity in obesity. PMID:27207557

Consequences of ChemR23 Heteromerization with the Chemokine Receptors CXCR4 and CCR7

PubMed Central

de Poorter, Cédric; Baertsoen, Kevin; Lannoy, Vincent; Parmentier, Marc; Springael, Jean-Yves

2013-01-01

Recent studies have shown that heteromerization of the chemokine receptors CCR2, CCR5 and CXCR4 is associated to negative binding cooperativity. In the present study, we build on these previous results, and investigate the consequences of chemokine receptor heteromerization with ChemR23, the receptor of chemerin, a leukocyte chemoattractant protein structurally unrelated to chemokines. We show, using BRET and HTRF assays, that ChemR23 forms homomers, and provide data suggesting that ChemR23 also forms heteromers with the chemokine receptors CCR7 and CXCR4. As previously described for other chemokine receptor heteromers, negative binding cooperativity was detected between ChemR23 and chemokine receptors, i.e. the ligands of one receptor competed for the binding of a specific tracer of the other. We also showed, using mouse bone marrow-derived dendritic cells prepared from wild-type and ChemR23 knockout mice, that ChemR23-specific ligands cross-inhibited CXCL12 binding on CXCR4 in a ChemR23-dependent manner, supporting the relevance of the ChemR23/CXCR4 interaction in native leukocytes. Finally, and in contrast to the situation encountered for other previously characterized CXCR4 heteromers, we showed that the CXCR4-specific antagonist AMD3100 did not cross-inhibit chemerin binding in cells co-expressing ChemR23 and CXCR4, demonstrating that cross-regulation by AMD3100 depends on the nature of receptor partners with which CXCR4 is co-expressed. PMID:23469143

Consequences of ChemR23 heteromerization with the chemokine receptors CXCR4 and CCR7.

PubMed

de Poorter, Cédric; Baertsoen, Kevin; Lannoy, Vincent; Parmentier, Marc; Springael, Jean-Yves

2013-01-01

Recent studies have shown that heteromerization of the chemokine receptors CCR2, CCR5 and CXCR4 is associated to negative binding cooperativity. In the present study, we build on these previous results, and investigate the consequences of chemokine receptor heteromerization with ChemR23, the receptor of chemerin, a leukocyte chemoattractant protein structurally unrelated to chemokines. We show, using BRET and HTRF assays, that ChemR23 forms homomers, and provide data suggesting that ChemR23 also forms heteromers with the chemokine receptors CCR7 and CXCR4. As previously described for other chemokine receptor heteromers, negative binding cooperativity was detected between ChemR23 and chemokine receptors, i.e. the ligands of one receptor competed for the binding of a specific tracer of the other. We also showed, using mouse bone marrow-derived dendritic cells prepared from wild-type and ChemR23 knockout mice, that ChemR23-specific ligands cross-inhibited CXCL12 binding on CXCR4 in a ChemR23-dependent manner, supporting the relevance of the ChemR23/CXCR4 interaction in native leukocytes. Finally, and in contrast to the situation encountered for other previously characterized CXCR4 heteromers, we showed that the CXCR4-specific antagonist AMD3100 did not cross-inhibit chemerin binding in cells co-expressing ChemR23 and CXCR4, demonstrating that cross-regulation by AMD3100 depends on the nature of receptor partners with which CXCR4 is co-expressed.

Functional characterization of CCR in birch (Betula platyphylla × Betula pendula) through overexpression and suppression analysis.

PubMed

Zhang, Wenbo; Wei, Rui; Chen, Su; Jiang, Jing; Li, Huiyu; Huang, Haijiao; Yang, Guang; Wang, Shuo; Wei, Hairong; Liu, Guifeng

2015-06-01

We cloned a Cinnamoyl-CoA Reductase gene (BpCCR1) from an apical meristem and first internode of Betula platyphylla and characterized its functions in lignin biosynthesis, wood formation and tree growth through transgenic approaches. We generated overexpression and suppression transgenic lines and analyzed them in comparison with the wild-type in terms of lignin content, anatomical characteristics, height and biomass. We found that BpCCR1 overexpression could increase lignin content up to 14.6%, and its underexpression decreased lignin content by 6.3%. Surprisingly, modification of BpCCR1 expression led to conspicuous changes in wood characteristics, including xylem vessel number and arrangement, and secondary wall thickness. The growth of transgenic trees in terms of height was also significantly influenced by the modification of BpCCR1 genes. We discuss the functions of BpCCR1 in the context of a phylogenetic tree built with CCR genes from multiple species. © 2014 Scandinavian Plant Physiology Society.

Polymorphisms in chemokine and receptor genes and gastric cancer risk and survival in a high risk Polish population.

PubMed

Gawron, Andrew J; Fought, Angela J; Lissowska, Jolanta; Ye, Weimin; Zhang, Xiao; Chow, Wong-Ho; Beane Freeman, Laura E; Hou, Lifang

2011-03-01

To examine if genetic variations in chemokine receptor and ligand genes are associated with gastric cancer risk and survival. The study included 298 cases and 417 controls from a population-based study of gastric cancer conducted in Warsaw, Poland in 1994-1996. We investigated seven single nucleotide polymorphisms in a chemokine ligand (CXCL12) and chemokine receptor (CCR2, CCR5, CX3CR1) genes and one frameshift deletion (CCR5) in blood leukocyte DNA in relation to gastric cancer risk and survival. Genotyping was conducted at the NCI Core Genotyping Facility. Odds ratios and 95% confidence intervals were computed using univariate and multivariate logistic regression models. Survival analysis was performed using Cox proportional hazards models. Gastric cancer risk was not associated with single chemokine polymorphisms. A CCR5 haplotype that contained the common alleles of IVS1+151 G>T (rs2734648), IVS2+80 C>T (rs1800024) and minor allele of IVS1+246 A>G (rs1799987) was associated with a borderline significantly increased risk (OR = 1.5, 95% CI: 1.0?2.2). For gastric cancer cases, there was a greater risk of death for carriers of the minor alleles of CCR2 Ex2+241 G>A (rs1799864) (HR = 1.5, 95% CI: 1.1-2.1) and CCR5 IVS2+80 C>T (rs1800024) (HR = 1.5, 95% CI: 1.1-2.1). Carriers of the CCR5 minor allele of IVS1+151 G>T (rs2734648) had a decreased risk of death compared to homozygote carriers of the common allele (HR = 0.8, 95% CI: 0.6-1.0). Our findings do not support an association between gastric cancer risk and single chemokine genetic variation. The observed associations between cancer risk and a CCR5 haplotype and between survival and polymorphisms in CCR2 and CCR5 need replication in future studies.

Transcription Factor SOX5 Promotes the Migration and Invasion of Fibroblast-Like Synoviocytes in Part by Regulating MMP-9 Expression in Collagen-Induced Arthritis.

PubMed

Shi, Yumeng; Wu, Qin; Xuan, Wenhua; Feng, Xiaoke; Wang, Fang; Tsao, Betty P; Zhang, Miaojia; Tan, Wenfeng

2018-01-01

Fibroblast-like synoviocytes (FLS) exhibit a unique aggressive phenotype in rheumatoid arthritis (RA). Increased FLS migration and subsequent invasion of the extracellular matrix are essential to joint destruction in RA. Our previous research reported that transcription factor SOX5 was highly expressed in RA-FLS. Here, the effects of SOX5 in RA-FLS migration and invasion will be investigated. The migration and invasion of RA-FLS were evaluated using a transwell chamber assay. The expression of several potential SOX5-targeted genes, including matrix metalloproteinases (MMP-1, 2, 3 and 9), chemokines (CCL4, CCL2, CCR5 and CCR2), and pro-inflammatory cytokines (TNF-α and IL-6), were examined in RA-FLS using SOX5 gain- and loss-of-function study. The molecular mechanisms of SOX5-mediated MMP-9 expressions were assayed by luciferase reporter gene and chromatin immunoprecipitation (ChIP) studies. The in vivo effect of SOX5 on FLS migration and invasion was examined using collagen-induced arthritis (CIA) in DBA/1J mice. Knockdown SOX5 decreased lamellipodium formation, migration, and invasion of RA-FLS. The expression of MMP-9 was the only gene tested to be concomitantly affected by silencing or overexpressing SOX5. ChIP assay revealed that SOX5 was bound to the MMP-9 promoter in RA-FLS. The overexpression of SOX5 markedly enhanced the MMP-9 promoter activity, and specific deletion of a putative SOX5-binding site in MMP-9 promoter diminished this promoter-driven transcription in FLS. Locally knocked down SOX5 inhibited MMP-9 expression in the joint tissue and reduced pannus migration and invasion into the cartilage in CIA mice. SOX5 plays a novel role in mediating migration and invasion of FLS in part by regulating MMP-9 expression in RA.

CCR6 and NK1.1 distinguish between IL-17A and IFN-gamma-producing gammadelta effector T cells.

PubMed

Haas, Jan D; González, Frano H Malinarich; Schmitz, Susanne; Chennupati, Vijaykumar; Föhse, Lisa; Kremmer, Elisabeth; Förster, Reinhold; Prinz, Immo

2009-12-01

Gammadelta T cells are a potent source of innate IL-17A and IFN-gamma, and they acquire the capacity to produce these cytokines within the thymus. However, the precise stages and required signals that guide this differentiation are unclear. Here we show that the CD24(low) CD44(high) effector gammadelta T cells of the adult thymus are segregated into two lineages by the mutually exclusive expression of CCR6 and NK1.1. Only CCR6+ gammadelta T cells produced IL-17A, while NK1.1+ gammadelta T cells were efficient producers of IFN-gamma but not of IL-17A. Their effector phenotype correlated with loss of CCR9 expression, particularly among the NK1.1+ gammadelta T cells. Accordingly, both gammadelta T-cell subsets were rare in gut-associated lymphoid tissues, but abundant in peripheral lymphoid tissues. There, they provided IL-17A and IFN-gamma in response to TCR-specific and TCR-independent stimuli. IL-12 and IL-18 induced IFN-gamma and IL-23 induced IL-17A production by NK1.1+ or CCR6+ gammadelta T cells, respectively. Importantly, we show that CCR6+ gammadelta T cells are more responsive to TCR stimulation than their NK1.1+ counterparts. In conclusion, our findings support the hypothesis that CCR6+ IL-17A-producing gammadelta T cells derive from less TCR-dependent selection events than IFN-gamma-producing NK1.1+ gammadelta T cells.

An Improved X-Band Maser System for Deep Space Network Applications

NASA Astrophysics Data System (ADS)

Britcliffe, M.; Hanson, T.; Fernandez, J.

2000-01-01

An 8450-MHz (X-band) maser system utilizing a commercial Gifford--McMahon (GM) closed-cycle cryocooler (CCR) was designed, fabricated, and demonstrated. The CCR system was used to cool a maser operating at 8450 MHz. The prototype GM CCR system meets or exceeds all Deep Space Network requirements for maser performance. The two-stage GM CCR operates at 4.2 K; for comparison, the DSN's current three-stage cryocooler, which uses a Joule--Thompson cooling stage in addition to GM cooling, operates at 4.5 K. The new CCR withstands heat loads of 1.5 W at 4.2 K as compared to 1 W at 4.5 K for the existing DSN cryocooler used for cooling masers. The measured noise temperature, T_e, of the maser used for these tests is defined at the ambient connection to the antenna feed system. The T_e measured 5.0 K at a CCR temperature of 4.5 K, about 1.5 K higher than the noise temperature of a typical DSN Block II-A X-band traveling-wave maser (TWM). Reducing the temperature of the CCR significantly lowers the maser noise temperature and increases maser gain and bandwidth. The new GM CCR gives future maser systems significant operational advantages, including reduced maintenance time and logistics requirements. The results of a demonstration of this new system are presented. Advantages of using a GM-cooled maser and the effects of the reduced CCR temperature on maser performance are discussed.

Functional Mimetics of the HIV-1 CCR5 Co-Receptor Displayed on the Surface of Magnetic Liposomes.

PubMed

Kuzmina, Alona; Vaknin, Karin; Gdalevsky, Garik; Vyazmensky, Maria; Marks, Robert S; Taube, Ran; Engel, Stanislav

2015-01-01

Chemokine G protein coupled receptors, principally CCR5 or CXCR4, function as co-receptors for HIV-1 entry into CD4+ T cells. Initial binding of the viral envelope glycoprotein (Env) gp120 subunit to the host CD4 receptor induces a cascade of structural conformational changes that lead to the formation of a high-affinity co-receptor-binding site on gp120. Interaction between gp120 and the co-receptor leads to the exposure of epitopes on the viral gp41 that mediates fusion between viral and cell membranes. Soluble CD4 (sCD4) mimetics can act as an activation-based inhibitor of HIV-1 entry in vitro, as it induces similar structural changes in gp120, leading to increased virus infectivity in the short term but to virus Env inactivation in the long term. Despite promising clinical implications, sCD4 displays low efficiency in vivo, and in multiple HIV strains, it does not inhibit viral infection. This has been attributed to the slow kinetics of the sCD4-induced HIV Env inactivation and to the failure to obtain sufficient sCD4 mimetic levels in the serum. Here we present uniquely structured CCR5 co-receptor mimetics. We hypothesized that such mimetics will enhance sCD4-induced HIV Env inactivation and inhibition of HIV entry. Co-receptor mimetics were derived from CCR5 gp120-binding epitopes and functionalized with a palmitoyl group, which mediated their display on the surface of lipid-coated magnetic beads. CCR5-peptidoliposome mimetics bound to soluble gp120 and inhibited HIV-1 infectivity in a sCD4-dependent manner. We concluded that CCR5-peptidoliposomes increase the efficiency of sCD4 to inhibit HIV infection by acting as bait for sCD4-primed virus, catalyzing the premature discharge of its fusion potential.

Functional Mimetics of the HIV-1 CCR5 Co-Receptor Displayed on the Surface of Magnetic Liposomes

PubMed Central

Kuzmina, Alona; Vaknin, Karin; Gdalevsky, Garik; Vyazmensky, Maria; Marks, Robert S.; Taube, Ran

2015-01-01

Chemokine G protein coupled receptors, principally CCR5 or CXCR4, function as co-receptors for HIV-1 entry into CD4+ T cells. Initial binding of the viral envelope glycoprotein (Env) gp120 subunit to the host CD4 receptor induces a cascade of structural conformational changes that lead to the formation of a high-affinity co-receptor-binding site on gp120. Interaction between gp120 and the co-receptor leads to the exposure of epitopes on the viral gp41 that mediates fusion between viral and cell membranes. Soluble CD4 (sCD4) mimetics can act as an activation-based inhibitor of HIV-1 entry in vitro, as it induces similar structural changes in gp120, leading to increased virus infectivity in the short term but to virus Env inactivation in the long term. Despite promising clinical implications, sCD4 displays low efficiency in vivo, and in multiple HIV strains, it does not inhibit viral infection. This has been attributed to the slow kinetics of the sCD4-induced HIV Env inactivation and to the failure to obtain sufficient sCD4 mimetic levels in the serum. Here we present uniquely structured CCR5 co-receptor mimetics. We hypothesized that such mimetics will enhance sCD4-induced HIV Env inactivation and inhibition of HIV entry. Co-receptor mimetics were derived from CCR5 gp120-binding epitopes and functionalized with a palmitoyl group, which mediated their display on the surface of lipid-coated magnetic beads. CCR5-peptidoliposome mimetics bound to soluble gp120 and inhibited HIV-1 infectivity in a sCD4-dependent manner. We concluded that CCR5-peptidoliposomes increase the efficiency of sCD4 to inhibit HIV infection by acting as bait for sCD4-primed virus, catalyzing the premature discharge of its fusion potential. PMID:26629902

Vessel-Specific Reintroduction of CINNAMOYL-COA REDUCTASE1 (CCR1) in Dwarfed ccr1 Mutants Restores Vessel and Xylary Fiber Integrity and Increases Biomass1[OPEN

PubMed Central

Özparpucu, Merve

2018-01-01

Lignocellulosic biomass is recalcitrant toward deconstruction into simple sugars due to the presence of lignin. To render lignocellulosic biomass a suitable feedstock for the bio-based economy, plants can be engineered to have decreased amounts of lignin. However, engineered plants with the lowest amounts of lignin exhibit collapsed vessels and yield penalties. Previous efforts were not able to fully overcome this phenotype without settling in sugar yield upon saccharification. Here, we reintroduced CINNAMOYL-COENZYME A REDUCTASE1 (CCR1) expression specifically in the protoxylem and metaxylem vessel cells of Arabidopsis (Arabidopsis thaliana) ccr1 mutants. The resulting ccr1 ProSNBE:CCR1 lines had overcome the vascular collapse and had a total stem biomass yield that was increased up to 59% as compared with the wild type. Raman analysis showed that monolignols synthesized in the vessels also contribute to the lignification of neighboring xylary fibers. The cell wall composition and metabolome of ccr1 ProSNBE:CCR1 still exhibited many similarities to those of ccr1 mutants, regardless of their yield increase. In contrast to a recent report, the yield penalty of ccr1 mutants was not caused by ferulic acid accumulation but was (largely) the consequence of collapsed vessels. Finally, ccr1 ProSNBE:CCR1 plants had a 4-fold increase in total sugar yield when compared with wild-type plants. PMID:29158331

Identification of the Single Immunodominant Region of the Native Human CC Chemokine Receptor 6 Recognized by Mouse Monoclonal Antibodies.

PubMed

Dorgham, Karim; Dejou, Cécile; Piesse, Christophe; Gorochov, Guy; Pène, Jérôme; Yssel, Hans

2016-01-01

Chemokines and their receptors play an important role in cell trafficking and recruitment. The CCR6 chemokine receptor, selectively expressed on leukocyte populations, has been shown to play a deleterious role in the pathogenesis of various chronic inflammatory diseases and, as such, may constitute a prime target in the development of immunotherapeutic treatment. However, to date no neutralizing mouse monoclonal antibodies (mAbs) specific for this chemokine receptor have been reported, whereas information on small molecules capable of interfering with the interaction of CCR6 and its ligands is scant. Here, we report the failure to generate neutralizing mouse mAbs specific for human (hu)CCR6. Immunization of mice with peptides mimicking extracellular domains, potentially involved in CCR6 function, failed to induce Abs reactive with the native receptor. Although the use of NIH-3T3 cells expressing huCCR6 resulted in the isolation of mAbs specific for this receptor, they were not able to block the interaction between huCCR6 and huCCL20. Investigation of the anti-huCCR6 mAbs generated in the present study, as well as those commercially available, show that all mAbs invariably recognize a unique, non-neutralizing, immunodominant region in the first part of its N-terminal domain. Together, these results indicate that the generation of potential neutralizing anti-huCCR6 mAbs in the mouse is unlikely to succeed and that alternative techniques, such as the use of other animal species for immunization, might constitute a better approach to generate such a potentially therapeutic tool for the treatment of inflammatory disease.

Genetic Susceptibility to Cardiac and Digestive Clinical Forms of Chronic Chagas Disease: Involvement of the CCR5 59029 A/G Polymorphism.

PubMed

de Oliveira, Amanda Priscila; Bernardo, Cássia Rubia; Camargo, Ana Vitória da Silveira; Ronchi, Luiz Sérgio; Borim, Aldenis Albaneze; de Mattos, Cinara Cássia Brandão; de Campos Júnior, Eumildo; Castiglioni, Lílian; Netinho, João Gomes; Cavasini, Carlos Eugênio; Bestetti, Reinaldo Bulgarelli; de Mattos, Luiz Carlos

2015-01-01

The clinical manifestations of chronic Chagas disease include the cardiac form of the disease and the digestive form. Not all the factors that act in the variable clinical course of this disease are known. This study investigated whether the CCR5Δ32 (rs333) and CCR5 59029 A/G (promoter region--rs1799987) polymorphisms of the CCR5 gene are associated with different clinical forms of chronic Chagas disease and with the severity of left ventricular systolic dysfunction in patients with chronic Chagas heart disease (CCHD). The antibodies anti-T. cruzi were identified by ELISA. PCR and PCR-RFLP were used to identify the CCR5Δ32 and CCR5 59029 A/G polymorphisms. The chi-square test was used to compare variables between groups. There was a higher frequency of the AA genotype in patients with CCHD compared with patients with the digestive form of the disease and the control group. The results also showed a high frequency of the AG genotype in patients with the digestive form of the disease compared to the other groups. The results of this study show that the CCR5Δ32 polymorphism does not seem to influence the different clinical manifestations of Chagas disease but there is involvement of the CCR5 59029 A/G polymorphism in susceptibility to the different forms of chronic Chagas disease. Besides, these polymorphisms do not influence left ventricular systolic dysfunction in patients with CCHD.

A novel dual luciferase assay for the simultaneous monitoring of HIV infection and cell viability.

PubMed

Mitsuki, Yu-Ya; Yamamoto, Takuya; Mizukoshi, Fuminori; Momota, Masatoshi; Terahara, Kazutaka; Yoshimura, Kazuhisa; Harada, Shigeyoshi; Tsunetsugu-Yokota, Yasuko

2016-05-01

Human immunodeficiency virus type 1 (HIV-1) reporter cell lines are critical tools for drug development. However, one disadvantage of HIV-1 reporter cell lines is that reductions in reporter gene activity need to be normalized to cytotoxicity, i.e., live cell numbers. Here, we developed a dual luciferase assay based on a R. reniformis luciferase (hRLuc)-expressing R5-type HIV-1 (NLAD8-hRLuc) and a CEM cell line expressing CCR5 and firefly luciferase (R5CEM-FiLuc). The NLAD8-hRLuc reporter virus was replication competent in peripheral blood mononuclear cells. The level of hRLuc was correlated with p24 antigen levels (p<0.001, R=0.862). The target cell line, R5CEM-FiLuc, stably expressed the firefly luciferase (FiLuc) reporter gene and allowed the simultaneous monitoring of compound cytotoxicity. The dual reporter assay combining a NLAD8-hRLuc virus with R5CEM-FiLuc cells permitted the accurate determination of drug susceptibility for entry, reverse transcriptase, integrase, and protease inhibitors at different multiplicities of infection. This dual reporter assay provides a rapid and direct method for the simultaneous monitoring of HIV infection and cell viability. Copyright © 2016 Elsevier B.V. All rights reserved.

Cortisol increases CXCR4 expression but does not affect CD62L and CCR7 levels on specific T cell subsets in humans.

PubMed

Besedovsky, Luciana; Linz, Barbara; Dimitrov, Stoyan; Groch, Sabine; Born, Jan; Lange, Tanja

2014-06-01

Glucocorticoids are well known to affect T cell migration, leading to a redistribution of the cells from blood to the bone marrow, accompanied by a concurrent suppression of lymph node homing. Despite numerous studies in this context, with most of them employing synthetic glucocorticoids in nonphysiological doses, the mechanisms of this redistribution are not well understood. Here, we investigated in healthy men the impact of cortisol at physiological concentrations on the expression of different migration molecules on eight T cell subpopulations in vivo and in vitro. Hydrocortisone (cortisol, 22 mg) infused during nocturnal rest when endogenous cortisol levels are low, compared with placebo, differentially reduced numbers of T cell subsets, with naive CD4(+) and CD8(+) subsets exhibiting the strongest reduction. Hydrocortisone in vivo and in vitro increased CXCR4 expression, which presumably mediates the recruitment of T cells to the bone marrow. Expression of the lymph node homing receptor CD62L on total CD3(+) and CD8(+) T cells appeared reduced following hydrocortisone infusion. However, this was due to a selective extravasation of CD62L(+) T cell subsets, as hydrocortisone affected neither CD62L expression on a subpopulation level nor CD62L expression in vitro. Corresponding results in the opposite direction were observed after blocking of endogenous cortisol synthesis by metyrapone. CCR7, another lymph node homing receptor, was also unaffected by hydrocortisone in vitro. Thus, cortisol seems to redirect T cells to the bone marrow by upregulating their CXCR4 expression, whereas its inhibiting effect on T cell homing to lymph nodes is apparently regulated independently of the expression of classical homing receptors. Copyright © 2014 the American Physiological Society.

Relationship of Genetic Polymorphisms of the Chemokine, CCL5, and Its Receptor, CCR5, with Coronary Artery Disease in Taiwan

PubMed Central

Ting, Ke-Hsin; Ueng, Kwo-Chang; Chiang, Whei-Ling; Chou, Ying-Erh; Yang, Shun-Fa; Wang, Po-Hui

2015-01-01

The chemokine receptor CCR5 polymorphism, which confers resistance to HIV infection, has been associated with reduced risk of cardiovascular disease. However, the association of the chemokine, CCL5, and its receptor, CCR5, polymorphism and coronary artery disease (CAD) in the Taiwanese has not been studied. In this study, 483 subjects who received elective coronary angiography were recruited from Chung Shan Medical University Hospital. CCL5-403 and CCR5-59029 were determined by polymerase chain reaction-restriction fragment length polymorphism. We found that CCL5-403 with TT genotype frequencies was significantly associated with the risk of CAD group (odds ratio = 3.063 and p = 0.012). Moreover, the frequencies of CCR5-59029 with GG or GA genotype were higher than AA genotype in acute coronary syndrome individuals (odds ratio = 1.853, CI = 1.176–2.921, p = 0.008). In conclusion, we found that CCL5-403 polymorphism may increase genetic susceptibility of CAD. CCL5-403 or CCR5-59029 single nucleotide polymorphism may include genotype score and it may predict cardiovascular event. PMID:26688689

Circulating precursor CCR7(lo)PD-1(hi) CXCR5⁺ CD4⁺ T cells indicate Tfh cell activity and promote antibody responses upon antigen reexposure.

PubMed

He, Jing; Tsai, Louis M; Leong, Yew Ann; Hu, Xin; Ma, Cindy S; Chevalier, Nina; Sun, Xiaolin; Vandenberg, Kirsten; Rockman, Steve; Ding, Yan; Zhu, Lei; Wei, Wei; Wang, Changqi; Karnowski, Alexander; Belz, Gabrielle T; Ghali, Joanna R; Cook, Matthew C; Riminton, D Sean; Veillette, André; Schwartzberg, Pamela L; Mackay, Fabienne; Brink, Robert; Tangye, Stuart G; Vinuesa, Carola G; Mackay, Charles R; Li, Zhanguo; Yu, Di

2013-10-17

Follicular B helper T (Tfh) cells support high affinity and long-term antibody responses. Here we found that within circulating CXCR5⁺ CD4⁺ T cells in humans and mice, the CCR7(lo)PD-1(hi) subset has a partial Tfh effector phenotype, whereas CCR7(hi)PD-1(lo) cells have a resting phenotype. The circulating CCR7(lo)PD-1(hi) subset was indicative of active Tfh differentiation in lymphoid organs and correlated with clinical indices in autoimmune diseases. Thus the CCR7(lo)PD-1(hi) subset provides a biomarker to monitor protective antibody responses during infection or vaccination and pathogenic antibody responses in autoimmune diseases. Differentiation of both CCR7(hi)PD-1(lo) and CCR7(lo)PD-1(hi) subsets required ICOS and BCL6, but not SAP, suggesting that circulating CXCR5⁺ helper T cells are primarily generated before germinal centers. Upon antigen reencounter, CCR7(lo)PD-1(hi) CXCR5⁺ precursors rapidly differentiate into mature Tfh cells to promote antibody responses. Therefore, circulating CCR7(lo)PD-1(hi) CXCR5⁺ CD4⁺ T cells are generated during active Tfh differentiation and represent a new mechanism of immunological early memory. Copyright © 2013 Elsevier Inc. All rights reserved.

Influence of the CCR-5/MIP-1 α Axis in the Pathogenesis of Rocio Virus Encephalitis in a Mouse Model

PubMed Central

Chávez, Juliana H.; França, Rafael F. O.; Oliveira, Carlo J. F.; de Aquino, Maria T. P.; Farias, Kleber J. S.; Machado, Paula R. L.; de Oliveira, Thelma F. M.; Yokosawa, Jonny; Soares, Edson G.; da Silva, João S.; da Fonseca, Benedito A. L.; Figueiredo, Luiz T. M.

2013-01-01

Rocio virus (ROCV) caused an outbreak of human encephalitis during the 1970s in Brazil and its immunopathogenesis remains poorly understood. CC-chemokine receptor 5 (CCR5) is a chemokine receptor that binds to macrophage inflammatory protein (MIP-1 α). Both molecules are associated with inflammatory cells migration during infections. In this study, we demonstrated the importance of the CCR5 and MIP-1 α, in the outcome of viral encephalitis of ROCV-infected mice. CCR5 and MIP-1 α knockout mice survived longer than wild-type (WT) ROCV-infected animals. In addition, knockout mice had reduced inflammation in the brain. Assessment of brain viral load showed mice virus detection five days post-infection in wild-type and CCR5−/− mice, while MIP-1 α−/− mice had lower viral loads seven days post-infection. Knockout mice required a higher lethal dose than wild-type mice as well. The CCR5/MIP-1 α axis may contribute to migration of infected cells to the brain and consequently affect the pathogenesis during ROCV infection. PMID:24080631

Increased circulating fibrocytes are associated with higher reticulocyte percent in children with sickle cell anemia.

PubMed

Karafin, Matthew S; Dogra, Shibani; Rodeghier, Mark; Burdick, Marie; Mehrad, Borna; Rose, C Edward; Strieter, Robert M; DeBaun, Michael R; Strunk, Robert C; Field, Joshua J

2016-03-01

Interstitial lung disease is common in patients with sickle cell anemia (SCA). Fibrocytes are circulating cells implicated in the pathogenesis of pulmonary fibrosis and airway remodeling in asthma. In this study, we tested the hypotheses that fibrocyte levels are: (1) increased in children with SCA compared to healthy controls, and (2) associated with pulmonary disease. Cross-sectional cohort study of children with SCA who participated in the Sleep Asthma Cohort Study. Fibrocyte levels were obtained from 45 children with SCA and 24 controls. Mean age of SCA cases was 14 years and 53% were female. In children with SCA, levels of circulating fibrocytes were greater than controls (P < 0.01). The fibrocytes expressed a hierarchy of chemokine receptors, with CXCR4 expressed on the majority of cells and CCR2 and CCR7 expressed on a smaller subset. Almost half of fibrocytes demonstrated α-smooth muscle actin activation. Increased fibrocyte levels were associated with a higher reticulocyte count (P = 0.03) and older age (P = 0.048) in children with SCA. However, children with increased levels of fibrocytes were not more likely to have asthma or lower percent predicted forced expiratory volume in 1 sec/forced vital capacity (FEV1 /FVC) or FEV1 than those with lower fibrocyte levels. Higher levels of fibrocytes in children with SCA compared to controls may be due to hemolysis. Longitudinal studies may be able to better assess the relationship between fibrocyte level and pulmonary dysfunction. © 2015 Wiley Periodicals, Inc.

Polymorphisms in chemokine and receptor genes and gastric cancer risk and survival in a high risk Polish population

PubMed Central

Gawron, Andrew J.; Fought, Angela J.; Lissowska, Jolanta; Ye, Weimin; Zhang, Xiao; Chow, Wong-Ho; Freeman, Laura E. Beane; Hou, Lifang

2010-01-01

Objective To examine if genetic variations in chemokine receptor and ligand genes are associated with gastric cancer risk and survival. Methods The study included 298 cases and 417 controls from a population-based study of gastric cancer conducted in Warsaw, Poland in 1994–1996. We investigated seven single nucleotide polymorphisms in a chemokine ligand (CXCL12) and chemokine receptor (CCR2, CCR5, CX3CR1) genes and one frameshift deletion (CCR5) in blood leukocyte DNA in relation to gastric cancer risk and survival. Genotyping was conducted at the NCI Core Genotyping Facility. Odds ratios and 95% confidence intervals were computed using univariate and multivariate logistic regression models. Survival analysis was performed using Cox proportional hazards models. Results Gastric cancer risk was not associated with single chemokine polymorphisms. A CCR5 haplotype that contained the common alleles of IVS1+151 G>T (rs2734648), IVS2+80 C>T (rs1800024) and minor allele of IVS1+246 A>G (rs1799987) was associated with a borderline significantly increased risk (OR = 1.5, 95% CI: 1.0–2.2). For gastric cancer cases, there was a greater risk of death for carriers of the minor alleles of CCR2 Ex2+241 G>A (rs1799864) (HR = 1.5, 95% CI: 1.1–2.1) and CCR5 IVS2+80 C>T (rs1800024) (HR = 1.5, 95% CI: 1.1–2.1). Carriers of the CCR5 minor allele of IVS1+151 G>T (rs2734648) had a decreased risk of death compared to homozygote carriers of the common allele (HR = 0.8, 95% CI: 0.6–1.0). Conclusions Our findings do not support an association between gastric cancer risk and single chemokine genetic variation. The observed associations between cancer risk and a CCR5 haplotype and between survival and polymorphisms in CCR2 and CCR5 need replication in future studies. PMID:21091093

Frequency analysis of the delta32ccr5 HIV resistance allele in a medieval plague mass grave.

PubMed

Kremeyer, Barbara; Hummel, Susanne; Herrmann, Bernd

2005-03-01

The 32 basepair deletion in the gene for the human chemokine receptor CCR5 (delta32ccr5) conferring resistance against HIV-1 infection is present in Caucasian populations. The mutant allele is believed to have originated by a single mutational event in historic times and to have reached its present population frequency of an average 10 % in Europe through selective pressure by a pathogenic agent. Because of their great impact on European populations, the medieval Plague epidemics have been considered as a possible candidate. To test this hypothesis, we studied the delta32ccr5-frequency in 35 individuals from a mass grave containing victims of the 14th century Plague pandemic in Lübeck, Northern Germany, and compared them to the frequency in a control group from the same burial site, dating from the time before the first Plague pandemic. If the delta32ccr5 allele conferred an at least partial resistance against the medieval Plague, its frequency would be expected to be lower in those that died in the pandemic, than it was in the local population before the arrival of the Plague. The CCR5 locus could be typed successfully for 14 Plague victims and for 20 individuals from the medieval control group. We found a delta32ccr5 allelic frequency of 14.2% and 12.5%, respectively. The difference between these figures is not statistically significant. Furthermore, they are comparable to the delta32ccr5 frequency for nowadays Northern Europe. We therefore conclude that the medieval Plague pandemic has not contributed to an increase in the allelic frequency of the mutant delta32ccr5 allele and that, if there has been a positive selection of this allele, it is likely to have occurred before the 14th century and thus before the arrival of the Plague in Europe.

Genetic Susceptibility to Cardiac and Digestive Clinical Forms of Chronic Chagas Disease: Involvement of the CCR5 59029 A/G Polymorphism

PubMed Central

de Oliveira, Amanda Priscila; Bernardo, Cássia Rubia; Camargo, Ana Vitória da Silveira; Ronchi, Luiz Sérgio; Borim, Aldenis Albaneze; Brandão de Mattos, Cinara Cássia; de Campos Júnior, Eumildo; Castiglioni, Lílian; Netinho, João Gomes; Cavasini, Carlos Eugênio; Bestetti, Reinaldo Bulgarelli; de Mattos, Luiz Carlos

2015-01-01

The clinical manifestations of chronic Chagas disease include the cardiac form of the disease and the digestive form. Not all the factors that act in the variable clinical course of this disease are known. This study investigated whether the CCR5Δ32 (rs333) and CCR5 59029 A/G (promoter region—rs1799987) polymorphisms of the CCR5 gene are associated with different clinical forms of chronic Chagas disease and with the severity of left ventricular systolic dysfunction in patients with chronic Chagas heart disease (CCHD). The antibodies anti-T. cruzi were identified by ELISA. PCR and PCR-RFLP were used to identify the CCR5Δ32 and CCR5 59029 A/G polymorphisms. The chi-square test was used to compare variables between groups. There was a higher frequency of the AA genotype in patients with CCHD compared with patients with the digestive form of the disease and the control group. The results also showed a high frequency of the AG genotype in patients with the digestive form of the disease compared to the other groups. The results of this study show that the CCR5Δ32 polymorphism does not seem to influence the different clinical manifestations of Chagas disease but there is involvement of the CCR5 59029 A/G polymorphism in susceptibility to the different forms of chronic Chagas disease. Besides, these polymorphisms do not influence left ventricular systolic dysfunction in patients with CCHD. PMID:26599761

The Frequency of CCR5del32 Mutation in Populations of Russians, Tatars and Bashkirs of Chelyabinsk Region, Russia.

PubMed

Govorovskaya, Irina; Khromova, Elena; Suslova, Tatiana; Alexeev, Leonid; Kofiadi, Ilya

2016-12-01

The distribution of genetic variants associated with natural resistance to viral infections can vary among human ethnic groups due to evolutionary factors, defining the different epidemiologic background of world populations. The polymorphisms, defining the natural resistance to HIV-infection and the rate of progression up to AIDS, are very important since epidemic is still on rise. We have studied the distribution of allele and genotype frequencies of CCR5delta32 mutation in major populations inhabiting Chelyabinsk region of the Russian Federation. Genetic survey included the population of 509 potential blood marrow donors: Russians (N = 300), Bashkirs (N = 118) and Tatars (N = 91). The genotyping assay was performed using real-time polymerase chain reaction (real-time PCR). The genotypes were defined by melting curve analysis. The CCR5delta32 allele and CCR5delta32/delta32 genotype are presented in population of Russians in Chelyabinsk region with the frequencies of F x  = 10.83% and P x  = 1.67, for the CCR5delta32 allele and its homozygosity, respectively. In populations of Bashkirs and Tatars CCR5delta32 allele and CCR5delta32/delta32 genotype are presented at lower frequencies of F x  = 6.36%/P x  = 0.85 and F x  = 7.14%/P x  = 1.10, respectively. These data are consistent with the theory of northern origin of the CCR5delta32 mutation.

A CCL chemokine-derived peptide (CDIP-2) exerts anti-inflammatory activity via CCR1, CCR2 and CCR3 chemokine receptors: Implications as a potential therapeutic treatment of asthma.

PubMed

Méndez-Enríquez, E; Medina-Tamayo, J; Soldevila, G; Fortoul, T I; Anton, B; Flores-Romo, L; García-Zepeda, E A

2014-05-01

Allergic asthma is a chronic inflammatory disease characterized by the accumulation of eosinophils, Th2 cells and mononuclear cells in the airways, leading to changes in lung architecture and subsequently reduced respiratory function. We have previously demonstrated that CDIP-2, a chemokine derived peptide, reduced in vitro chemotaxis and decreased cellular infiltration in a murine model of allergic airway inflammation. However, the mechanisms involved in this process have not been identified yet. Now, we found that CDIP-2 reduces chemokine-mediated functions via interactions with CCR1, CCR2 and CCR3. Moreover, using bone marrow-derived eosinophils, we demonstrated that CDIP-2 modifies the calcium fluxes induced by CCL11 and down-modulated CCR3 expression. Finally, CDIP-2 treatment in a murine model of OVA-induced allergic airway inflammation reduced leukocyte recruitment and decreases production of cytokines. These data suggest that chemokine-derived peptides represent new therapeutic tools to generate more effective antiinflammatory drugs. Copyright © 2014 Elsevier B.V. All rights reserved.

Inflammatory Monocytes Orchestrate Innate Antifungal Immunity in the Lung

PubMed Central

Dutta, Orchi; Kasahara, Shinji; Donnelly, Robert; Du, Peicheng; Rosenfeld, Jeffrey; Leiner, Ingrid; Chen, Chiann-Chyi; Ron, Yacov; Hohl, Tobias M.; Rivera, Amariliz

2014-01-01

Aspergillus fumigatus is an environmental fungus that causes invasive aspergillosis (IA) in immunocompromised patients. Although -CC-chemokine receptor-2 (CCR2) and Ly6C-expressing inflammatory monocytes (CCR2+Mo) and their derivatives initiate adaptive pulmonary immune responses, their role in coordinating innate immune responses in the lung remain poorly defined. Using conditional and antibody-mediated cell ablation strategies, we found that CCR2+Mo and monocyte-derived dendritic cells (Mo-DCs) are essential for innate defense against inhaled conidia. By harnessing fluorescent Aspergillus reporter (FLARE) conidia that report fungal cell association and viability in vivo, we identify two mechanisms by which CCR2+Mo and Mo-DCs exert innate antifungal activity. First, CCR2+Mo and Mo-DCs condition the lung inflammatory milieu to augment neutrophil conidiacidal activity. Second, conidial uptake by CCR2+Mo temporally coincided with their differentiation into Mo-DCs, a process that resulted in direct conidial killing. Our findings illustrate both indirect and direct functions for CCR2+Mo and their derivatives in innate antifungal immunity in the lung. PMID:24586155

Carbon catabolite regulation in Streptomyces: new insights and lessons learned.

PubMed

Romero-Rodríguez, Alba; Rocha, Diana; Ruiz-Villafán, Beatriz; Guzmán-Trampe, Silvia; Maldonado-Carmona, Nidia; Vázquez-Hernández, Melissa; Zelarayán, Augusto; Rodríguez-Sanoja, Romina; Sánchez, Sergio

2017-09-01

One of the most significant control mechanisms of the physiological processes in the genus Streptomyces is carbon catabolite repression (CCR). This mechanism controls the expression of genes involved in the uptake and utilization of alternative carbon sources in Streptomyces and is mostly independent of the phosphoenolpyruvate phosphotransferase system (PTS). CCR also affects morphological differentiation and the synthesis of secondary metabolites, although not all secondary metabolite genes are equally sensitive to the control by the carbon source. Even when the outcome effect of CCR in bacteria is the same, their essential mechanisms can be rather different. Although usually, glucose elicits this phenomenon, other rapidly metabolized carbon sources can also cause CCR. Multiple efforts have been put through to the understanding of the mechanism of CCR in this genus. However, a reasonable mechanism to explain the nature of this process in Streptomyces does not yet exist. Several examples of primary and secondary metabolites subject to CCR will be examined in this review. Additionally, recent advances in the metabolites and protein factors involved in the Streptomyces CCR, as well as their mechanisms will be described and discussed in this review.

Insights into the binding modes of CC chemokine receptor 4 (CCR4) inhibitors: a combined approach involving homology modelling, docking, and molecular dynamics simulation studies.

PubMed

Gadhe, Changdev G; Kim, Mi-hyun

2015-02-01

CC chemokine receptor 4 (CCR4), a G protein-coupled receptor (GPCR), plays a vital role in the progression of asthma, T-cell lymphoma, inflammation, and Alzheimer's disease. To date, the structure of CCR4 has not been determined. Therefore, the nature of the interactions between inhibitors and CCR4 is not well known. In this study, we used CCR5 as a template to model the structure of CCR4. Docking studies were performed for four naphthalene-sulphonamide derivatives and crucial ligand-protein interactions were analysed. Molecular dynamics (MD) simulations of these complexes (100 ns each) were carried out to gain insights into the interactions between ligands and CCR4. MD simulations revealed that the residues identified by the docking were displaced and new residues were inserted near the ligands. Results of a principal component analysis (PCA) suggested that CCR4 unfolds at the extracellular site surrounding the ligands. Our simulations identified crucial residues involved in CCR4 antagonism, which were supported by previous mutational studies. Additionally, we identified Ser3.29, Leu3.33, Ser5.39, Phe6.47, Ile7.35, Thr7.38, Thr7.40, and Ala7.42 as residues that play crucial roles in CCR4 antagonism. Mutational studies will help elucidate the significance of these residues in CCR4 antagonism. An understanding of ligand-CCR4 interactions might aid in the design of novel CCR4 inhibitors.

CCR5 polymorphism and plague resistance in natural populations of the black rat in Madagascar.

PubMed

Tollenaere, C; Rahalison, L; Ranjalahy, M; Rahelinirina, S; Duplantier, J-M; Brouat, C

2008-12-01

Madagascar remains one of the world's largest plague foci. The black rat, Rattus rattus, is the main reservoir of plague in rural areas. This species is highly susceptible to plague in plague-free areas (low-altitude regions), whereas rats from the plague focus areas (central highlands) have evolved a disease-resistance polymorphism. We used the candidate gene CCR5 to investigate the genetic basis of plague resistance in R. rattus. We found a unique non-synonymous substitution (H184R) in a functionally important region of the gene. We then compared (i) CCR5 genotypes of dying and surviving plague-challenged rats and (ii) CCR5 allelic frequencies in plague focus and plague-free populations. Our results suggested a higher prevalence of the substitution in resistant animals compared to susceptible individuals, and a tendency for higher frequencies in plague focus areas compared to plague-free areas. Therefore, the CCR5 polymorphism may be involved in Malagasy black rat plague resistance. CCR5 and other undetermined plague resistance markers may provide useful biological information about host evolution and disease dynamics.

Chemokine GPCR Signaling Inhibits β-Catenin during Zebrafish Axis Formation

PubMed Central

Wu, Shu-Yu; Shin, Jimann; Sepich, Diane S.; Solnica-Krezel, Lilianna

2012-01-01

Embryonic axis formation in vertebrates is initiated by the establishment of the dorsal Nieuwkoop blastula organizer, marked by the nuclear accumulation of maternal β-catenin, a transcriptional effector of canonical Wnt signaling. Known regulators of axis specification include the canonical Wnt pathway components that positively or negatively affect β-catenin. An involvement of G-protein coupled receptors (GPCRs) was hypothesized from experiments implicating G proteins and intracellular calcium in axis formation, but such GPCRs have not been identified. Mobilization of intracellular Ca2+ stores generates Ca2+ transients in the superficial blastomeres of zebrafish blastulae when the nuclear accumulation of maternal β-catenin marks the formation of the Nieuwkoop organizer. Moreover, intracellular Ca2+ downstream of non-canonical Wnt ligands was proposed to inhibit β-catenin and axis formation, but mechanisms remain unclear. Here we report a novel function of Ccr7 GPCR and its chemokine ligand Ccl19.1, previously implicated in chemotaxis and other responses of dendritic cells in mammals, as negative regulators of β-catenin and axis formation in zebrafish. We show that interference with the maternally and ubiquitously expressed zebrafish Ccr7 or Ccl19.1 expands the blastula organizer and the dorsoanterior tissues at the expense of the ventroposterior ones. Conversely, Ccr7 or Ccl19.1 overexpression limits axis formation. Epistatic analyses demonstrate that Ccr7 acts downstream of Ccl19.1 ligand and upstream of β-catenin transcriptional targets. Moreover, Ccl19/Ccr7 signaling reduces the level and nuclear accumulation of maternal β-catenin and its axis-inducing activity and can also inhibit the Gsk3β -insensitive form of β-catenin. Mutational and pharmacologic experiments reveal that Ccr7 functions during axis formation as a GPCR to inhibit β-catenin, likely by promoting Ca2+ transients throughout the blastula. Our study delineates a novel negative, Gsk3β-independent control mechanism of β-catenin and implicates Ccr7 as a long-hypothesized GPCR regulating vertebrate axis formation. PMID:23055828

CCR7(lo)PD-1(hi) CXCR5(+) CD4(+) T cells are positively correlated with levels of IL-21 in active and transitional cystic echinococcosis patients.

PubMed

Zhang, Fengbo; Pang, Nannan; Zhu, Yuejie; Zhou, Dexian; Zhao, Hui; Hu, Jinwei; Ma, Xiumin; Li, Jun; Wen, Hao; Samten, Buka; Fan, Haining; Ding, Jianbing

2015-10-26

In our study, we investigated whether circulating T follicular helper (Tfh) and the related cytokines are involved in human cystic echinococcosis (CE). A total of 64 patients with CE and 30 healthy controls were enrolled in this study. Percentages of CCR7(lo)PD-1(hi) cells within CXCR5(+) CD4(+) T cells (circulating Tfh cells) were detected by flow cytometry. Levels of IL-21 and IL-4 in peripheral blood were detected by cytometric bead array. The mRNA expression of IL-21, IL-4, Bcl-6, and Blimp-1 in peripheral blood mononuclear cells (PBMCs) were measured by real-time PCR. Levels of IgG1, IgG2, IgG3, and IgG4 in the patients' sera were measured using enzyme-linked immunosorbent assay. Percentages of circulating Tfh cells were significantly increased in the CE1, CE2, and CE3 groups (p < 0.05). The concentrations of IL-21 and IL-4 in the serum were significantly increased in CE1, CE2, and CE3 groups (p < 0.05). IL-21 was positively correlated with circulating Tfh cells in CE3 group (r = 0.779, p < 0.05). The mRNA levels of IL-21, IL-4, and Bcl-6 were increased in CE1, CE2, and CE3 groups. Levels of IgG1 and IgG4 in patients' sera were increased in CE1, CE2, and CE3 groups. Levels of IgG2 and IgG3 were increased in CE4-5 group. Additionally, after stimulation with hydatid fluid in vitro, the levels of circulating Tfh cells, IL-21 and IL-4 in PBMCs isolated from CE patients were significantly increased (p < 0.05). The levels of circulating Tfh and related cytokines were significantly increased in CE patients, suggesting that they are involved in human CE.

Different Pathogenesis of CCR5-Using Primary HIV-1 Isolates from Non-Switch and Switch Virus Patients in Human Lymphoid Tissue Ex Vivo

NASA Technical Reports Server (NTRS)

Iarlsson, Ingrid; Grivel, Jean-Charles; Chen. Silvia; Karlsson, Anders; Albert, Jan; Fenyol, Eva Maria; Margolis, Leonid B.

2005-01-01

CCR5-utilizing HIV-1 variants (R5) typically transmit infection and dominate its early stages, whereas emergence of CXCR4-using (X4 or R5X4) HIV-1 is often associated with disease progression. However, such a switch in co-receptor usage can only be detected in approximately onehalf of HIV-infected patients (switch virus patients), and progression to immunodeficiency may also occur in patients without detectable switch in co-receptor usage (non-switch virus patients). Here, we used a system of ex vivo-infected tonsillar tissue to compare the pathogenesis of sequential primary R5 HIV-1 isolates from the switch and non-switch patients. Inoculation of ex vivo tissue with these R5 isolates resulted in viral replication and CCR5(+)CD4(+) T cell depletion. The levels of such depletion by HIV-1 isolated from non-switch virus patients were significantly higher than those by R5 HIV-1 isolates from switch virus patients. T cell depletion seemed to be controlled by viral factors and did not significantly vary between tissues from different donors. In contrast, viral replication did not correlate with the switch status of the patients; in tissues fiom different donors it varied 30-fold and seemed to be controlled by a combination of viral and tissue factors. Nevertheless, replication-level hierarchy among sequential isolates remained constant in tissues from various donors. Viral load in vivo was higher in switch virus patients compared to non-switch virus patients. The high cytopathogenicity of CCR5(+)CD4(+) T cells by R5 HIV-1 isolates from non-switch virus patients may explain the steady decline of CD4(+) T cells in the absence of CXCR4 using virus; elimination of target cells by these isolates may limit their own replication in vivo.

The Role of Microglial Subsets in Regulating Traumatic Brain Injury

DTIC Science & Technology

2012-07-01

22 Ccr7 Ccr7 ~ <u ~ ’ log2 FC .M2 Ill anti-inflammatory -3 0 3 .M1 ~ infla to mma ry 0 M2andM1 • chemotaxis I1IJ wound healing Figure 3...Representative Macs Macs Genes ’"’-::n -1 0 1 log2 FC Cc/7, Cxc/1, Ppap2b, Mt2, Sema3c, C1cn7 Cd83, Cybb, Ciita, Nfkb1, Cc/5 11111ra1, l/1rl2... Barcelona , October 26-30, 2010. CONCLUSIONS Our studies have shown that resting microglia do not express markers for an M1 or an M2 phenotype, as

Development of an inflammation imaging tracer, 111In-DOTA-DAPTA, targeting chemokine receptor CCR5 and preliminary evaluation in an ApoE-/- atherosclerosis mouse model.

PubMed

Wei, Lihui; Petryk, Julia; Gaudet, Chantal; Kamkar, Maryam; Gan, Wei; Duan, Yin; Ruddy, Terrence D

2018-02-07

Chemokine receptor 5 (CCR5) plays an important role in atherosclerosis. Our objective was to develop a SPECT tracer targeting CCR5 for imaging plaque inflammation by radiolabeling D-Ala-peptide T-amide (DAPTA), a CCR5 antagonist, with 111 In. 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) conjugated DAPTA (DOTA-DAPTA) was labeled with 111 In. Cell uptake studies were conducted in U87-CD4-CCR5 and U87-MG cells. Biodistribution was determined in C57BL/6 mice. Autoradiography, en face and Oil Red O (ORO) imaging studies were performed in ApoE -/- mice. DOTA-DAPTA was radiolabeled with 111 In with high radiochemical purity (> 98%) and specific activity (70 MBq·nmol). 111 In-DOTA-DAPTA exhibited fast blood and renal clearance and high spleen uptake. The U87-CD4-CCR5 cells had significantly higher uptake in comparison to the U87-MG cells. The cell uptake was reduced by three times with DAPTA, indicating the receptor specificity of the uptake. Autoradiographic images showed significantly higher lesion uptake of 111 In-DOTA-DAPTA in ApoE -/- mice than that in C57BL/6 mice. The tracer uptake in 4 month old ApoE -/- high fat diet (HFD) mice with blocking agent was twofold lower than the same mice without the blocking agent, demonstrating the specificity of the tracer for the CCR5 receptor. 111 In-DOTA-DAPTA, specifically targeting chemokine receptor CCR5, is a potential SPECT agent for imaging inflammation in atherosclerosis.

Ancient DNA Investigation of a Medieval German Cemetery Confirms Long-Term Stability of CCR5-Δ32 Allele Frequencies in Central Europe.

PubMed

Bouwman, Abigail; Shved, Natallia; Akgül, Gülfirde; Rühli, Frank; Warinner, Christina

2017-04-01

The CCR5-Δ32 mutation present in European populations is among the most prominently debated cases of recent positive selection in humans. This allele, a 32-bp deletion that renders the T-cell CCR5 receptor nonfunctional, has important epidemiological and public health significance, as homozygous carriers are resistant to several HIV strains. However, although the function of this allele in preventing HIV infection is now well described, its human evolutionary origin is poorly understood. Initial attempts to determine the emergence of the CCR5-Δ32 allele pointed to selection during the 14th-century Black Death pandemic; however, subsequent analyses suggest that the allele rose in frequency more than 5,000 years ago, possibly through drift. Recently, three studies have identified populations predating the 14th century CE that are positive for the CCR5-Δ32 allele, supporting the claim for a more ancient origin. However, these studies also suggest poorly understood regional differences in the recent evolutionary history of the CCR5-Δ32 allele. Here a new hydrolysis-probe-based real-time PCR assay was designed to ascertain CCR5 allele frequency in 53 individuals from a 10th- to 12th-century CE church and convent complex in central Germany that predates outbreaks of the Black Death pandemic. High-confidence genotypes were obtained for 32 individuals, and results show that CCR5-Δ32 allele frequency has remained unchanged in this region of Central Europe over the last millennium, suggesting that there has been no strong positive selective pressure over this time period and confirming a more ancient origin for the allele.

Functional impact of HIV coreceptor-binding site mutations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Biscone, Mark J.; Miamidian, John L.; Muchiri, John M.

2006-07-20

The bridging sheet region of the gp120 subunit of the HIV-1 Env protein interacts with the major virus coreceptors, CCR5 and CXCR4. We examined the impact of mutations in and adjacent to the bridging sheet region of an X4 tropic HIV-1 on membrane fusion and entry inhibitor susceptibility. When the V3-loop of this Env was changed so that CCR5 was used, the effects of these same mutations on CCR5 use were assayed as well. We found that coreceptor-binding site mutations had greater effects on CXCR4-mediated fusion and infection than when CCR5 was used as a coreceptor, perhaps related to differencesmore » in coreceptor affinity. The mutations also reduced use of the alternative coreceptors CCR3 and CCR8 to varying degrees, indicating that the bridging sheet region is important for the efficient utilization of both major and minor HIV coreceptors. As seen before with a primary R5 virus strain, bridging sheet mutations increased susceptibility to the CCR5 inhibitor TAK-779, which correlated with CCR5 binding efficiency. Bridging sheet mutations also conferred increased susceptibility to the CXCR4 ligand AMD-3100 in the context of the X4 tropic Env. However, these mutations had little effect on the rate of membrane fusion and little effect on susceptibility to enfuvirtide, a membrane fusion inhibitor whose activity is dependent in part on the rate of Env-mediated membrane fusion. Thus, mutations that reduce coreceptor binding and enhance susceptibility to coreceptor inhibitors can affect fusion and enfuvirtide susceptibility in an Env context-dependent manner.« less

Short communication: evidence of HIV type 1 clade C env clones containing low V3 loop charge obtained from an AIDS patient in India that uses CXCR6 and CCR8 for entry in addition to CCR5.

PubMed

Gharu, Lavina; Ringe, Rajesh; Satyakumar, Anupindi; Patil, Ajit; Bhattacharya, Jayanta

2011-02-01

Abstract HIV-1 clade C is the major subtype circulating in India and preferentially uses CCR5 during the entire disease course. We have recently shown that env clones from an Indian patient; NARI-VB105 uses multiple coreceptors for entry and was presented with an unusual V3 loop sequence giving rise to high net V3 loop positive charges. Here we show that env clones belonging to subtype C obtained from an AIDS patient, NARI-VB52, use CXCR6 and CCR8 in addition to CCR5 for entry. However, unlike the NARI-105 patient, the env clones contained a low V3 loop net charge of +3 with a conserved GPGQ motif typical of CCR5 using subtype C strains, indicating that residues outside the V3 loop contributed to extended coreceptor use in this particular patient.

[CCR5, CCR2, apoe, p53, ITGB3 and HFE gene polymorphism in Western Siberia long-livers].

PubMed

Ivanoshchuk, D E; Mikhaĭlova, S V; Kulikov, I V; Maksimov, V N; Voevoda, M I; Romashchenko, A G

2012-01-01

In order to estimate the distribution of some polymorphisms for the CCR5, CCR2, apoE, p53, ITGB3, and HFE genes in Russian long-livers from Western Siberia, a sample of 271 individuals (range 90-105 years) was examined. It was demonstrated that carriage of the delta32 polymorphism for the CCR5 gene, V64/polymorphism for the CCR2 gene, e2/e3/e4 for the apoE gene, L33P for the ITGB3 gene, as well as H63D and S65C polymorphisms for the HFE gene does not influence on predisposition to the longevity; carriage of the 282 Y allele for the HFE gene negatively influences on the longevity; carriage of the heterozygous genotype for the R72P polymorphism for the p53 gene correlates with the longevity of elderly people.

Lung dendritic cells imprint T cell lung homing and promote lung immunity through the chemokine receptor CCR4

PubMed Central

Strassner, James P.

2013-01-01

T cell trafficking into the lung is critical for lung immunity, but the mechanisms that mediate T cell lung homing are not well understood. Here, we show that lung dendritic cells (DCs) imprint T cell lung homing, as lung DC–activated T cells traffic more efficiently into the lung in response to inhaled antigen and at homeostasis compared with T cells activated by DCs from other tissues. Consequently, lung DC–imprinted T cells protect against influenza more effectively than do gut and skin DC–imprinted T cells. Lung DCs imprint the expression of CCR4 on T cells, and CCR4 contributes to T cell lung imprinting. Lung DC–activated, CCR4-deficient T cells fail to traffic into the lung as efficiently and to protect against influenza as effectively as lung DC–activated, CCR4-sufficient T cells. Thus, lung DCs imprint T cell lung homing and promote lung immunity in part through CCR4. PMID:23960189

High-dose bee venom exposure induces similar tolerogenic B-cell responses in allergic patients and healthy beekeepers.

PubMed

Boonpiyathad, T; Meyer, N; Moniuszko, M; Sokolowska, M; Eljaszewicz, A; Wirz, O F; Tomasiak-Lozowska, M M; Bodzenta-Lukaszyk, A; Ruxrungtham, K; van de Veen, W

2017-03-01

The involvement of B cells in allergen tolerance induction remains largely unexplored. This study investigates the role of B cells in this process, by comparing B-cell responses in allergic patients before and during allergen immunotherapy (AIT) and naturally exposed healthy beekeepers before and during the beekeeping season. Circulating B cells were characterized by flow cytometry. Phospholipase A2 (PLA)-specific B cells were identified using dual-color staining with fluorescently labeled PLA. Expression of regulatory B-cell-associated surface markers, interleukin-10, chemokine receptors, and immunoglobulin heavy-chain isotypes, was measured. Specific and total IgG1, IgG4, IgA, and IgE from plasma as well as culture supernatants of PLA-specific cells were measured by ELISA. Strikingly, similar responses were observed in allergic patients and beekeepers after venom exposure. Both groups showed increased frequencies of plasmablasts, PLA-specific memory B cells, and IL-10-secreting CD73 - CD25 + CD71 + B R 1 cells. Phospholipase A2-specific IgG4-switched memory B cells expanded after bee venom exposure. Interestingly, PLA-specific B cells showed increased CCR5 expression after high-dose allergen exposure while CXCR4, CXCR5, CCR6, and CCR7 expression remained unaffected. This study provides the first detailed characterization of allergen-specific B cells before and after bee venom tolerance induction. The observed B-cell responses in both venom immunotherapy-treated patients and naturally exposed beekeepers suggest a similar functional immunoregulatory role for B cells in allergen tolerance in both groups. These findings can be investigated in other AIT models to determine their potential as biomarkers of early and successful AIT responses. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Chemokine receptors CCR6 and CXCR3 are necessary for CD4(+) T cell mediated ocular surface disease in experimental dry eye disease.

PubMed

Coursey, Terry G; Gandhi, Niral B; Volpe, Eugene A; Pflugfelder, Stephen C; de Paiva, Cintia S

2013-01-01

CD4(+) T cells are essential to pathogenesis of ocular surface disease in dry eye. Two subtypes of CD4(+) T cells, Th1 and Th17 cells, function concurrently in dry eye to mediate disease. This occurs in spite of the cross-regulation of IFN-γ and IL-17A, the prototypical cytokines Th1 and Th17 cells, respectively. Essential to an effective immune response are chemokines that direct and summon lymphocytes to specific tissues. T cell trafficking has been extensively studied in other models, but this is the first study to examine the role of chemokine receptors in ocular immune responses. Here, we demonstrate that the chemokine receptors, CCR6 and CXCR3, which are expressed on Th17 and Th1 cells, respectively, are required for the pathogenesis of dry eye disease, as CCR6KO and CXCR3KO mice do not develop disease under desiccating stress. CD4(+) T cells from CCR6KO and CXCR3KO mice exposed to desiccating stress (DS) do not migrate to the ocular surface, but remain in the superficial cervical lymph nodes. In agreement with this, CD4(+) T cells from CCR6 and CXCR3 deficient donors exposed to DS, when adoptively transferred to T cell deficient recipients manifest minimal signs of dry eye disease, including significantly less T cell infiltration, goblet cell loss, and expression of inflammatory cytokine and matrix metalloproteinase expression compared to wild-type donors. These findings highlight the important interaction of chemokine receptors on T cells and chemokine ligand expression on epithelial cells of the cornea and conjunctiva in dry eye pathogenesis and reveal potential new therapeutic targets for dry eye disease.

Chemokine Receptors CCR6 and CXCR3 Are Necessary for CD4+ T Cell Mediated Ocular Surface Disease in Experimental Dry Eye Disease

PubMed Central

Coursey, Terry G.; Gandhi, Niral B.; Volpe, Eugene A.; Pflugfelder, Stephen C.; de Paiva, Cintia S.

2013-01-01

CD4+ T cells are essential to pathogenesis of ocular surface disease in dry eye. Two subtypes of CD4+ T cells, Th1 and Th17 cells, function concurrently in dry eye to mediate disease. This occurs in spite of the cross-regulation of IFN-γ and IL-17A, the prototypical cytokines Th1 and Th17 cells, respectively. Essential to an effective immune response are chemokines that direct and summon lymphocytes to specific tissues. T cell trafficking has been extensively studied in other models, but this is the first study to examine the role of chemokine receptors in ocular immune responses. Here, we demonstrate that the chemokine receptors, CCR6 and CXCR3, which are expressed on Th17 and Th1 cells, respectively, are required for the pathogenesis of dry eye disease, as CCR6KO and CXCR3KO mice do not develop disease under desiccating stress. CD4+ T cells from CCR6KO and CXCR3KO mice exposed to desiccating stress (DS) do not migrate to the ocular surface, but remain in the superficial cervical lymph nodes. In agreement with this, CD4+ T cells from CCR6 and CXCR3 deficient donors exposed to DS, when adoptively transferred to T cell deficient recipients manifest minimal signs of dry eye disease, including significantly less T cell infiltration, goblet cell loss, and expression of inflammatory cytokine and matrix metalloproteinase expression compared to wild-type donors. These findings highlight the important interaction of chemokine receptors on T cells and chemokine ligand expression on epithelial cells of the cornea and conjunctiva in dry eye pathogenesis and reveal potential new therapeutic targets for dry eye disease. PMID:24223818

The Yeast PUF Protein Puf5 Has Pop2-Independent Roles in Response to DNA Replication Stress

PubMed Central

Traven, Ana; Lo, Tricia L.; Lithgow, Trevor; Heierhorst, Jörg

2010-01-01

PUFs are RNA binding proteins that promote mRNA deadenylation and decay and inhibit translation. Yeast Puf5 is the prototype for studying PUF-dependent gene repression. Puf5 binds to the Pop2 subunit of the Ccr4-Pop2-NOT mRNA deadenylase, recruiting the deadenylase and associated translational repressors to mRNAs. Here we used yeast genetics to show that Puf5 has additional roles in vivo that do not require Pop2. Deletion of PUF5 caused increased sensitivity to DNA replication stress in cells lacking Pop2, as well as in cells mutated for two activities recruited to mRNAs by the Puf5-Pop2 interaction, the deadenylase Ccr4 and the translational repressor Dhh1. A functional Puf5 RNA binding domain was required, and Puf5 cytoplasmic localisation was sufficient for resistance to replication stress, indicating posttranscriptional gene expression control is involved. In contrast to DNA replication stress, in response to the cell wall integrity pathway activator caffeine, PUF5 and POP2 acted in the same genetic pathway, indicating that functions of Puf5 in the caffeine response are mediated by Pop2-dependent gene repression. Our results support a model in which Puf5 uses multiple, Pop2-dependent and Pop2-independent mechanisms to control mRNA expression. The Pop2-independent roles for Puf5 could involve spatial control of gene expression, a proposition supported by our data indicating that the active form of Puf5 is localised to cytoplasmic foci. PMID:20498834

Defining the chemokine basis for leukocyte recruitment during viral encephalitis.

PubMed

Michlmayr, Daniela; McKimmie, Clive S; Pingen, Marieke; Haxton, Ben; Mansfield, Karen; Johnson, Nicholas; Fooks, Anthony R; Graham, Gerard J

2014-09-01

The encephalitic response to viral infection requires local chemokine production and the ensuing recruitment of immune and inflammatory leukocytes. Accordingly, chemokine receptors present themselves as plausible therapeutic targets for drugs aimed at limiting encephalitic responses. However, it remains unclear which chemokines are central to this process and whether leukocyte recruitment is important for limiting viral proliferation and survival in the brain or whether it is predominantly a driver of coincident inflammatory pathogenesis. Here we examine chemokine expression and leukocyte recruitment in the context of avirulent and virulent Semliki Forest virus (SFV) as well as West Nile virus infection and demonstrate rapid and robust expression of a variety of inflammatory CC and CXC chemokines in all models. On this basis, we define a chemokine axis involved in leukocyte recruitment to the encephalitic brain during SFV infection. CXCR3 is the most active; CCR2 is also active but less so, and CCR5 plays only a modest role in leukocyte recruitment. Importantly, inhibition of each of these receptors individually and the resulting suppression of leukocyte recruitment to the infected brain have no effect on viral titer or survival following infection with a virulent SFV strain. In contrast, simultaneous blockade of CXCR3 and CCR2 results in significantly reduced mortality in response to virulent SFV infection. In summary, therefore, our data provide an unprecedented level of insight into chemokine orchestration of leukocyte recruitment in viral encephalitis. Our data also highlight CXCR3 and CCR2 as possible therapeutic targets for limiting inflammatory damage in response to viral infection of the brain. Brain inflammation (encephalitis) in response to viral infection can lead to severe illness and even death. This therefore represents an important clinical problem and one that requires the development of new therapeutic approaches. Central to the pathogenesis of encephalitis is the recruitment of inflammatory leukocytes to the infected brain, a process driven by members of the chemokine family. Here we provide an in-depth analysis of the chemokines involved in leukocyte recruitment to the virally infected brain and demonstrate that simultaneous blockade of two of these receptors, namely, CXCR3 and CCR2, does not alter viral titers within the brain but markedly reduces inflammatory leukocyte recruitment and enhances survival in a murine model of lethal viral encephalitis. Our results therefore highlight chemokine receptors as plausible therapeutic targets in treating viral encephalitis. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

CCR2 antagonist CCX140-B provides renal and glycemic benefits in diabetic transgenic human CCR2 knockin mice

PubMed Central

Sullivan, Timothy; Miao, Zhenhua; Dairaghi, Daniel J.; Krasinski, Antoni; Wang, Yu; Zhao, Bin N.; Baumgart, Trageen; Ertl, Linda S.; Pennell, Andrew; Seitz, Lisa; Powers, Jay; Zhao, Ruiping; Ungashe, Solomon; Wei, Zheng; Boring, Landin; Tsou, Chia-Lin; Charo, Israel; Schall, Thomas J.; Jaen, Juan C.

2013-01-01

Chemokine (C-C motif) receptor 2 (CCR2) is central for the migration of monocytes into inflamed tissues. The novel CCR2 antagonist CCX140-B, which is currently in two separate phase 2 clinical trials in diabetic nephropathy, has recently been shown to reduce hemoglobin A1c and fasting blood glucose levels in type 2 diabetics. In this report, we describe the effects of this compound on glycemic and renal function parameters in diabetic mice. Since CCX140-B has a low affinity for mouse CCR2, transgenic human CCR2 knockin mice were generated and rendered diabetic with either a high-fat diet (diet-induced obesity) or by deletion of the leptin receptor gene (db/db). CCX140-B treatment in both models resulted in decreased albuminuria, which was associated with decreased glomerular hypertrophy and increased podocyte density. Moreover, treatment of diet-induced obese mice with CCX140-B resulted in decreased levels of fasting blood glucose and insulin, normalization of homeostatic model assessment of insulin resistance values, and decreased numbers of adipose tissue inflammatory macrophages. Unlike other CCR2 antagonists, CCX140-B had no effect on plasma levels of the CCR2 ligand CCL2 or on the numbers of blood monocytes. These results support the ongoing evaluation of this molecule in diabetic subjects with impaired renal function. PMID:23986513

A Simplified Technique for Evaluating Human "CCR5" Genetic Polymorphism

ERIC Educational Resources Information Center

Falteisek, Lukáš; Cerný, Jan; Janštová, Vanda

2013-01-01

To involve students in thinking about the problem of AIDS (which is important in the view of nondecreasing infection rates), we established a practical lab using a simplified adaptation of Thomas's (2004) method to determine the polymorphism of HIV co-receptor CCR5 from students' own epithelial cells. CCR5 is a receptor involved in inflammatory…

Monocyte profile in peripheral blood of gestational diabetes mellitus patients.

PubMed

Angelo, Ana G S; Neves, Carla T C; Lobo, Thalita F; Godoy, Ramon V C; Ono, Érika; Mattar, Rosiane; Daher, Silvia

2018-07-01

Gestational diabetes Mellitus has been considered an inflammatory disease involving different cells and mediators in its development. The role of innate immune cells in GDM physiopathology remains unclear, therefore this study was conducted to assess monocyte profile in GDM patients. This was a case-control study including 20 glucose-tolerant pregnant women (controls) and 18 GDM patients. Flow cytometry was used to assess peripheral blood monocytes subsets (classical, intermediate, non-classical), the expression of TLR4 and CCR2 chemokine receptor (CD192) and cytokines (TNFA, IL6, IL10) secretion by monocytes subsets. In addition, sCD14 serum levels were evaluated by ELISA. We observed increased percentage of CD14 + cells, decreased frequency of intermediate monocytes (CD14 + CD16 + ), and lower percentage of circulating monocytes (classical, intermediate and non-classical) that express TLR4 in the diabetic group compared to controls. Soluble CD14 + serum levels were higher in GDM patients compared to controls. There were no differences in the expression of the CCR2 chemokine receptor and cytokines (TNFA, IL6 and IL10) secretion between the studied groups. Our results demonstrated that GDM patients present impaired monocyte profile in the peripheral blood, suggesting that these cells are involved in GDM physiopathology. Copyright © 2017 Elsevier Ltd. All rights reserved.

CCR7 Maintains Nonresolving Lymph Node and Adipose Inflammation in Obesity.

PubMed

Hellmann, Jason; Sansbury, Brian E; Holden, Candice R; Tang, Yunan; Wong, Blenda; Wysoczynski, Marcin; Rodriguez, Jorge; Bhatnagar, Aruni; Hill, Bradford G; Spite, Matthew

2016-08-01

Accumulation of immune cells in adipose tissue promotes insulin resistance in obesity. Although innate and adaptive immune cells contribute to adipose inflammation, the processes that sustain these interactions are incompletely understood. Here we show that obesity promotes the accumulation of CD11c(+) adipose tissue immune cells that express C-C chemokine receptor 7 (CCR7) in mice and humans, and that CCR7 contributes to chronic inflammation and insulin resistance. We identified that CCR7(+) macrophages and dendritic cells accumulate in adipose tissue in close proximity to lymph nodes (LNs) (i.e., perinodal) and visceral adipose. Consistent with the role of CCR7 in regulating the migration of immune cells to LNs, obesity promoted the accumulation of CD11c(+) cells in LNs, which was prevented by global or hematopoietic deficiency of Ccr7 Obese Ccr7(-/-) mice had reduced accumulation of CD8(+) T cells, B cells, and macrophages in adipose tissue, which was associated with reduced inflammatory signaling. This reduction in maladaptive inflammation translated to increased insulin signaling and improved glucose tolerance in obesity. Therapeutic administration of an anti-CCR7 antibody phenocopied the effects of genetic Ccr7 deficiency in mice with established obesity. These results suggest that CCR7 plays a causal role in maintaining innate and adaptive immunity in obesity. © 2016 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.

Haplotypes in CCR5-CCR2, CCL3 and CCL5 are associated with natural resistance to HIV-1 infection in a Colombian cohort.

PubMed

Vega, Jorge A; Villegas-Ospina, Simón; Aguilar-Jiménez, Wbeimar; Rugeles, María T; Bedoya, Gabriel; Zapata, Wildeman

2017-06-01

Variants in genes encoding for HIV-1 co-receptors and their natural ligands have been individually associated to natural resistance to HIV-1 infection. However, the simultaneous presence of these variants has been poorly studied. To evaluate the association of single and multilocus haplotypes in genes coding for the viral co-receptors CCR5 and CCR2, and their ligands CCL3 and CCL5, with resistance or susceptibility to HIV-1 infection. Nine variants in CCR5-CCR2, two SNPs in CCL3 and two in CCL5 were genotyped by PCR-RFLP in 35 seropositive (cases) and 49 HIV-1-exposed seronegative Colombian individuals (controls). Haplotypes were inferred using the Arlequin software, and their frequency in individual or combined loci was compared between cases and controls by the chi-square test. A p' value ;0.05 after Bonferroni correction was considered significant. Homozygosis of the human haplogroup (HH) E was absent in controls and frequent in cases, showing a tendency to susceptibility. The haplotypes C-C and T-T in CCL3 were associated with susceptibility (p'=0.016) and resistance (p';0.0001) to HIV-1 infection, respectively. Finally, in multilocus analysis, the haplotype combinations formed by HHC in CCR5-CCR2, T-T in CCL3 and G-C in CCL5 were associated with resistance (p'=0.006). Our results suggest that specific combinations of variants in genes from the same signaling pathway can define an HIV-1 resistant phenotype. Despite our small sample size, our statistically significant associations suggest strong effects; however, these results should be further validated in larger cohorts.

Structure of the CCR5 Chemokine Receptor-HIV Entry Inhibitor Maraviroc Complex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tan, Qiuxiang; Zhu, Ya; Li, Jian

2013-10-21

The CCR5 chemokine receptor acts as a co-receptor for HIV-1 viral entry. Here we report the 2.7 angstrom–resolution crystal structure of human CCR5 bound to the marketed HIV drug maraviroc. The structure reveals a ligand-binding site that is distinct from the proposed major recognition sites for chemokines and the viral glycoprotein gp120, providing insights into the mechanism of allosteric inhibition of chemokine signaling and viral entry. A comparison between CCR5 and CXCR4 crystal structures, along with models of co-receptor–gp120-V3 complexes, suggests that different charge distributions and steric hindrances caused by residue substitutions may be major determinants of HIV-1 co-receptor selectivity.more » These high-resolution insights into CCR5 can enable structure-based drug discovery for the treatment of HIV-1 infection.« less

HTLV-1 induces a Th1-like state in CD4+CCR4+ T cells

PubMed Central

Araya, Natsumi; Sato, Tomoo; Ando, Hitoshi; Tomaru, Utano; Yoshida, Mari; Coler-Reilly, Ariella; Yagishita, Naoko; Yamauchi, Junji; Hasegawa, Atsuhiko; Kannagi, Mari; Hasegawa, Yasuhiro; Takahashi, Katsunori; Kunitomo, Yasuo; Tanaka, Yuetsu; Nakajima, Toshihiro; Nishioka, Kusuki; Utsunomiya, Atae; Jacobson, Steven; Yamano, Yoshihisa

2014-01-01

Human T-lymphotropic virus type 1 (HTLV-1) is linked to multiple diseases, including the neuroinflammatory disease HTLV-1–associated myelopathy/tropical spastic paraparesis (HAM/TSP) and adult T cell leukemia/lymphoma. Evidence suggests that HTLV-1, via the viral protein Tax, exploits CD4+ T cell plasticity and induces transcriptional changes in infected T cells that cause suppressive CD4+CD25+CCR4+ Tregs to lose expression of the transcription factor FOXP3 and produce IFN-γ, thus promoting inflammation. We hypothesized that transformation of HTLV-1–infected CCR4+ T cells into Th1-like cells plays a key role in the pathogenesis of HAM/TSP. Here, using patient cells and cell lines, we demonstrated that Tax, in cooperation with specificity protein 1 (Sp1), boosts expression of the Th1 master regulator T box transcription factor (T-bet) and consequently promotes production of IFN-γ. Evaluation of CSF and spinal cord lesions of HAM/TSP patients revealed the presence of abundant CD4+CCR4+ T cells that coexpressed the Th1 marker CXCR3 and produced T-bet and IFN-γ. Finally, treatment of isolated PBMCs and CNS cells from HAM/TSP patients with an antibody that targets CCR4+ T cells and induces cytotoxicity in these cells reduced both viral load and IFN-γ production, which suggests that targeting CCR4+ T cells may be a viable treatment option for HAM/TSP. PMID:24960164

Caveolin-1 regulates chemokine receptor 5-mediated contribution of bone marrow-derived cells to dermal fibrosis

PubMed Central

Lee, Rebecca; Perry, Beth; Heywood, Jonathan; Reese, Charles; Bonner, Michael; Hatfield, Corey M.; Silver, Richard M.; Visconti, Richard P.; Hoffman, Stanley; Tourkina, Elena

2014-01-01

In fibrotic diseases caveolin-1 underexpression in fibroblasts results in collagen overexpression and in monocytes leads to hypermigration. These profibrotic behaviors are blocked by the caveolin-1 scaffolding domain peptide (CSD) which compensates for caveolin-1 deficiency. Monocytes and fibroblasts are related in that monocytes are the progenitors of fibrocytes (CD45+/Collagen I+ cells) that, in turn, are the progenitors of many fibroblasts in fibrotic tissues. In an additional anti-fibrotic activity, CSD blocks monocyte differentiation into fibrocytes. We studied a mouse fibrosis model (Pump Model) involving systemic bleomycin delivery that closely models scleroderma (SSc) in several ways, the most important of which for this study is that fibrosis is observed in the lungs, skin, and internal organs. We show here that dermal thickness is increased 2-fold in the Pump Model and that this effect is almost completely blocked by CSD (p < 0.001). Concomitantly, the subcutaneous fat layer becomes >80% thinner. This effect is also blocked by CSD (p < 0.001). Even in mice receiving vehicle instead of bleomycin, CSD increases the thickness of the fat layer. To study the mechanisms of action of bleomycin and CSD, we examined the accumulation of the chemokine receptor CCR5 and its ligands MIP1α and MIP1β in fibrotic tissue and their roles in monocyte migration. Fibrocytes and other leukocytes expressing CCR5 and its ligands were present at high levels in the fibrotic dermis of SSc patients and Pump Model mice while CSD blocked their accumulation in mouse dermis. Migration toward CCR5 ligands of SSc monocytes and Pump Model bone marrow cells was 3-fold greater than cells from control subjects. This enhanced migration was almost completely blocked by CSD. These results suggest that low monocyte caveolin-1 promotes fibrosis by enhancing the recruitment of fibrocytes and their progenitors into affected tissue. PMID:24966836

Methionine-supplemented diet affects the expression of cardiovascular disease-related genes and increases inflammatory cytokines in mice heart and liver.

PubMed

Aissa, Alexandre Ferro; Amaral, Catia Lira do; Venancio, Vinicius Paula; Machado, Carla da Silva; Hernandes, Lívia Cristina; Santos, Patrick Wellington da Silva; Curi, Rui; Bianchi, Maria de Lourdes Pires; Antunes, Lusânia Maria Greggi

2017-01-01

Some important environmental factors that influence the development of cardiovascular diseases (CVD) include tobacco, excess alcohol, and unhealthy diet. Methionine obtained from the diet participates in the synthesis of DNA, proteins, lipids and affects homocysteine levels, which is associated with the elevated risk for CVD development. Therefore, the aim of this study was to investigate the manner in which dietary methionine might affect cellular mechanisms underlying CVD occurrence. Swiss albino mice were fed either control (0.3% DL-methionine), methionine-supplemented (2% DL-methionine), or a methionine-deprived diet (0% DL-methionine) over a 10-week period. The parameters measured included plasma homocysteine concentrations, oxidative stress by reduced glutathione (GSH)/oxidized glutathione (GSSG) ratio, levels of inflammatory cytokines IL-1ß, TNF-α, and IL-6, as well as expression of genes associated with CVD. The levels of apolipoprotein A5 (APOA5), a regulator of plasma triglycerides, were measured. The methionine-supplemented diet increased oxidative stress by lowering the GSH/GSSG ratio in heart tissues and decreased expression of the genes Apob, Ctgf, Serpinb2, Spp1, Il1b, and Sell, but elevated expression of Thbs4, Tgfb2, Ccr1, and Vegfa. Methionine-deprived diet reduced expression of Col3a1, Cdh5, Fabp3, Bax, and Hbegf and increased expression of Sell, Ccl5, Itga2, Birc3, Msr1, Bcl2a1a, Il1r2, and Selp. Methionine-deprived diet exerted pro-inflammatory consequences as evidenced by elevated levels of cytokines IL-1ß, TNF-α, and IL-6 noted in liver. Methionine-supplemented diet increased hepatic IL-6 and cardiac TNF-α. Both methionine supplementation and deprivation lowered hepatic levels of APOA5. In conclusion, data demonstrated that a methionine-supplemented diet modulated important biological processes associated with high risk of CVD development.

Microbiota induces tonic CCL2 systemic levels that control pDC trafficking in steady state.

PubMed

Swiecki, M; Miller, H L; Sesti-Costa, R; Cella, M; Gilfillan, S; Colonna, M

2017-07-01

Plasmacytoid dendritic cells (pDCs) detect viruses initiating antiviral type I interferon responses. The microbiota is known to shape immune responses, but whether it influences pDC homeostasis and/or function is poorly understood. By comparing pDCs in germ-free and specific pathogen-free mice, we found that the microbiota supports homeostatic trafficking by eliciting constitutive levels of the chemokine CCL2 that engages CCR2. Mononuclear phagocytes were required for tonic CCL2 levels. CCL2 was particularly important for trafficking of a CCR2 hi subset of pDCs that produced proinflammatory cytokines and was prone to apoptosis. We further demonstrated that CCR2 was also essential for pDC migration during inflammation. Wild-type (WT):Ccr2 -/- mixed bone marrow chimeras revealed that CCR2 promotes pDC migration in a cell-intrinsic manner. Overall, we identify a novel role for the microbiota in shaping immunity, which includes induction of CCL2 levels that control homeostatic trafficking of pDCs.

Gene editing of CCR5 in autologous CD4 T cells of persons infected with HIV.

PubMed

Tebas, Pablo; Stein, David; Tang, Winson W; Frank, Ian; Wang, Shelley Q; Lee, Gary; Spratt, S Kaye; Surosky, Richard T; Giedlin, Martin A; Nichol, Geoff; Holmes, Michael C; Gregory, Philip D; Ando, Dale G; Kalos, Michael; Collman, Ronald G; Binder-Scholl, Gwendolyn; Plesa, Gabriela; Hwang, Wei-Ting; Levine, Bruce L; June, Carl H

2014-03-06

CCR5 is the major coreceptor for human immunodeficiency virus (HIV). We investigated whether site-specific modification of the gene ("gene editing")--in this case, the infusion of autologous CD4 T cells in which the CCR5 gene was rendered permanently dysfunctional by a zinc-finger nuclease (ZFN)--is safe. We enrolled 12 patients in an open-label, nonrandomized, uncontrolled study of a single dose of ZFN-modified autologous CD4 T cells. The patients had chronic aviremic HIV infection while they were receiving highly active antiretroviral therapy. Six of them underwent an interruption in antiretroviral treatment 4 weeks after the infusion of 10 billion autologous CD4 T cells, 11 to 28% of which were genetically modified with the ZFN. The primary outcome was safety as assessed by treatment-related adverse events. Secondary outcomes included measures of immune reconstitution and HIV resistance. One serious adverse event was associated with infusion of the ZFN-modified autologous CD4 T cells and was attributed to a transfusion reaction. The median CD4 T-cell count was 1517 per cubic millimeter at week 1, a significant increase from the preinfusion count of 448 per cubic millimeter (P<0.001). The median concentration of CCR5-modified CD4 T cells at 1 week was 250 cells per cubic millimeter. This constituted 8.8% of circulating peripheral-blood mononuclear cells and 13.9% of circulating CD4 T cells. Modified cells had an estimated mean half-life of 48 weeks. During treatment interruption and the resultant viremia, the decline in circulating CCR5-modified cells (-1.81 cells per day) was significantly less than the decline in unmodified cells (-7.25 cells per day) (P=0.02). HIV RNA became undetectable in one of four patients who could be evaluated. The blood level of HIV DNA decreased in most patients. CCR5-modified autologous CD4 T-cell infusions are safe within the limits of this study. (Funded by the National Institute of Allergy and Infectious Diseases and others; ClinicalTrials.gov number, NCT00842634.).

Protection of macaques from vaginal SHIV challenge by vaginally delivered inhibitors of virus-cell fusion.

PubMed

Veazey, Ronald S; Klasse, Per Johan; Schader, Susan M; Hu, Qinxue; Ketas, Thomas J; Lu, Min; Marx, Preston A; Dufour, Jason; Colonno, Richard J; Shattock, Robin J; Springer, Martin S; Moore, John P

2005-11-03

Human immunodeficiency virus type 1 (HIV-1) continues to spread, principally by heterosexual sex, but no vaccine is available. Hence, alternative prevention methods are needed to supplement educational and behavioural-modification programmes. One such approach is a vaginal microbicide: the application of inhibitory compounds before intercourse. Here, we have evaluated the microbicide concept using the rhesus macaque 'high dose' vaginal transmission model with a CCR5-receptor-using simian-human immunodeficiency virus (SHIV-162P3) and three compounds that inhibit different stages of the virus-cell attachment and entry process. These compounds are BMS-378806, a small molecule that binds the viral gp120 glycoprotein and prevents its attachment to the CD4 and CCR5 receptors, CMPD167, a small molecule that binds to CCR5 to inhibit gp120 association, and C52L, a bacterially expressed peptide inhibitor of gp41-mediated fusion. In vitro, all three compounds inhibit infection of T cells and cervical tissue explants, and C52L acts synergistically with CMPD167 or BMS-378806 to inhibit infection of cell lines. In vivo, significant protection was achieved using each compound alone and in combinations. CMPD167 and BMS-378806 were protective even when applied 6 h before challenge.

Infection of rhesus macaques with a pool of simian immunodeficiency virus with the envelope genes from acute HIV-1 infections.

PubMed

Krebs, Kendall C; Tian, Meijuan; Asmal, Mohammed; Ling, Binhua; Nelson, Kenneth; Henry, Kenneth; Gibson, Richard; Li, Yuejin; Han, Weining; Shattock, Robin J; Veazey, Ronald S; Letvin, Norman; Arts, Eric J; Gao, Yong

2016-11-25

New simian-human immunodeficiency chimeric viruses with an HIV-1 env (SHIVenv) are critical for studies on HIV pathogenesis, vaccine development, and microbicide testing. Macaques are typically exposed to single CCR5-using SHIVenv which in most instances does not reflect the conditions during acute/early HIV infection (AHI) in humans. Instead of individual and serial testing new SHIV constructs, a pool of SHIVenv_B derived from 16 acute HIV-1 infections were constructed using a novel yeast-based SHIV cloning approach and then used to infect macaques. Even though none of the 16 SHIVenvs contained the recently reported mutations in env genes that could significantly enhance their binding affinity to RhCD4, one SHIVenv (i.e. SHIVenv_B3-PRB926) established infection in macaques exposed to this pool. AHI SHIVenv_B viruses as well as their HIVenv_B counterparts were analyzed for viral protein content, function, and fitness to identify possible difference between SHIVenv_B3-PRB926 and the other 15 SHIVenvs in the pool. All of the constructs produced SHIV or HIV chimeric with wild type levels of capsid (p27 and p24) content, reverse transcriptase (RT) activity, and expressed envelope glycoproteins that could bind to cell receptors CD4/CCR5 and mediate virus entry. HIV-1env_B chimeric viruses were propagated in susceptible cell lines but the 16 SHIVenv_B variants showed only limited replication in macaque peripheral blood mononuclear cells (PBMCs) and 174×CEM.CCR5 cell line. AHI chimeric viruses including HIVenv_B3 showed only minor variations in cell entry efficiency and kinetics as well as replicative fitness in human PBMCs. Reduced number of N-link glycosylation sites and slightly greater CCR5 affinity/avidity was the only distinguishing feature of env_B3 versus other AHI env's in the pool, a feature also observed in the HIV establishing new infections in humans. Despite the inability to propagate in primary cells and cell lines, a pool of 16 SHIVenv viruses could establish infection but only one virus, SHIVenv_B3 was isolated in the macaque and then shown to repeatedly infected macaques. This SHIVenv_B3 virus did not show any distinct phenotypic property from the other 15 SHIVenv viruses but did have the fewest N-linked glycosylation sites.

T-lymphocyte and cytokine expression in human inflammatory periapical lesions.

PubMed

de Brito, Luciana Carla Neves; Teles, Flávia Rocha Fonseca; Teles, Ricardo Palmier; Totola, Antônio Helvécio; Vieira, Leda Quércia; Sobrinho, Antônio Paulino Ribeiro

2012-04-01

Lymphocytes, among many cells, express different sets of cytokines, chemokines, and receptors, which are considered important mediators of periapical immune response to infection. The aim of this study was to evaluate the mRNA expression of CD4(+)CD28(+) and CD8(+) T genes and the gene expression of interferon-γ, tumor necrosis factor-α, interleukin (IL)-1β, IL-17A, IL-10, CCL2/MCP-1, CCL4, CCL5, CXCR4, CCR5, and receptor activator for nuclear factor kappa B ligand (RANKL) in periapical interstitial fluid from human root canal infections. The samples were collected immediately after root canal cleaning and 7 days later (restrained root canal bacterial load) to characterize those gene expressions. Real-time polymerase chain reaction demonstrated significantly higher levels of CD4(+)CD28(+) and CD8(+) T-cell markers in the former root canal condition and an increase of IL-10 and CXCR4, followed by a decrease of proinflammatory cytokines such as RANKL, interferon-γ, IL-1β, and CCL5. Analyses of T-lymphocyte and cytokine expression in periapical area were able to show that distinct root canal conditions might play regulatory roles in controlling local immune/inflammatory processes. Copyright © 2012 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

[CCL21 promotes the metastasis of human pancreatic cancer Panc-1 cells via epithelial- mesenchymal transition].

PubMed

Liu, Qing; Chen, Fangfang; Duan, Tanghai; Zhu, Haitao; Xie, Xiaodong; Wu, Yingying; Zhang, Zhijian; Wang, Dongqing

2015-01-01

To investigate the mechanism underlying that chemokine (C-C motif) ligand 21 (CCL21) promotes the metastasis ability of human pancreatic cancer Panc-1 cells. Transwell(TM) was used to access the chemotaxis effect of CCL21 on Panc-1 cells. Real-time quantitative PCR was performed to detect the expression of C-C chemokine receptor type 7 (CCR7) mRNA in the upper and lower chambers. Immunofluorescence staining and Western blotting were employed to examine the expressions of the epithelial-mesenchymal transition (EMT)-related proteins and CD133 of Panc-1 cells in the lower chamber, which were compared with those of the upper chamber as the control. The numbers of the Panc-1 cells induced by 0, 50, 100, 200 ng/mL CCL21 were 13.00 ± 3.00, 78.00 ± 9.00, 161.00 ± 11.00, 281.00 ± 17.00, respectively; with the increase of the concentration of CCL21, there were more cells migrating from the upper to the lower chamber; and the cells in the lower chamber expressed higher level of CCR7 mRNA than the ones staying in the upper chamber. The relative protein expressions of MMP-9, vimentin, E-cadherin and CD133 in the lower chamber were 0.42 ± 0.04, 0.36 ± 0.03, 0.12 ± 0.02, 0.46 ± 0.03, respectively, which were statistically significantly different from those in the upper chamber (0.15 ± 0.02, 0.25 ± 0.02, 0.25 ± 0.03, 0.13 ± 0.02, respectively). CCL21/CCR7 axis maybe play an important role in the metastasis of pancreatic cancer stem cells by EMT and up-regulation of MMP-9.

CCL2 and CCR2 regulate pain-related behaviour and early gene expression in post-traumatic murine osteoarthritis but contribute little to chondropathy.

PubMed

Miotla Zarebska, J; Chanalaris, A; Driscoll, C; Burleigh, A; Miller, R E; Malfait, A M; Stott, B; Vincent, T L

2017-03-01

The role of inflammation in structural and symptomatic osteoarthritis (OA) remains unclear. One key mediator of inflammation is the chemokine CCL2, primarily responsible for attracting monocytes to sites of injury. We investigated the role of CCL2 and its receptor CCR2 in experimental OA. OA was induced in 10 weeks old male wild type (WT), Ccl2 -/- and Ccr2 -/- mice, by destabilisation of the medial meniscus (DMM). RNA was extracted from whole joints at 6 h and 7 days post-surgery and examined by reverse transcription polymerase chain reaction (RT-PCR). Gene expression changes between naïve and DMM-operated mice were compared. Chondropathy scores, from mice at 8, 12, 16 and 20 weeks post DMM were calculated using modified Osteoarthritis Research Society International (OARSI) grading systems. Changes in hind paw weight distribution, as a measure of pain, were assessed by Linton incapacitance. Absence of CCL2 strongly suppressed (>90%) selective inflammatory response genes in the joint 6 h post DMM, including arginase 1, prostaglandin synthase 2, nitric oxide synthase 2 and inhibin A. IL6, MMP3 and tissue inhibitor of metalloproteinase 1 were also significantly suppressed. Similar trends were also observed in the absence of CCR2. A lower average chondropathy score was observed in both Ccl2 -/- and Ccr2 -/- mice at 12, 16 and 20 weeks post DMM compared with WT mice, but this was only statistically significant at 20 weeks in Ccr2 -/- mice. Pain-related behaviour in Ccl2 -/- and Ccr2 -/- mice post DMM was delayed in onset. The CCL2/CCR2 axis plays an important role in the development of pain in murine OA, but contributes little to cartilage damage. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

Increase in activated Treg in TIL in lung cancer and in vitro depletion of Treg by ADCC using an antihuman CCR4 mAb (KM2760).

PubMed

Kurose, Koji; Ohue, Yoshihiro; Sato, Eiichi; Yamauchi, Akira; Eikawa, Shingo; Isobe, Midori; Nishio, Yumi; Uenaka, Akiko; Oka, Mikio; Nakayama, Eiichi

2015-01-01

Tregs infiltrate tumors and inhibit immune responses against them. We investigated subpopulations of Foxp3 CD4 T cells previously defined by Miyara et al. (Immunity 30, 899-911, 2009) in peripheral blood mononuclear cells (PBMCs) and tumor infiltrating lymphocytes (TILs) in lung cancer. We also showed that Tregs in healthy donors that express CCR4 could be efficiently eliminated in vitro by cotreatment with antihuman (h) CCR4 mAb (KM2760) and NK cells. In lung cancer, the number of activated/effector Tregs and non-Tregs, but not resting/naive Tregs, was increased in TILs compared with the number of those cells in PBMCs. The non-Treg population contained Th2 and Th17. CCR4 expression on activated/effector Tregs and non-Tregs in TILs was down-regulated compared with that on those cells in PBMCs. Chemokinetic migration of CD25 CD4 T cells containing the Treg population sorted from the PBMCs of healthy donors to CCL22/MDC was abrogated by pretreatment with anti-hCCR4 mAb (KM2760). The inhibitory activity of CD25 CD127 CD4 Tregs on the proliferative response of CD4 and CD8 T cells stimulated with anti-CD3/CD28 coated beads was abrogated by adding an anti-hCCR4 mAb (KM2760) and CD56 NK cells to the culture. The findings suggested the CCR4 on activated/effector Tregs and non-Tregs was functionally involved in the chemokinetic migration and accumulation of those cells to the tumor site. In vitro findings of efficient elimination of Tregs may give the basis for implementation of a clinical trial to investigate Treg depletion by administration of an anti-hCCR4 mAb to solid cancer patients.

HIV-1 with Multiple CCR5/CXCR4 Chimeric Receptor Use Is Predictive of Immunological Failure in Infected Children

PubMed Central

Cavarelli, Mariangela; Karlsson, Ingrid; Zanchetta, Marisa; Antonsson, Liselotte; Plebani, Anna; Giaquinto, Carlo; Fenyö, Eva Maria; De Rossi, Anita; Scarlatti, Gabriella

2008-01-01

Background HIV-1 R5 viruses are characterized by a large phenotypic variation, that is reflected by the mode of coreceptor use. The ability of R5 HIV-1 to infect target cells expressing chimeric receptors between CCR5 and CXCR4 (R5broad viruses), was shown to correlate with disease stage in HIV-1 infected adults. Here, we ask the question whether phenotypic variation of R5 viruses could play a role also in mother-to-child transmission (MTCT) of HIV-1 and pediatric disease progression. Methodology/Principal Findings Viral isolates obtained from a total of 59 HIV-1 seropositive women (24 transmitting and 35 non transmitting) and 28 infected newborn children, were used to infect U87.CD4 cells expressing wild type or six different CCR5/CXCR4 chimeric receptors. HIV-1 isolates obtained from newborn infants had predominantly R5narrow phenotype (n = 20), but R5broad and R5X4 viruses were also found in seven and one case, respectively. The presence of R5broad and R5X4 phenotypes correlated significantly with a severe decline of the CD4+ T cells (CDC stage 3) or death within 2 years of age. Forty-three percent of the maternal R5 isolates displayed an R5broad phenotype, however, the presence of the R5broad virus was not predictive for MTCT of HIV-1. Of interest, while only 1 of 5 mothers with an R5X4 virus transmitted the dualtropic virus, 5 of 6 mothers carrying R5broad viruses transmitted viruses with a similar broad chimeric coreceptor usage. Thus, the maternal R5broad phenotype was largely preserved during transmission and could be predictive of the phenotype of the newborn's viral variant. Conclusions/Significance Our results show that R5broad viruses are not hampered in transmission. When transmitted, immunological failure occurs earlier than in children infected with HIV-1 of R5narrow phenotype. We believe that this finding is of utmost relevance for therapeutic interventions in pediatric HIV-1 infection. PMID:18820725

HIV-1 with multiple CCR5/CXCR4 chimeric receptor use is predictive of immunological failure in infected children.

PubMed

Cavarelli, Mariangela; Karlsson, Ingrid; Zanchetta, Marisa; Antonsson, Liselotte; Plebani, Anna; Giaquinto, Carlo; Fenyö, Eva Maria; De Rossi, Anita; Scarlatti, Gabriella

2008-09-29

HIV-1 R5 viruses are characterized by a large phenotypic variation, that is reflected by the mode of coreceptor use. The ability of R5 HIV-1 to infect target cells expressing chimeric receptors between CCR5 and CXCR4 (R5(broad) viruses), was shown to correlate with disease stage in HIV-1 infected adults. Here, we ask the question whether phenotypic variation of R5 viruses could play a role also in mother-to-child transmission (MTCT) of HIV-1 and pediatric disease progression. Viral isolates obtained from a total of 59 HIV-1 seropositive women (24 transmitting and 35 non transmitting) and 28 infected newborn children, were used to infect U87.CD4 cells expressing wild type or six different CCR5/CXCR4 chimeric receptors. HIV-1 isolates obtained from newborn infants had predominantly R5(narrow) phenotype (n = 20), but R5(broad) and R5X4 viruses were also found in seven and one case, respectively. The presence of R5(broad) and R5X4 phenotypes correlated significantly with a severe decline of the CD4+ T cells (CDC stage 3) or death within 2 years of age. Forty-three percent of the maternal R5 isolates displayed an R5(broad) phenotype, however, the presence of the R5(broad) virus was not predictive for MTCT of HIV-1. Of interest, while only 1 of 5 mothers with an R5X4 virus transmitted the dualtropic virus, 5 of 6 mothers carrying R5(broad) viruses transmitted viruses with a similar broad chimeric coreceptor usage. Thus, the maternal R5(broad) phenotype was largely preserved during transmission and could be predictive of the phenotype of the newborn's viral variant. Our results show that R5(broad) viruses are not hampered in transmission. When transmitted, immunological failure occurs earlier than in children infected with HIV-1 of R5(narrow) phenotype. We believe that this finding is of utmost relevance for therapeutic interventions in pediatric HIV-1 infection.

Nonproductive human immunodeficiency virus type 1 infection of human fetal astrocytes: independence from CD4 and major chemokine receptors.

PubMed

Sabri, F; Tresoldi, E; Di Stefano, M; Polo, S; Monaco, M C; Verani, A; Fiore, J R; Lusso, P; Major, E; Chiodi, F; Scarlatti, G

1999-11-25

Human immunodeficiency virus type 1 (HIV-1) infection of the brain is associated with neurological manifestations both in adults and in children. The primary target for HIV-1 infection in the brain is the microglia, but astrocytes can also be infected. We tested 26 primary HIV-1 isolates for their capacity to infect human fetal astrocytes in culture. Eight of these isolates, independent of their biological phenotype and chemokine receptor usage, were able to infect astrocytes. Although no sustained viral replication could be demonstrated, the virus was recovered by coculture with receptive cells such as macrophages or on stimulation with interleukin-1beta. To gain knowledge into the molecular events that regulate attachment and penetration of HIV-1 in astrocytes, we investigated the expression of several chemokine receptors. Fluorocytometry and calcium-mobilization assay did not provide evidence of expression of any of the major HIV-1 coreceptors, including CXCR4, CCR5, CCR3, and CCR2b, as well as the CD4 molecule on the cell surface of human fetal astrocytes. However, mRNA transcripts for CXCR4, CCR5, Bonzo/STRL33/TYMSTR, and APJ were detected by RT-PCR. Furthermore, infection of astrocytes by HIV-1 isolates with different chemokine receptor usage was not inhibited by the chemokines SDF-1beta, RANTES, MIP-1beta, or MCP-1 or by antibodies directed against the third variable region or the CD4 binding site of gp120. These data show that astrocytes can be infected by primary HIV-1 isolates via a mechanism independent of CD4 or major chemokine receptors. Furthermore, astrocytes are potential carriers of latent HIV-1 and on activation may be implicated in spreading the infection to other neighbouring cells, such as microglia or macrophages. Copyright 1999 Academic Press.

Chemokine-like factor 1 (CLFK1) is over-expressed in patients with atopic dermatitis.

PubMed

Yang, Gao-Yun; Chen, Xue; Sun, Ya-Chun; Ma, Chen-Li; Qian, Ge

2013-01-01

Human chemokine-like factor 1 (CKLF1), a recently discovered chemokine, has a broad spectrum of biological functions in immune-mediated diseases. It is highly expressed on Th2 lymphocytes and is a functional ligand for human CCR4. CKLF1 has a major role in the recruitment and activation of leucocytes, which plays an important role in the pathogenesis of allergic diseases. The present study was designed to determine the expression of CKLF1 in skin and serum in patients with atopic dermatitis (AD). The CKLF1 protein expression in skin lesion was analyzed by immunohistochemistry and ELISA. The mRNA expression of CKLF1 in skin lesion was detected by Real-time PCR. The serum levels of CKLF1, IgE, eotaxin, IL-4, IL-5, and IL-13 were measured by ELISA. Histopathological changes in the skin of AD patients showed local inflammation with epidermal thickening and significant inflammatory cellular infiltration. Immunohistochemistry results demonstrated that CKLF1-staining positive cells were located in the epidermal and dermis, and that the CKLF1 expression in AD patients was significantly higher than that in normal control. The CKLF1 mRNA expression in AD patients was significantly higher than that in healthy controls. Serum CKLF1 and IgE levels were significantly increased in AD patients, as were the serum levels of IL-4, IL-5, IL-13 and eotaxin. Both CKLF1 protien and mRNA levels are overexpressed in the skin lesion of AD patients, along with an increase in serum CKLF1 level, indicating that CKLF1 may play an important role in the development of atopic dermatitis.

Eotaxin increases monolayer permeability of human coronary artery endothelial cells.

PubMed

Jamaluddin, Md Saha; Wang, Xinwen; Wang, Hao; Rafael, Cubas; Yao, Qizhi; Chen, Changyi

2009-12-01

The objective of this study was to determine the effects and molecular mechanisms of eotaxin, a newly discovered chemokine (CCL11), on endothelial permeability in the human coronary artery endothelial cells (HCAECs). Cells were treated with eotaxin, and the monolayer permeability was studied by using a costar transwell system with a Texas Red-labeled dextran tracer. Eotaxin significantly increased monolayer permeability in a concentration-dependent manner. In addition, eotaxin treatment significantly decreased the mRNA and protein levels of endothelial junction molecules including zonula occludens-1 (ZO-1), occludin, and claudin-1 in a concentration-dependent manner as determined by real-time RT-PCR and Western blot analysis, respectively. Increased oxidative stress was observed in eotaxin-treated HCAECs by analysis of cellular glutathione levels. Furthermore, eotaxin treatment substantially activated the phosphorylation of MAPK p38. HCAECs expressed CCR3. Consequently, antioxidants (ginkgolide B and MnTBAP), specific p38 inhibitor SB203580, and anti-CCR3 antibody effectively blocked the eotaxin-induced permeability increase in HCAECs. Eotaxin also increased the phosphorylation of Stat3 and nuclear translocation of NF-kappaB in HCAECs. Eotaxin increases vascular permeability through CCR3, the downregulation of tight junction proteins, increase of oxidative stress, and activation of MAPK p38, Stat3, and NF-kB pathways in HCAECs.

Diversity of staphylococcal cassette chromosome mec elements in nosocomial multiresistant Staphylococcus haemolyticus isolates.

PubMed

Szczuka, Ewa; Krajewska, Magdalena; Lijewska, Dagmara; Bosacka, Karolina; Kaznowski, Adam

2016-11-01

Staphylococcus haemolyticus is the second, most frequently isolated coagulase-negative staphyloccus (CoNS) from patients with hospital-acquired infections, and it is usually resistant to methicillin and other semisynthetic penicillins. The purpose of this study was to characterize staphylococcal cassette chromosome mec (SCCmec) elements and assess the in-vitro activity of antibiotics against 60 S. haemolyticus strains recovered from hospitalized patients. All these strains expressed methicillin resistance and carried a mecA gene. Moreover, all strains possessed a multiresistant phenotype, i.e., exhibited resistance to more than three classes of antibiotics. Eleven strains (18 %) harbored the SCCmec type V, containing ccrC and mec complex C. Three isolates harboring the ccrC gene did not contain a known mec complex. One strain positive for mec complex C was not typeable for ccr. This suggests that ccrC and mec complex C may exist autonomously. Only four strains carried mec complex B, whereas none of the S. haemolyticus harboured mec complex A. A new combination, which is mec complex B-ccrAB ship , was found in S. haemolitycus. The ccrAB ship was also identified in two strains of S. haemolitycus in which the mec gene complex was not identified. The results of the present study indicate that in S. haemolyticus the mec gene complex and the ccr genes are highly divergent. However, ccr sequence analysis does not allow the identification of a new allotype, based on a cut-off value of 85 % identity. The ccr genes in the S. haemolitycus strain showed ≥96 % sequence identity to the ccrAB2 genes.

CCR5 receptor antagonists: discovery and SAR study of guanylhydrazone derivatives.

PubMed

Wei, Robert G; Arnaiz, Damian O; Chou, Yuo-Ling; Davey, Dave; Dunning, Laura; Lee, Wheeseong; Lu, Shou-Fu; Onuffer, James; Ye, Bin; Phillips, Gary

2007-01-01

High throughput screening (HTS) led to the identification of the guanylhydrazone of 2-(4-chlorobenzyloxy)-5-bromobenzaldehyde as a CCR5 receptor antagonist. Initial modifications of the guanylhydrazone series indicated that substitution of the benzyl group at the para-position was well tolerated. Substitution at the 5-position of the central phenyl ring was critical for potency. Replacement of the guanylhydrazone group led to the discovery of a novel series of CCR5 antagonists.

Comparison of the editing patterns and editing efficiencies of TALEN and CRISPR-Cas9 when targeting the human CCR5 gene.

PubMed

Nerys-Junior, Arildo; Braga-Dias, Luciene P; Pezzuto, Paula; Cotta-de-Almeida, Vinícius; Tanuri, Amilcar

2018-01-01

The human C-C chemokine receptor type-5 (CCR5) is the major transmembrane co-receptor that mediates HIV-1 entry into target CD4+ cells. Gene therapy to knock-out the CCR5 gene has shown encouraging results in providing a functional cure for HIV-1 infection. In gene therapy strategies, the initial region of the CCR5 gene is a hotspot for producing functional gene knock-out. Such target gene editing can be done using programmable endonucleases such as transcription activator-like effector nucleases (TALEN) or clustered regularly interspaced short palindromic repeats (CRISPR-Cas9). These two gene editing approaches are the most modern and effective tools for precise gene modification. However, little is known of potential differences in the efficiencies of TALEN and CRISPR-Cas9 for editing the beginning of the CCR5 gene. To examine which of these two methods is best for gene therapy, we compared the patterns and amount of editing at the beginning of the CCR5 gene using TALEN and CRISPR-Cas9 followed by DNA sequencing. This comparison revealed that CRISPR-Cas9 mediated the sorting of cells that contained 4.8 times more gene editing than TALEN+ transfected cells.

Comparison of the editing patterns and editing efficiencies of TALEN and CRISPR-Cas9 when targeting the human CCR5 gene

PubMed Central

Nerys-Junior, Arildo; Braga-Dias, Luciene P.; Pezzuto, Paula; Cotta-de-Almeida, Vinícius; Tanuri, Amilcar

2018-01-01

Abstract The human C-C chemokine receptor type-5 (CCR5) is the major transmembrane co-receptor that mediates HIV-1 entry into target CD4+ cells. Gene therapy to knock-out the CCR5 gene has shown encouraging results in providing a functional cure for HIV-1 infection. In gene therapy strategies, the initial region of the CCR5 gene is a hotspot for producing functional gene knock-out. Such target gene editing can be done using programmable endonucleases such as transcription activator-like effector nucleases (TALEN) or clustered regularly interspaced short palindromic repeats (CRISPR-Cas9). These two gene editing approaches are the most modern and effective tools for precise gene modification. However, little is known of potential differences in the efficiencies of TALEN and CRISPR-Cas9 for editing the beginning of the CCR5 gene. To examine which of these two methods is best for gene therapy, we compared the patterns and amount of editing at the beginning of the CCR5 gene using TALEN and CRISPR-Cas9 followed by DNA sequencing. This comparison revealed that CRISPR-Cas9 mediated the sorting of cells that contained 4.8 times more gene editing than TALEN+ transfected cells. PMID:29583154

Closely related, yet unique: Distinct homo- and heterodimerization patterns of G protein coupled chemokine receptors and their fine-tuning by cholesterol

PubMed Central

Gahbauer, Stefan; Pluhackova, Kristyna

2018-01-01

Chemokine receptors, a subclass of G protein coupled receptors (GPCRs), play essential roles in the human immune system, they are involved in cancer metastasis as well as in HIV-infection. A plethora of studies show that homo- and heterodimers or even higher order oligomers of the chemokine receptors CXCR4, CCR5, and CCR2 modulate receptor function. In addition, membrane cholesterol affects chemokine receptor activity. However, structural information about homo- and heterodimers formed by chemokine receptors and their interplay with cholesterol is limited. Here, we report homo- and heterodimer configurations of the chemokine receptors CXCR4, CCR5, and CCR2 at atomistic detail, as obtained from thousands of molecular dynamics simulations. The observed homodimerization patterns were similar for the closely related CC chemokine receptors, yet they differed significantly between the CC receptors and CXCR4. Despite their high sequence identity, cholesterol modulated the CC homodimer interfaces in a subtype-specific manner. Chemokine receptor heterodimers display distinct dimerization patterns for CXCR4/CCR5 and CXCR4/CCR2. Furthermore, associations between CXCR4 and CCR5 reveal an increased cholesterol-sensitivity as compared to CXCR4/CCR2 heterodimerization patterns. This work provides a first comprehensive structural overview over the complex interaction network between chemokine receptors and indicates how heterodimerization and the interaction with the membrane environment diversifies the function of closely related GPCRs. PMID:29529028

HIV-1 gp120 as well as alcohol affect blood-brain barrier permeability and stress fiber formation: involvement of reactive oxygen species.

PubMed

Shiu, Carlum; Barbier, Elisabeth; Di Cello, Francescopaolo; Choi, Hee Jung; Stins, Monique

2007-01-01

HIV-1 infection commonly leads to serious HIV-1-associated neurological disorders, such as HIV-1-associated encephalopathy and dementia. In addition, alcohol is commonly used and/or abused among AIDS patients, but it is unclear whether alcohol affects the disease progression and if it affects it, how this occurs. We hypothesized that alcohol could affect the blood-brain barrier (BBB) integrity and thus could affect the onset and/or progression of HIV-associated neurological disorders. Human brain microvascular endothelial cells (HBMEC) in a BBB model system were pretreated with alcohol (17 and 68 mM) and subsequently coexposed with HIV-1 gp120. Expression of chemokine receptors CCR3, CCR5, and CXCR4 was assessed by enzyme-linked immunosorbent assay and real-time polymerase chain reaction. Changes in the permeability of the HBMEC monolayer were assessed using paracellular markers [(3)H]inulin or propidium iodide. Actin rearrangements in HBMEC were visualized by fluorescence microscopy and viability assessed using Live/Dead stain. Both gp120 and alcohol increased the permeability of the BBB model by up to 141%, without affecting HBMEC viability. Cotreatment with alcohol and gp120 did not result in a significant synergistic effect. Gp120 permeability involved chemokine receptor CCR5. Alcohol did not affect chemokine receptor expression on brain endothelial cells. Both gp120 and alcohol reorganized the cytoskeleton and induced stress fiber formation. Inhibition of reactive oxygen species (ROS) formation through NADPH blocked the effects of both gp120 and alcohol on permeability and stress fiber formation. These results indicate that both HIV-1 gp120 and alcohol induce stress fibers, causing increased permeability of the human BBB endothelium. Alcohol (68 mM)-mediated permeability increase was linked to ROS formation. The alcohol-mediated physiological changes in the HBMEC monolayers may increase diffusion of plasma components and viral penetration across the BBB. This suggests that alcohol, especially at levels attained in heavy drinkers, can potentially contribute in a negative fashion to HIV-1 neuropathogenesis.

IL-22 promotes Fas expression in oligodendrocytes and inhibits FOXP3 expression in T cells by activating the NF-κB pathway in multiple sclerosis.

PubMed

Zhen, Jin; Yuan, Jun; Fu, Yongwang; Zhu, Runxiu; Wang, Meiling; Chang, Hong; Zhao, Yan; Wang, Dong; Lu, Zuneng

2017-02-01

Multiple sclerosis (MS) is characterized by an increase in interleukin-22 and Fas, and a decrease in FOXP3, among other factors. In this study, we examined patients with MS and healthy control subjects and used the experimental autoimmune encephalomyelitis (EAE) animal model to identify the effects of IL-22 on oligodendrocytes and T cells in MS development. In MS, the expression of Fas in oligodendrocytes and IL-22 in CD4 + CCR4 + CCR6 + CCR10 + T cells was enhanced. Ikaros and FOXP3 were both decreased in T cells. Depending on exogenous IL-22, Fas increased the phosphorylation of mitogen- and stress-activated protein kinase 1 and activated the nuclear factor-κB pathway in oligodendrocytes, leading to an increase in Fas and oligodendrocyte apoptosis. IL-22 decreased FOXP3 expression by activating NF-κB, and it further inhibited PTEN and Ikaros expression. Tregs reversed the functions of IL-22. Taken together, these findings help to elucidate the mechanisms of IL-22 in MS development. Copyright © 2016 Elsevier Ltd. All rights reserved.

CCR2 V64I polymorphism in rifampicin resistant tuberculosis patients in Moewardi General Hospital Surakarta, Indonesia

NASA Astrophysics Data System (ADS)

Marwoto; Agung Prasetyo, Afiono; Reviono; Suradi

2018-05-01

CC chemokine receptor-2 (CCR2) play important roles in inflammation. The CCR2 V64I polymorphism already reported associated with many diseases; however, the association of CCR2 V64I polymorphism with tuberculosis is still unknown. Also, there is no report about the presentation of CCR2 V64I polymorphisms in Indonesian tuberculosis patients with rifampicin-mono resistant status has ever been published, to the best of our knowledge. This study evaluated the presence of CCR2 V64I polymorphisms in Javanese rifampicin-mono resistant tuberculosis patients. In an ongoing molecular epidemiology study of human genomic polymorphisms and infection, 51 Javanese rifampicin-mono resistant tuberculosis patients in Dr. Moewardi General Hospital in Surakarta were enrolled in the study. The blood samples were aliquoted and fractionated. The nucleic acids were extracted from all blood samples and subjected to the CCR2 V64I polymorphisms detection by a polymerase chain reaction-sequence-specific primer (PCR-SSP) technique. PCR products were analyzed in 3% agarose. CCR2 64V and 64I homozygote were found in 23.5% (12/51) and 23.5% (12/51) blood samples, respectively. The CCR2 VI genotype was found in 52.9% (27/51) blood samples. The CCR2 VI genotype was found predominant in Javanese rifampicin-mono resistant tuberculosis patients and may have anassociation with the clinical progression.

The molecular mechanism of human resistance to HIV-1 infection in persistently infected individuals--a review, hypothesis and implications.

PubMed

Becker, Yechiel

2005-08-01

Resistance to HIV-1 infection in Europeans is associated with a mutation in the gene that codes for the CCR5 protein that is present in Th2 cells and serves as a coreceptor for HIV-1 R5 strain. A deletion of 32 amino acids from the cytokine receptor prevents infection. This mutation prevails in Europeans and is absent in Africans. However, duplication of a gene that codes for a chemokine that binds to the CCR5 was discovered in Africans (mean gene copy 6 while in non-Africans the mean gene copy is 3). Higher expression of these genes protects T cells against HIV-1 infection in vitro. It should be noted that resistance to HIV-1 R5 variant does not protect against HIV-1 R4 variant. It was reported that a minority of highly HIV-1 exposed African professional sex workers (APSW) were resistant to the virus infection during a 10 years period. Recently, the analysis of the cytokines in the serum of the persistently infected seronegative women revealed that the latter hypo-expresses the cytokine IL-4. Since the molecular events during HIV-1 infection are associated with a marked increase in the levels of IL-4 and IgE in the sera of the infected individuals, it suggests that AIDS is an allergy. Thus, a very low level of IL-4 production may abrogate the virus infection. Studies on the human IL-4 gene revealed that together with the IL-4 mRNA a spliced variant with a deletion of exon 2 is synthesized. The latter is a natural antagonist of IL-4 and when expressed in an individual at a level higher than IL-4, the person will resist a microbial infection (e.g. Mycobacterium tuberculosis) or asthma. The present hypothesis suggests that the HIV-1 resistant APSWs produce more IL-4 delta 2 molecules than IL-4 molecules. The binding of IL-4 delta 2 to IL-4 receptors on T and B cells prevents their functions and the infection by HIV-1. The implications of these studies are that treatment of HIV-1 infected people with drugs that will block the IL-4 receptors will stop HIV-1 infections and the determination of the levels of IL-4 and IL-4 delta 2 in the sera of HIV-1+ patients will enable to identify the individuals that have a natural resistance to HIV-l/AIDS and those who need treatments.

Biological and Structural Characterization of Rotamers of C-C Chemokine Receptor Type 5 (CCR5) Inhibitor GSK214096.

PubMed

Kazmierski, Wieslaw M; Danehower, Susan; Duan, Maosheng; Ferris, Robert G; Elitzin, Vassil; Minick, Douglas; Sharp, Matthew; Stewart, Eugene; Villeneuve, Manon

2014-12-11

We recently reported the discovery of preclinical CCR5 inhibitor GSK214096, 1 (J. Med. Chem. 2011, 54, 756). Detailed characterization of 1 revealed that it exists as a mixture of four separable atropisomers A-D. The two slow-interconverting pairs of rotamers A + B and C + D were separated and further characterized. HIV and CCR5-mediated chemotaxis data strongly suggest that the antiviral potency of 1 is due to rotamers A + B and not C + D. Furthermore, integrated UV, vibrational circular dichroism VCD and computational approach allowed to determine the M chirality in C + D (and P chirality in A + B). These findings imply additional avenues to be pursued toward new CCR5 antagonists.

P2X1 Receptor Antagonists Inhibit HIV-1 Fusion by Blocking Virus-Coreceptor Interactions

PubMed Central

Giroud, Charline; Marin, Mariana; Hammonds, Jason; Spearman, Paul

2015-01-01

ABSTRACT HIV-1 Env glycoprotein-mediated fusion is initiated upon sequential binding of Env to CD4 and the coreceptor CXCR4 or CCR5. Whereas these interactions are thought to be necessary and sufficient to promote HIV-1 fusion, other host factors can modulate this process. Previous studies reported potent inhibition of HIV-1 fusion by selective P2X1 receptor antagonists, including NF279, and suggested that these receptors play a role in HIV-1 entry. Here we investigated the mechanism of antiviral activity of NF279 and found that this compound does not inhibit HIV-1 fusion by preventing the activation of P2X1 channels but effectively blocks the binding of the virus to CXCR4 or CCR5. The notion of an off-target effect of NF279 on HIV-1 fusion is supported by the lack of detectable expression of P2X1 receptors in cells used in fusion experiments and by the fact that the addition of ATP or the enzymatic depletion of ATP in culture medium does not modulate viral fusion. Importantly, NF279 fails to inhibit HIV-1 fusion with cell lines and primary macrophages when added at an intermediate stage downstream of Env-CD4-coreceptor engagement. Conversely, in the presence of NF279, HIV-1 fusion is arrested downstream of CD4 binding but prior to coreceptor engagement. NF279 also antagonizes the signaling function of CCR5, CXCR4, and another chemokine receptor, as evidenced by the suppression of calcium responses elicited by specific ligands and by recombinant gp120. Collectively, our results demonstrate that NF279 is a dual HIV-1 coreceptor inhibitor that interferes with the functional engagement of CCR5 and CXCR4 by Env. IMPORTANCE Inhibition of P2X receptor activity suppresses HIV-1 fusion and replication, suggesting that P2X signaling is involved in HIV-1 entry. However, mechanistic experiments conducted in this study imply that P2X1 receptor is not expressed in target cells or involved in viral fusion. Instead, we found that inhibition of HIV-1 fusion by a specific P2X1 receptor antagonist, NF279, is due to the blocking of virus interactions with both the CXCR4 and CCR5 coreceptors. The ability of NF279 to abrogate cellular calcium signaling induced by the respective chemokines showed that this compound acts as a dual-coreceptor antagonist. P2X1 receptor antagonists could thus represent a new class of dual-coreceptor inhibitors with a structure and a mechanism of action that are distinct from those of known HIV-1 coreceptor antagonists. PMID:26136569

P2X1 Receptor Antagonists Inhibit HIV-1 Fusion by Blocking Virus-Coreceptor Interactions.

PubMed

Giroud, Charline; Marin, Mariana; Hammonds, Jason; Spearman, Paul; Melikyan, Gregory B

2015-09-01

HIV-1 Env glycoprotein-mediated fusion is initiated upon sequential binding of Env to CD4 and the coreceptor CXCR4 or CCR5. Whereas these interactions are thought to be necessary and sufficient to promote HIV-1 fusion, other host factors can modulate this process. Previous studies reported potent inhibition of HIV-1 fusion by selective P2X1 receptor antagonists, including NF279, and suggested that these receptors play a role in HIV-1 entry. Here we investigated the mechanism of antiviral activity of NF279 and found that this compound does not inhibit HIV-1 fusion by preventing the activation of P2X1 channels but effectively blocks the binding of the virus to CXCR4 or CCR5. The notion of an off-target effect of NF279 on HIV-1 fusion is supported by the lack of detectable expression of P2X1 receptors in cells used in fusion experiments and by the fact that the addition of ATP or the enzymatic depletion of ATP in culture medium does not modulate viral fusion. Importantly, NF279 fails to inhibit HIV-1 fusion with cell lines and primary macrophages when added at an intermediate stage downstream of Env-CD4-coreceptor engagement. Conversely, in the presence of NF279, HIV-1 fusion is arrested downstream of CD4 binding but prior to coreceptor engagement. NF279 also antagonizes the signaling function of CCR5, CXCR4, and another chemokine receptor, as evidenced by the suppression of calcium responses elicited by specific ligands and by recombinant gp120. Collectively, our results demonstrate that NF279 is a dual HIV-1 coreceptor inhibitor that interferes with the functional engagement of CCR5 and CXCR4 by Env. Inhibition of P2X receptor activity suppresses HIV-1 fusion and replication, suggesting that P2X signaling is involved in HIV-1 entry. However, mechanistic experiments conducted in this study imply that P2X1 receptor is not expressed in target cells or involved in viral fusion. Instead, we found that inhibition of HIV-1 fusion by a specific P2X1 receptor antagonist, NF279, is due to the blocking of virus interactions with both the CXCR4 and CCR5 coreceptors. The ability of NF279 to abrogate cellular calcium signaling induced by the respective chemokines showed that this compound acts as a dual-coreceptor antagonist. P2X1 receptor antagonists could thus represent a new class of dual-coreceptor inhibitors with a structure and a mechanism of action that are distinct from those of known HIV-1 coreceptor antagonists. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Development and Characterization of a Novel Single-Cycle Recombinant-Virus Assay To Determine Human Immunodeficiency Virus Type 1 Coreceptor Tropism▿

PubMed Central

Whitcomb, Jeannette M.; Huang, Wei; Fransen, Signe; Limoli, Kay; Toma, Jonathan; Wrin, Terri; Chappey, Colombe; Kiss, Linda D. B.; Paxinos, Ellen E.; Petropoulos, Christos J.

2007-01-01

Most human immunodeficiency virus type 1 (HIV-1) strains require either the CXCR4 or CCR5 chemokine receptor to efficiently enter cells. Blocking viral binding to these coreceptors is an attractive therapeutic target. Currently, several coreceptor antagonists are being evaluated in clinical trials that require characterization of coreceptor tropism for enrollment. In this report, we describe the development of an automated and accurate procedure for determining HIV-1 coreceptor tropism (Trofile) and its validation for routine laboratory testing. HIV-1 pseudoviruses are generated using full-length env genes derived from patient virus populations. Coreceptor tropism is determined by measuring the abilities of these pseudovirus populations to efficiently infect CD4+/U87 cells expressing either the CXCR4 or CCR5 coreceptor. Viruses exclusively and efficiently infecting CXCR4+/CD4+/U87 cells are designated X4-tropic. Conversely, viruses exclusively and efficiently infecting CCR5+/CD4+/U87 cells are designated R5-tropic. Viruses capable of infecting both CXCR4+/CD4+/U87 and CCR5+/CD4+/U87 cells are designated dual/mixed-tropic. Assay accuracy and reproducibility were established by evaluating the tropisms of well-characterized viruses and the variability among replicate results from samples tested repeatedly. The viral subtype, hepatitis B virus or hepatitis C virus coinfection, and the plasma viral load did not affect assay performance. Minority subpopulations with alternate tropisms were reliably detected when present at 5 to 10%. The plasma viral load above which samples can be amplified efficiently in the Trofile assay is 1,000 copies per ml of plasma. Trofile has been automated for high-throughput use; it can be used to identify patients most likely to benefit from treatment regimens that include a coreceptor inhibitor and to monitor patients on treatment for the emergence of resistant virus populations that switch coreceptor tropism. PMID:17116663

Cystatin C-Based Equation Does Not Accurately Estimate the Glomerular Filtration in Japanese Living Kidney Donors.

PubMed

Tsujimura, Kazuma; Ota, Morihito; Chinen, Kiyoshi; Adachi, Takayuki; Nagayama, Kiyomitsu; Oroku, Masato; Nishihira, Morikuni; Shiohira, Yoshiki; Iseki, Kunitoshi; Ishida, Hideki; Tanabe, Kazunari

2017-06-23

BACKGROUND Precise evaluation of a living donor's renal function is necessary to ensure adequate residual kidney function after donor nephrectomy. Our aim was to evaluate the feasibility of estimating glomerular filtration rate (GFR) using serum cystatin-C prior to kidney transplantation. MATERIAL AND METHODS Using the equations of the Japanese Society of Nephrology, we calculated the GFR using serum creatinine (eGFRcre) and cystatin C levels (eGFRcys) for 83 living kidney donors evaluated between March 2010 and March 2016. We compared eGFRcys and eGFRcre values against the creatinine clearance rate (CCr). RESULTS The study population included 27 males and 56 females. The mean eGFRcys, eGFRcre, and CCr were, 91.4±16.3 mL/min/1.73 m² (range, 59.9-128.9 mL/min/1.73 m²), 81.5±14.2 mL/min/1.73 m² (range, 55.4-117.5 mL/min/1.73 m²) and 108.4±21.6 mL/min/1.73 m² (range, 63.7-168.7 mL/min/1.73 m²), respectively. eGFRcys was significantly lower than CCr (p<0.001). The correlation coefficient between eGFRcys and CCr values was 0.466, and the mean difference between the two values was -17.0 (15.7%), with a root mean square error of 19.2. Thus, eGFRcre was significantly lower than CCr (p<0.001). The correlation coefficient between eGFRcre and CCr values was 0.445, and the mean difference between the two values was -26.9 (24.8%), with a root mean square error of 19.5. CONCLUSIONS Although eGFRcys provided a better estimation of GFR than eGFRcre, eGFRcys still did not provide an accurate measure of kidney function in Japanese living kidney donors.

[Maraviroc: clinical trials results].

PubMed

Chidiac, C; Katlama, C; Yeni, P

2008-03-01

Just over a decade after identification of chemokine receptors CCR5 and CXCR4 as coreceptors for HIV, maraviroc (Celsentri), the first CCR5 antagonist, has recently obtained its Marketing Authorization in the United States and Europe, for treatment of treatment-experienced adult patients infected with only CCR5-tropic HIV-1 detectable. CCR5 antagonists, after fusion inhibitor enfuvirtide available since 2003, also belong to entry inhibitors. These molecules, unlike previous antiretrovirals, do not target the virus but its target cell by blocking viral penetration. Maraviroc has shown its clinical efficacy in patients failing other antiretroviral classes. Its safety profile was similar to placebo in two large phase III trials. However, careful assessment of both hepatic and immunologic safety of this new therapeutic class is needed. Viral tropism testing has to be investigated before using maraviroc in the clinic, because CCR5 antagonists are not active against CXCR4 viruses. For the moment indicated for the treatment-experienced patient population, maraviroc could in the future benefit to other types of patients, depending on ongoing trials results.

C-C chemokine receptor type five (CCR5): An emerging target for the control of HIV infection.

PubMed

Barmania, Fatima; Pepper, Michael S

2013-12-01

When HIV was initially discovered as the causative agent of AIDS, many expected to find a vaccine within a few years. This has however proven to be elusive; it has been approximately 30 years since HIV was first discovered, and a suitable vaccine is still not in effect. In 2009, a paper published by Hutter et al. reported on a bone marrow transplant performed on an HIV positive individual using stem cells that were derived from a donor who was homozygous for a mutation in the CCR5 gene known as CCR5 delta-32 (Δ32) (Hütter et al., 2009). The HIV positive individual became HIV negative and remained free of viral detection after transplantation despite having halted anti-retroviral (ARV) treatment. This review will focus on CCR5 as a key component in HIV immunity and will discuss the role of CCR5 in the control of HIV infection.

CCR6+ Th cells in the cerebrospinal fluid of persons with multiple sclerosis are dominated by pathogenic non-classic Th1 cells and GM-CSF-only-secreting Th cells.

PubMed

Restorick, S M; Durant, L; Kalra, S; Hassan-Smith, G; Rathbone, E; Douglas, M R; Curnow, S J

2017-08-01

Considerable attention has been given to CCR6 + IL-17-secreting CD4 + T cells (Th17) in the pathology of a number of autoimmune diseases including multiple sclerosis (MS). However, other Th subsets also play important pathogenic roles, including those that secrete IFNγ and GM-CSF. CCR6 expression by Th17 cells allows their migration across the choroid plexus into the cerebrospinal fluid (CSF), where they are involved in the early phase of experimental autoimmune encephalomyelitis (EAE), and in MS these cells are elevated in the CSF during relapses and contain high frequencies of autoreactive cells. However, the relatively low frequency of Th17 cells suggests they cannot by themselves account for the high percentage of CCR6 + cells in MS CSF. Here we identify the dominant CCR6 + T cell subsets in both the blood and CSF as non-classic Th1 cells, including many that secrete GM-CSF, a key encephalitogenic cytokine. In addition, we show that Th cells secreting GM-CSF but not IFNγ or IL-17, a subset termed GM-CSF-only-secreting Th cells, also accumulate in the CSF. Importantly, in MS the proportion of IFNγ- and GM-CSF-secreting T cells expressing CCR6 was significantly enriched in the CSF, and was elevated in MS, suggesting these cells play a pathogenic role in this disease. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Alternatively activated macrophages upregulate mesothelial expression of P-selectin to enhance adhesion of ovarian cancer cells.

PubMed

Carroll, Molly J; Fogg, Kaitlin C; Patel, Harin A; Krause, Harris B; Mancha, Anne-Sophie; Patankar, Manish S; Weisman, Paul S; Barroilhet, Lisa; Kreeger, Pamela K

2018-05-08

Peritoneal metastasis of high-grade serous ovarian cancer (HGSOC) occurs when tumor cells suspended in ascites adhere to mesothelial cells. Despite the strong relationship between metastatic burden and prognosis in HGSOC, there are currently no therapies specifically targeting the metastatic process. We utilized a co-culture model and multivariate analysis to examine how interactions between tumor cells, mesothelial cells, and alternatively-activated macrophages (AAMs) influence the adhesion of tumor cells to mesothelial cells. We found that AAM-secreted MIP-1β activates CCR5/PI3K signaling in mesothelial cells, resulting in expression of P-selectin on the mesothelial cell surface. Tumor cells attached to this de novo P-selectin through CD24, resulting in increased tumor cell adhesion in static conditions and rolling under flow. C57/BL6 mice treated with MIP-1β exhibited increased P-selectin expression on mesothelial cells lining peritoneal tissues, which enhanced CaOV3 adhesion ex vivo and ID8 adhesion in vivo. Analysis of samples from HGSOC patients confirmed increased MIP-1β and P-selectin, suggesting that this novel multi-cellular mechanism could be targeted to slow or stop metastasis in HGSOC by repurposing anti- CCR5 and P-selectin therapies developed for other indications. Copyright ©2018, American Association for Cancer Research.

Sensitivity Changes over the Course of Infection Increases the Likelihood of Resistance Against Fusion but Not CCR5 Receptor Blockers

PubMed Central

Chatziandreou, Nikolaos; Arauz, Ana Belen; Freitas, Ines; Nyein, Phyu Hninn; Fenton, Gregory; Mehta, Shruti H.; Kirk, Gregory D.

2012-01-01

Abstract As HIV-1 evolves over the course of infection, resistance against antiretrovirals may arise in the absence of drug pressure, especially against receptor and fusion blockers because of the extensive changes observed in the envelope glycoprotein. Here we show that viruses from the chronic phase of disease are significantly less sensitive to CCR5 receptor and fusion blockers compared to early infection variants. Differences in susceptibility to CCR5 antagonists were observed in spite of no demonstrable CXCR4 receptor utilization. No significant sensitivity differences were observed to another entry blocker, soluble CD4, or to reverse transcriptase, protease, or integrase inhibitors. Chronic as compared to early phase variants demonstrated greater replication when passaged in the presence of subinhibitory concentrations of fusion but not CCR5 receptor inhibitors. Fusion antagonist resistance, however, emerged from only one chronic phase virus culture. Because sensitivity to receptor and fusion antagonists is correlated with receptor affinity and fusion capacity, respectively, changes that occur in the envelope glycoprotein over the course of infection confer greater ability to use the CCR5 receptor and increased fusion ability. Our in vitro passage studies suggest that these evolving phenotypes increase the likelihood of resistance against fusion but not CCR5 receptor blockers. PMID:22650962

CCL3L1-CCR5 genotype influences durability of immune recovery during antiretroviral therapy of HIV-1–infected individuals

PubMed Central

Ahuja, Sunil K; Kulkarni, Hemant; Catano, Gabriel; Agan, Brian K; Camargo, Jose F; He, Weijing; O'Connell, Robert J; Marconi, Vincent C; Delmar, Judith; Eron, Joseph; Clark, Robert A; Frost, Simon; Martin, Jeffrey; Ahuja, Seema S; Deeks, Steven G; Little, Susan; Richman, Douglas; Hecht, Frederick M; Dolan, Matthew J

2008-01-01

The basis for the extensive variability seen in the reconstitution of CD4+ T cell counts in HIV-infected individuals receiving highly active antiretroviral therapy (HAART) is not fully known. Here, we show that variations in CCL3L1 gene dose and CCR5 genotype, but not major histocompatibility complex HLA alleles, influence immune reconstitution, especially when HAART is initiated at <350 CD4+ T cells/mm3. The CCL3L1-CCR5 genotypes favoring CD4+ T cell recovery are similar to those that blunted CD4+ T cell depletion during the time before HAART became available (pre-HAART era), suggesting that a common CCL3L1-CCR5 genetic pathway regulates the balance between pathogenic and reparative processes from early in the disease course. Hence, CCL3L1-CCR5 variations influence HIV pathogenesis even in the presence of HAART and, therefore, may prospectively identify subjects in whom earlier initiation of therapy is more likely to mitigate immunologic failure despite viral suppression by HAART. Furthermore, as reconstitution of CD4+ cells during HAART is more sensitive to CCL3L1 dose than to CCR5 genotypes, CCL3L1 analogs might be efficacious in supporting immunological reconstitution. PMID:18376407

Expression of inflammation-related genes is altered in gastric tissue of patients with advanced stages of NAFLD.

PubMed

Mehta, Rohini; Birerdinc, Aybike; Neupane, Arpan; Shamsaddini, Amirhossein; Afendy, Arian; Elariny, Hazem; Chandhoke, Vikas; Baranova, Ancha; Younossi, Zobair M

2013-01-01

Obesity is associated with chronic low-grade inflammation perpetuated by visceral adipose. Other organs, particularly stomach and intestine, may also overproduce proinflammatory molecules. We examined the gene expression patterns in gastric tissue of morbidly obese patients with nonalcoholic fatty liver disease (NAFLD) and compared the changes in gene expression in different histological forms of NAFLD. Stomach tissue samples from 20 morbidly obese NAFLD patients who were undergoing sleeve gastrectomy were profiled using qPCR for 84 genes encoding inflammatory cytokines, chemokines, their receptors, and other components of inflammatory cascades. Interleukin 8 receptor-beta (IL8RB) gene overexpression in gastric tissue was correlated with the presence of hepatic steatosis, hepatic fibrosis, and histologic diagnosis of nonalcoholic steatohepatitis (NASH). Expression levels of soluble interleukin 1 receptor antagonist (IL1RN) were correlated with the presence of NASH and hepatic fibrosis. mRNA levels of interleukin 8 (IL8), chemokine (C-C motif) ligand 4 (CCL4), and its receptor chemokine (C-C motif) receptor type 5 (CCR5) showed a significant increase in patients with advanced hepatic inflammation and were correlated with the severity of the hepatic inflammation. The results of our study suggest that changes in expression patterns for inflammatory molecule encoding genes within gastric tissue may contribute to the pathogenesis of obesity-related NAFLD.

Expression of Inflammation-Related Genes Is Altered in Gastric Tissue of Patients with Advanced Stages of NAFLD

PubMed Central

Mehta, Rohini; Birerdinc, Aybike; Neupane, Arpan; Shamsaddini, Amirhossein; Afendy, Arian; Elariny, Hazem; Chandhoke, Vikas; Baranova, Ancha; Younossi, Zobair M.

2013-01-01

Obesity is associated with chronic low-grade inflammation perpetuated by visceral adipose. Other organs, particularly stomach and intestine, may also overproduce proinflammatory molecules. We examined the gene expression patterns in gastric tissue of morbidly obese patients with nonalcoholic fatty liver disease (NAFLD) and compared the changes in gene expression in different histological forms of NAFLD. Stomach tissue samples from 20 morbidly obese NAFLD patients who were undergoing sleeve gastrectomy were profiled using qPCR for 84 genes encoding inflammatory cytokines, chemokines, their receptors, and other components of inflammatory cascades. Interleukin 8 receptor-beta (IL8RB) gene overexpression in gastric tissue was correlated with the presence of hepatic steatosis, hepatic fibrosis, and histologic diagnosis of nonalcoholic steatohepatitis (NASH). Expression levels of soluble interleukin 1 receptor antagonist (IL1RN) were correlated with the presence of NASH and hepatic fibrosis. mRNA levels of interleukin 8 (IL8), chemokine (C-C motif) ligand 4 (CCL4), and its receptor chemokine (C-C motif) receptor type 5 (CCR5) showed a significant increase in patients with advanced hepatic inflammation and were correlated with the severity of the hepatic inflammation. The results of our study suggest that changes in expression patterns for inflammatory molecule encoding genes within gastric tissue may contribute to the pathogenesis of obesity-related NAFLD. PMID:23661906

A dual CCR2/CCR5 chemokine antagonist, BMS-813160? Evaluation of WO2011046916.

PubMed

Norman, Peter

2011-12-01

This application claims the dual CCR2/CCR5 antagonist (S)-1-[(1S,2R,4R)-4-isopropyl(methyl)amino)-2-propylcyclohexyl]-3-(6-(trifluoromethyl)quinazolin-4-ylamino)pyrrolidin-2-one, crystalline form N-1, a process for its preparation and therapeutic uses of it. The compound is shown to be a potent, well-absorbed, drug with a good duration of action. It shows better properties than similar compounds previously claimed by Bristol-Myers Squibb and appears to be a clinical development candidate.

Could FIV zoonosis responsible of the breakdown of the pathocenosis which has reduced the European CCR5-Delta32 allele frequencies?

PubMed Central

Faure, Eric

2008-01-01

Background In Europe, the north-south downhill cline frequency of the chemokine receptor CCR5 allele with a 32-bp deletion (CCR5-Δ32) raises interesting questions for evolutionary biologists. We had suggested first that, in the past, the European colonizers, principally Romans, might have been instrumental of a progressively decrease of the frequencies southwards. Indeed, statistical analyses suggested strong negative correlations between the allele frequency and historical parameters including the colonization dates by Mediterranean civilisations. The gene flows from colonizers to native populations were extremely low but colonizers are responsible of the spread of several diseases suggesting that the dissemination of parasites in naive populations could have induced a breakdown rupture of the fragile pathocenosis changing the balance among diseases. The new equilibrium state has been reached through a negative selection of the null allele. Results Most of the human diseases are zoonoses and cat might have been instrumental in the decrease of the allele frequency, because its diffusion through Europe was a gradual process, due principally to Romans; and that several cat zoonoses could be transmitted to man. The possible implication of a feline lentivirus (FIV) which does not use CCR5 as co-receptor is discussed. This virus can infect primate cells in vitro and induces clinical signs in macaque. Moreover, most of the historical regions with null or low frequency of CCR5-Δ32 allele coincide with historical range of the wild felid species which harbor species-specific FIVs. Conclusion We proposed the hypothesis that the actual European CCR5 allelic frequencies are the result of a negative selection due to a disease spreading. A cat zoonosis, could be the most plausible hypothesis. Future studies could provide if CCR5 can play an antimicrobial role in FIV pathogenesis. Moreover, studies of ancient DNA could provide more evidences regarding the implications of zoonoses in the actual CCR5-Δ32 distribution. PMID:18925940

Exclusive and persistent use of the entry coreceptor CXCR4 by human immunodeficiency virus type 1 from a subject homozygous for CCR5 delta32.

PubMed

Michael, N L; Nelson, J A; KewalRamani, V N; Chang, G; O'Brien, S J; Mascola, J R; Volsky, B; Louder, M; White, G C; Littman, D R; Swanstrom, R; O'Brien, T R

1998-07-01

Individuals who are homozygous for the 32-bp deletion in the gene coding for the chemokine receptor and major human immunodeficiency virus type 1 (HIV-1) coreceptor CCR5 (CCR5 -/-) lack functional cell surface CCR5 molecules and are relatively resistant to HIV-1 infection. HIV-1 infection in CCR5 -/- individuals, although rare, has been increasingly documented. We now report that the viral quasispecies from one such individual throughout disease is homogenous, T cell line tropic, and phenotypically syncytium inducing (SI); exclusively uses CXCR4; and replicates well in CCR5 -/- primary T cells. The recently discovered coreceptors BOB and Bonzo are not used. Although early and persistent SI variants have been described in longitudinal studies, this is the first demonstration of exclusive and persistent CXCR4 usage. With the caveat that the earliest viruses available from this subject were from approximately 4 years following primary infection, these data suggest that HIV-1 infection can be mediated and persistently maintained by viruses which exclusively utilize CXCR4. The lack of evolution toward the available minor coreceptors in this subject underscores the dominant biological roles of the major coreceptors CCR5 and CXCR4. This and two similar subjects (R. Biti, R. Ffrench, J. Young, B. Bennetts, G. Stewart, and T. Liang, Nat. Med. 3:252-253, 1997; I. Theodoreu, L. Meyer, M. Magierowska, C. Katlama, and C. Rouzioux, Lancet 349:1219-1220, 1997) showed relatively rapid CD4+ T-cell declines despite average or low initial viral RNA load. Since viruses which use CXCR4 exclusively cannot infect macrophages, these data have implications for the relative infection of the T-cell compartment versus the macrophage compartment in vivo and for the development of CCR5-based therapeutics.

Expression of human immunodeficiency virus (HIV)-binding lectin DC-SIGNR: Consequences for HIV infection and immunity.

PubMed

Soilleux, Elizabeth J; Morris, Lesley S; Rushbrook, Simon; Lee, Benhur; Coleman, Nicholas

2002-06-01

DC-SIGNR is a human immunodeficiency virus (HIV)-binding C-type lectin that is expressed on endothelium in the hepatic sinusoids, lymph node sinuses and placenta. Like closely related DC-SIGN, DC-SIGNR can bind both ICAM-3 and HIV and can potentiate HIV infection of T lymphocytes in trans. In the present study we have investigated reasons underlying the restricted distribution of DC-SIGNR and have examined DC-SIGNR expression in relation to HIV entry receptors. We show that DC-SIGNR expression does not depend on endothelial cell specialization or on activation state. DC-SIGNR-positive endothelium continues to express DC-SIGNR in conditions of hyperplasia, whereas the molecule is lost after neoplastic transformation, most likely as a result of changes in the microenvironment of the endothelial cells. We have further shown that CCR5, but not CD4, is coexpressed with DC-SIGNR on hepatic sinusoidal and placental capillary endothelial cells. However, CD4-positive CCR5-positive cells, such as hepatic Kupffer cells, placental Hofbauer cells, and CD4-positive T lymphocytes in lymph nodes, can be found adjacent to DC-SIGNR-positive endothelium. Therefore, DC-SIGNR may be able to mediate HIV infection of these cells in trans. Finally, we demonstrate that DC-SIGN and DC-SIGNR can be coexpressed on lymph node sinus endothelial cells, which may lead to modulation of the function of both molecules. Copyright 2002, Elsevier Science (USA). All rights reserved.

Associations of Circulating CXCR3-PD-1+CD4+T cells with Disease Activity of Systemic Lupus Erythematosus.

PubMed

Lei, Han; Xue, Yang; Yiyun, Yu; Weiguo, Wan; Ling, Lv; Zou, Hejian

2018-04-25

Which helper CD4 + T cell subset contributes to autoantibodies generation and severity of end-organ involvement in lupus patients remains to be explored. Our research aims to investigate the roles of circulating Tfh (cTfh) cell subsets and corresponding CXCR5 - Th cells in lupus patients and their correlation with SLEDAI. Peripheral blood mononuclear cells (PBMCs) were isolated from blood of SLE patients as well as healthy donors. The proportion of Th cell Subsets classified from cell surface markers (CD45RO, CXCR5, CXCR3, CCR6, PD-1, ICOS, and CCR7) is detected by flow cytometry. We found no difference in the frequency of CD45RO + CXCR5 + CD4 + T cells between SLE patients and health controls. As previous reported, SLE patients showed an increase in the percentage of CXCR5 + PD-1 + , CXCR5 + ICOS + PD-1 + and CXCR5 + CCR7 lo PD-1 hi cTfh subset, however, none of these populations had correlation with SLEDAI. Therefore, we further investigated the CXCR5 - subsets, and surprisingly we found that the frequency of CXCR3 - PD-1 + subset was correlated with SLEDAI, ds-DNA IgG, anti-nucleosome antibody, C3, and C4 independent of CXCR5. Consistently, CXCR3 - PD-1 + CD45RO + CD4 + T cells expressed factors associated with B-cell-help for the autoantibody production. CXCR3 - PD-1 + CD4 + T cells are a sensitive indicator to assess SLE disease activity and might contribute B cell help and the generation of autoantibodies in patients.

Expression of Ccl11 Associates with Immune Response Modulation and Protection against Neuroinflammation in Rats

PubMed Central

Zeitelhofer, Manuel; Hochmeister, Sonja; Beyeen, Amennai Daniel; Paulson, Atul; Gillett, Alan; Hedreul, Melanie Thessen; Covacu, Ruxandra; Lassmann, Hans; Olsson, Tomas; Jagodic, Maja

2012-01-01

Multiple sclerosis (MS) is a polygenic disease characterized by inflammation and demyelination in the central nervous system (CNS), which can be modeled in experimental autoimmune encephalomyelitis (EAE). The Eae18b locus on rat chromosome 10 has previously been linked to regulation of beta-chemokine expression and severity of EAE. Moreover, the homologous chemokine cluster in humans showed evidence of association with susceptibility to MS. We here established a congenic rat strain with Eae18b locus containing a chemokine cluster (Ccl2, Ccl7, Ccl11, Ccl12 and Ccl1) from the EAE- resistant PVG rat strain on the susceptible DA background and utilized myelin oligodendrocyte glycoprotein (MOG)-induced EAE to characterize the mechanisms underlying the genetic regulation. Congenic rats developed a milder disease compared to the susceptible DA strain, and this was reflected in decreased demyelination and in reduced recruitment of inflammatory cells to the brain. The congenic strain also showed significantly increased Ccl11 mRNA expression in draining lymph nodes and spinal cord after EAE induction. In the lymph nodes, macrophages were the main producers of CCL11, whereas macrophages and lymphocytes expressed the main CCL11 receptor, namely CCR3. Accordingly, the congenic strain also showed significantly increased Ccr3 mRNA expression in lymph nodes. In the CNS, the main producers of CCL11 were neurons, whereas CCR3 was detected on neurons and CSF producing ependymal cells. This corresponded to increased levels of CCL11 protein in the cerebrospinal fluid of the congenic rats. Increased intrathecal production of CCL11 in congenic rats was accompanied by a tighter blood brain barrier, reflected by more occludin+ blood vessels. In addition, the congenic strain showed a reduced antigen specific response and a predominant anti-inflammatory Th2 phenotype. These results indicate novel mechanisms in the genetic regulation of neuroinflammation. PMID:22815714

CCR6+ Th cell distribution differentiates systemic lupus erythematosus patients based on anti-dsDNA antibody status.

PubMed

Zhong, Wei; Jiang, Zhenyu; Wu, Jiang; Jiang, Yanfang; Zhao, Ling

2018-01-01

Systemic lupus erythematosus (SLE) disease has been shown to be associated with the generation of multiple auto-antibodies. Among these, anti-dsDNA antibodies (anti-DNAs) are specific and play a pathogenic role in SLE. Indeed, anti-DNA + SLE patients display a worse disease course. The generation of these pathogenic anti-DNAs has been attributed to the interaction between aberrant T helper (Th) cells and autoimmune B cells. Thus, in this study we have investigated whether CCR6 + Th cells have the ability to differentiate SLE patients based on anti-DNA status, and if their distribution has any correlation with disease activity. We recruited 25 anti-DNA + and 25 anti-DNA - treatment-naive onset SLE patients, matched for various clinical characteristics in our nested matched case-control study. CCR6 + Th cells and their additional subsets were analyzed in each patient by flow cytometry. Anti-DNA + SLE patients specifically had a higher percentage of Th cells expressing CCR6 and CXCR3. Further analysis of CCR6 + Th cell subsets showed that anti-DNA + SLE patients had elevated proportions of Th9, Th17, Th17.1 and CCR4/CXCR3 double-negative (DN) cells. However, the proportions of CCR6 - Th subsets, including Th1 and Th2 cells, did not show any association with anti-DNA status. Finally, we identified a correlation between CCR6 + Th subsets and clinical indicators, specifically in anti-DNA + SLE patients. Our data indicated that CCR6 + Th cells and their subsets were elevated and correlated with disease activity in anti-DNA + SLE patients. We speculated that CCR6 + Th cells may contribute to distinct disease severity in anti-DNA + SLE patients.

In vivo emergence of vicriviroc resistance in a human immunodeficiency virus type 1 subtype C-infected subject.

PubMed

Tsibris, Athe M N; Sagar, Manish; Gulick, Roy M; Su, Zhaohui; Hughes, Michael; Greaves, Wayne; Subramanian, Mani; Flexner, Charles; Giguel, Françoise; Leopold, Kay E; Coakley, Eoin; Kuritzkes, Daniel R

2008-08-01

Little is known about the in vivo development of resistance to human immunodeficiency virus type 1 (HIV-1) CCR5 antagonists. We studied 29 subjects with virologic failure from a phase IIb study of the CCR5 antagonist vicriviroc (VCV) and identified one individual with HIV-1 subtype C who developed VCV resistance. Studies with chimeric envelopes demonstrated that changes within the V3 loop were sufficient to confer VCV resistance. Resistant virus showed VCV-enhanced replication, cross-resistance to another CCR5 antagonist, TAK779, and increased sensitivity to aminooxypentane-RANTES and the CCR5 monoclonal antibody HGS004. Pretreatment V3 loop sequences reemerged following VCV discontinuation, implying that VCV resistance has associated fitness costs.

VEGF is a Promising Therapeutic Target for the Treatment of Clear Cell Carcinoma of the Ovary

PubMed Central

Mabuchi, Seiji; Kawase, Chiaki; Altomare, Deborah A.; Morishige, Kenichirou; Hayashi, Masami; Sawada, Kenjiro; Ito, Kimihiko; Terai, Yoshito; Nishio, Yukihiro; Klein-Szanto, Andres J.; Burger, Robert A.; Ohmichi, Masahide; Testa, Joseph R.; Kimura, Tadashi

2010-01-01

This study examined the role of VEGF as a therapeutic target in clear cell carcinoma (CCC) of the ovary, which has been regarded as a chemoresistant histological subtype. Immunohistochemical analysis using tissue microarrays of 98 primary ovarian cancers revealed that VEGF was strongly expressed both in early stage and advanced stage CCC of the ovary. In early stage CCCs, patients who had tumors with high levels of VEGF had significantly shorter survival than those with low levels of VEGF. In vitro experiments revealed that VEGF expression was significantly higher in cisplatin-refractory human clear cell carcinoma cells (RMG1-CR and KOC7C-CR), compared to the respective parental cells (RMG1 and KOC7C) in the presence of cisplatin. In vivo treatment with bevacizumab markedly inhibited the growth of both parental CCC cells-derived (RMG1 and KOC7C) and cisplatin-refractory CCC cells-derived (RMG1-CR and KOC7C-CR) tumors as a result of inhibition of tumor angiogenesis. The results of the current study indicate that VEGF is frequently expressed and can be a promising therapeutic target in the management of CCC. Bevacizumab may be efficacious not only as a first-line treatment but also as a second-line treatment of recurrent disease in patients previously treated with cisplatin. PMID:20663925

EBI1/CCR7 is a new member of dendritic cell chemokine receptor that is up-regulated upon maturation.

PubMed

Yanagihara, S; Komura, E; Nagafune, J; Watarai, H; Yamaguchi, Y

1998-09-15

Dendritic cells (DC) that are stimulated with inflammatory mediators can maturate and migrate from nonlymphoid tissues to lymphoid organs to initiate T cell-mediated immune responses. This migratory step is closely related to the maturation of the DC. In an attempt to identify chemokine receptors that might influence migration and are selectively expressed in mature DC, we have discovered that the chemokine receptor, EBI1/CCR7, is strikingly up-regulated upon maturation in three distinct culture systems: 1) mouse bone marrow-derived DC, 2) mouse epidermal Langerhans cells, and 3) human monocyte-derived DC. The EBI1/CCR7 expressed in mature DC is functional because ELC/MIP-3beta, recently identified as a ligand of EBI1/CCR7, induces a rise in intracellular free calcium concentrations and directional migration of human monocyte-derived mature DC (HLA-DRhigh, CD1a(low), CD14-, CD25+, CD83+, and CD86high) in a dose-dependent manner, but not of immature DC (HLA-DRlow, CD1a(high), CD14-, CD25-, CD83-, and CD86-). In contrast, macrophage inflammatory protein-1alpha (MIP-1alpha), monocyte chemotactic protein-3 (MCP-3), and RANTES are active on immature DC but not on mature DC. Thus, it seems likely that MIP-1alpha, MCP-3, and RANTES can mediate the migration of immature DC located in peripheral sites, whereas ELC/MIP-3beta can direct the migration of Ag-carrying DC from peripheral inflammatory sites, where DC are stimulated to up-regulate the expression of EBI1/CCR7, to lymphoid organs. It is postulated that different chemokines and chemokine receptors are involved in DC migration in vivo, depending on the maturation state of DC.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Takahashi, Akinori; Kikuguchi, Chisato; Morita, Masahiro

Highlights: Black-Right-Pointing-Pointer CNOT3 depletion increases the mitotic index. Black-Right-Pointing-Pointer CNOT3 inhibits the expression of MAD1. Black-Right-Pointing-Pointer CNOT3 destabilizes the MAD1 mRNA. Black-Right-Pointing-Pointer MAD1 knockdown attenuates the CNOT3 depletion-induced mitotic arrest. -- Abstract: The stability of mRNA influences the dynamics of gene expression. The CCR4-NOT complex, the major deadenylase in mammalian cells, shortens the mRNA poly(A) tail and contributes to the destabilization of mRNAs. The CCR4-NOT complex plays pivotal roles in various physiological functions, including cell proliferation, apoptosis, and metabolism. Here, we show that CNOT3, a subunit of the CCR4-NOT complex, is involved in the regulation of the spindle assembly checkpoint,more » suggesting that the CCR4-NOT complex also plays a part in the regulation of mitosis. CNOT3 depletion increases the population of mitotic-arrested cells and specifically increases the expression of MAD1 mRNA and its protein product that plays a part in the spindle assembly checkpoint. We showed that CNOT3 depletion stabilizes the MAD1 mRNA, and that MAD1 knockdown attenuates the CNOT3 depletion-induced increase of the mitotic index. Basing on these observations, we propose that CNOT3 is involved in the regulation of the spindle assembly checkpoint through its ability to regulate the stability of MAD1 mRNA.« less

Performance evaluation of continuity of care records (CCRs): parsing models in a mobile health management system.

PubMed

Chen, Hung-Ming; Liou, Yong-Zan

2014-10-01

In a mobile health management system, mobile devices act as the application hosting devices for personal health records (PHRs) and the healthcare servers construct to exchange and analyze PHRs. One of the most popular PHR standards is continuity of care record (CCR). The CCR is expressed in XML formats. However, parsing is an expensive operation that can degrade XML processing performance. Hence, the objective of this study was to identify different operational and performance characteristics for those CCR parsing models including the XML DOM parser, the SAX parser, the PULL parser, and the JSON parser with regard to JSON data converted from XML-based CCR. Thus, developers can make sensible choices for their target PHR applications to parse CCRs when using mobile devices or servers with different system resources. Furthermore, the simulation experiments of four case studies are conducted to compare the parsing performance on Android mobile devices and the server with large quantities of CCR data.

Immunological profile of periapical endodontic infections from HIV- and HIV+ patients.

PubMed

de Brito, L C N; Teles, F R; Teles, R P; Nogueira, P M; Vieira, L Q; Ribeiro Sobrinho, A P

2015-06-01

To evaluate CD4(+) CD28(+) and CD8(+) T-cell genes and the gene expression of IFN-γ, TNF-α, IL-1-β, IL-17A, IL-10, CCL-2/MCP-1, CCL-4, CCL-5 (RANTES), CXCR4, CCR5 and RANKL from cells in the periapical interstitial fluid from root canal infections in healthy patients (HIV-) and HIV-positive individuals (HIV+). Subjects included 20 HIV- and 23 HIV+ patients referred to the School of Dentistry at the Universidade Federal de Minas Gerais (Belo Horizonte, MG, Brazil). Almost all HIV+ patients were undergoing highly active antiretroviral therapy (HAART). Clinical samples were taken from teeth with pulp necrosis, and no patients had acute periapical symptoms at the time of the appointments. After cleaning and drying, 3 paper points were introduced into the root canal, passing passively through the root apex (2 mm) into the periapical tissues for 1 min. The samples were collected immediately after root canal cleaning and 7 days later (restrained root canal bacterial load) to characterize those gene expressions using real-time PCR. Significantly higher levels of CD4(+) CD28(+) and CD8(+) T cells in teeth with restrained bacterial loads (second collection) compared with the first collection were observed in both HIV- and HIV+ samples. In HIV- patients, an increase in IL-10 and CXCR4 expression was demonstrated as well as a decrease in pro-inflammatory cytokines such as RANKL, IFN-γ, IL1-β and CCL5. However, in HIV+ patients an increase in cytokines IFN-γ, IL-1-β, TNF-α and IL-17A, and chemokines CCL-2, CXCR4 and CCR5 were observed. The chemokine CCL-5 was not detected in HIV+ individuals. These findings suggest that after reducing the root canal bacterial load in HIV- individuals an anti-inflammatory response is generated whilst in HIV+ patients a pro-inflammatory response is sustained in the periapical area. © 2014 International Endodontic Journal. Published by John Wiley & Sons Ltd.

Optimization of RT-PCR reactions in studies with genes of lignin biosynthetic route in Saccharum spontaneum.

PubMed

Llerena, Juan P P; Araújo, Pedro; Mazzafera, Paulo

2018-01-01

Saccharum spontaneum has been used for the development of energy cane a crop aimed to be used for the production of second-generation ethanol, or lignocellulosic ethanol. Lignin is a main challenge in the conversion of cell wall sugars into ethanol. In our studies to isolate the genes the lignin biosynthesis in S. spontaneum we have had great difficulty in RT-PCR reactions. Thus, we evaluated the effectiveness of different additives in the amplification of these genes. While COMT and CCoAOMT genes did not need any additives for other genes there was no amplification (HCT, F5H, 4CL and CCR) or the yield was very low (CAD and C4H). The application of supplementary cDNA was enough to overcome the non-specificity and low yield for C4H and C3H, while the addition of 0.04% BSA + 2% formamide was effective to amplify 4CL, CCR, F5H and CCR. HCT was amplified only by addition of 0.04% BSA + 2% formamide + 0.1 M trehalose and amplification of PAL was possible with addition of 2% of DMSO. Besides optimization of expression assays, the results show that additives can act independently or synergistically.

HIV-1 Clinical Isolates Resistant to CCR5 Antagonists Exhibit Delayed Entry Kinetics That Are Corrected in the Presence of Drug

PubMed Central

Putcharoen, Opass; Lee, Sun Hee; Henrich, Timothy J.; Hu, Zixin; Vanichanan, Jakapat; Coakley, Eoin; Greaves, Wayne; Gulick, Roy M.; Kuritzkes, Daniel R.

2012-01-01

HIV CCR5 antagonists select for env gene mutations that enable virus entry via drug-bound coreceptor. To investigate the mechanisms responsible for viral adaptation to drug-bound coreceptor-mediated entry, we studied viral isolates from three participants who developed CCR5 antagonist resistance during treatment with vicriviroc (VCV), an investigational small-molecule CCR5 antagonist. VCV-sensitive and -resistant viruses were isolated from one HIV subtype C- and two subtype B-infected participants; VCV-resistant isolates had mutations in the V3 loop of gp120 and were cross-resistant to TAK-779, an investigational antagonist, and maraviroc (MVC). All three resistant isolates contained a 306P mutation but had variable mutations elsewhere in the V3 stem. We used a virus-cell β-lactamase (BlaM) fusion assay to determine the entry kinetics of recombinant viruses that incorporated full-length VCV-sensitive and -resistant envelopes. VCV-resistant isolates exhibited delayed entry rates in the absence of drug, relative to pretherapy VCV-sensitive isolates. The addition of drug corrected these delays. These findings were generalizable across target cell types with a range of CD4 and CCR5 surface densities and were observed when either population-derived or clonal envelopes were used to construct recombinant viruses. V3 loop mutations alone were sufficient to restore virus entry in the presence of drug, and the accumulation of V3 mutations during VCV therapy led to progressively higher rates of viral entry. We propose that the restoration of pre-CCR5 antagonist therapy HIV entry kinetics drives the selection of V3 loop mutations and may represent a common mechanism that underlies the emergence of CCR5 antagonist resistance. PMID:22090117

Simultaneous Knockout of CXCR4 and CCR5 Genes in CD4+ T Cells via CRISPR/Cas9 Confers Resistance to Both X4- and R5-Tropic Human Immunodeficiency Virus Type 1 Infection.

PubMed

Yu, Songlin; Yao, Yongchao; Xiao, Hongkui; Li, Jiaojiao; Liu, Quan; Yang, Yijun; Adah, Dickson; Lu, Junnan; Zhao, Siting; Qin, Li; Chen, Xiaoping

2018-01-01

Previous research has proven that disruption of either the CCR5 or the CXCR4 gene confers resistance to R5-tropic or X4-tropic human immunodeficiency virus type 1 (HIV-1) infection, respectively. However, the urgent need to ablate both of the co-receptors in individual post-thymic CD4+ T cells for dual protection remains. This study ablated the CCR5 and CXCR4 genes in human CD4+ cell lines and primary CD4+ T cells simultaneously using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9, a well-developed, highly efficient genetic engineering tool. The efficiency of gene modification is as high as 55% for CCR5 and 36% for CXCR4 in CD4+ cell lines through infection of a single lentiviral vector (LV-X4R5), which were markedly protected from both HIV-1 NL4-3 (X4-using strain) and HIV-1 YU-2 (R5-using strain) infection. Importantly, approximately 9% of the modified GHOST (3) CXCR4+CCR5+ cells harbor four bi-allelic gene disruptions in both the CXCR4 and CCR5 loci. Moreover, co-delivery of two single-guide RNAs loaded with Cas9: ribonucleoprotein (sgX4&R5 Cas9RNP) disrupted >12% of CCR5 and 10% of CXCR4 in primary human CD4+ T cells, which were rendered resistant to HIV-1 NL4-3 and HIV-1 YU-2 in vitro. Further, the modified cells do not show discernible mutagenesis in top-ranked off-target genes by the Surveyor assay and Sanger sequencing analysis. The results demonstrate the safety and efficacy of CRISPR/Cas9 in multiplex gene modification on peripherally circulating CD4+ T cells, which may promote a functional cure for HIV-1 infection.

Improved α-Amylase Production by Dephosphorylation Mutation of CreD, an Arrestin-Like Protein Required for Glucose-Induced Endocytosis of Maltose Permease and Carbon Catabolite Derepression in Aspergillus oryzae

PubMed Central

Tanaka, Mizuki; Hiramoto, Tetsuya; Tada, Hinako; Shintani, Takahiro

2017-01-01

ABSTRACT Aspergillus oryzae produces copious amount of amylolytic enzymes, and MalP, a major maltose permease, is required for the expression of amylase-encoding genes. The expression of these genes is strongly repressed by carbon catabolite repression (CCR) in the presence of glucose. MalP is transported from the plasma membrane to the vacuole by endocytosis, which requires the homolog of E6-AP carboxyl terminus ubiquitin ligase HulA, an ortholog of yeast Rsp5. In yeast, arrestin-like proteins mediate endocytosis as adaptors of Rsp5 and transporters. In the present study, we examined the involvement of CreD, an arrestin-like protein, in glucose-induced MalP endocytosis and CCR of amylase-encoding genes. Deletion of creD inhibited the glucose-induced endocytosis of MalP, and CreD showed physical interaction with HulA. Phosphorylation of CreD was detected by Western blotting, and two serine residues were determined as the putative phosphorylation sites. However, the phosphorylation state of the serine residues did not regulate MalP endocytosis and its interaction with HulA. Although α-amylase production was significantly repressed by creD deletion, both phosphorylation and dephosphorylation mimics of CreD had a negligible effect on α-amylase activity. Interestingly, dephosphorylation of CreD was required for CCR relief of amylase genes that was triggered by disruption of the deubiquitinating enzyme-encoding gene creB. The α-amylase activity of the creB mutant was 1.6-fold higher than that of the wild type, and the dephosphorylation mimic of CreD further improved the α-amylase activity by 2.6-fold. These results indicate that a combination of the dephosphorylation mutation of CreD and creB disruption increased the production of amylolytic enzymes in A. oryzae. IMPORTANCE In eukaryotes, glucose induces carbon catabolite repression (CCR) and proteolytic degradation of plasma membrane transporters via endocytosis. Glucose-induced endocytosis of transporters is mediated by their ubiquitination, and arrestin-like proteins act as adaptors of transporters and ubiquitin ligases. In this study, we showed that CreD, an arrestin-like protein, is involved in glucose-induced endocytosis of maltose permease and carbon catabolite derepression of amylase gene expression in Aspergillus oryzae. Dephosphorylation of CreD was required for CCR relief triggered by the disruption of creB, which encodes a deubiquitinating enzyme; a combination of the phosphorylation-defective mutation of CreD and creB disruption dramatically improved α-amylase production. This study shows the dual function of an arrestin-like protein and provides a novel approach for improving the production of amylolytic enzymes in A. oryzae. PMID:28455339

Improved α-Amylase Production by Dephosphorylation Mutation of CreD, an Arrestin-Like Protein Required for Glucose-Induced Endocytosis of Maltose Permease and Carbon Catabolite Derepression in Aspergillus oryzae.

PubMed

Tanaka, Mizuki; Hiramoto, Tetsuya; Tada, Hinako; Shintani, Takahiro; Gomi, Katsuya

2017-07-01

Aspergillus oryzae produces copious amount of amylolytic enzymes, and MalP, a major maltose permease, is required for the expression of amylase-encoding genes. The expression of these genes is strongly repressed by carbon catabolite repression (CCR) in the presence of glucose. MalP is transported from the plasma membrane to the vacuole by endocytosis, which requires the homolog of E6-AP carboxyl terminus ubiquitin ligase HulA, an ortholog of yeast Rsp5. In yeast, arrestin-like proteins mediate endocytosis as adaptors of Rsp5 and transporters. In the present study, we examined the involvement of CreD, an arrestin-like protein, in glucose-induced MalP endocytosis and CCR of amylase-encoding genes. Deletion of creD inhibited the glucose-induced endocytosis of MalP, and CreD showed physical interaction with HulA. Phosphorylation of CreD was detected by Western blotting, and two serine residues were determined as the putative phosphorylation sites. However, the phosphorylation state of the serine residues did not regulate MalP endocytosis and its interaction with HulA. Although α-amylase production was significantly repressed by creD deletion, both phosphorylation and dephosphorylation mimics of CreD had a negligible effect on α-amylase activity. Interestingly, dephosphorylation of CreD was required for CCR relief of amylase genes that was triggered by disruption of the deubiquitinating enzyme-encoding gene creB The α-amylase activity of the creB mutant was 1.6-fold higher than that of the wild type, and the dephosphorylation mimic of CreD further improved the α-amylase activity by 2.6-fold. These results indicate that a combination of the dephosphorylation mutation of CreD and creB disruption increased the production of amylolytic enzymes in A. oryzae IMPORTANCE In eukaryotes, glucose induces carbon catabolite repression (CCR) and proteolytic degradation of plasma membrane transporters via endocytosis. Glucose-induced endocytosis of transporters is mediated by their ubiquitination, and arrestin-like proteins act as adaptors of transporters and ubiquitin ligases. In this study, we showed that CreD, an arrestin-like protein, is involved in glucose-induced endocytosis of maltose permease and carbon catabolite derepression of amylase gene expression in Aspergillus oryzae Dephosphorylation of CreD was required for CCR relief triggered by the disruption of creB , which encodes a deubiquitinating enzyme; a combination of the phosphorylation-defective mutation of CreD and creB disruption dramatically improved α-amylase production. This study shows the dual function of an arrestin-like protein and provides a novel approach for improving the production of amylolytic enzymes in A. oryzae . Copyright © 2017 American Society for Microbiology.

The expression of the genes involved in leucine catabolism of Pseudomonas aeruginosa is controlled by the transcriptional regulator LiuR and by the CbrAB/Crc system.

PubMed

Díaz-Pérez, Alma Laura; Núñez, Cinthia; Meza Carmen, Victor; Campos-García, Jesús

2018-05-19

Pseudomonas aeruginosa metabolizes leucine through the leucine/isovalerate utilization pathway, whose enzymes are encoded in the liuRABCDE gene cluster (liu). In this study, we investigated the role of the LiuR protein in the liu cluster regulation. Our results indicated that liu expression is regulated at the transcriptional level by LiuR. Mobility shift assays using purified recombinant His-tagged LiuR showed that it was able to bind at the promoter region of liuR, in a dose-dependent manner. Results revealed that expression of the liu operon is subjected to carbon catabolite repression control (CCR); protein LiuD was strongly expressed in the presence of leucine, but it was repressed in the presence of glucose or succinate. Furthermore, this CCR control was dependent on LiuR as in the liuR - mutant the LiuD protein was strongly expressed in all the carbon sources tested. In agreement with this result, in the absence of the Crc protein, LiuD was expressed independently of the carbon source used, whereas in a cbrB - mutant its expression was severely impaired. The results indicated that the liu cluster is subjected to a coordinated transcriptional and translational regulation by the LiuR repressor and by the CbrAB/Crc system, respectively, in response to the available carbon source. Copyright © 2018 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

CCR presentations at AACR | Center for Cancer Research

Cancer.gov

CCR presentations at AACR Several CCR scientists will present their research at the AACR Annual Meeting in Washington, D.C., between April 1-5, 2017. Selected oral presentations are listed below. A full list of abstracts can be found on the AACR website.

The Not4 E3 Ligase and CCR4 Deadenylase Play Distinct Roles in Protein Quality Control

PubMed Central

Halter, David; Collart, Martine A.; Panasenko, Olesya O.

2014-01-01

Eukaryotic cells control their proteome by regulating protein production and protein clearance. Protein production is determined to a large extent by mRNA levels, whereas protein degradation depends mostly upon the proteasome. Dysfunction of the proteasome leads to the accumulation of non-functional proteins that can aggregate, be toxic for the cell, and, in extreme cases, lead to cell death. mRNA levels are controlled by their rates of synthesis and degradation. Recent evidence indicates that these rates have oppositely co-evolved to ensure appropriate mRNA levels. This opposite co-evolution has been correlated with the mutations in the Ccr4-Not complex. Consistently, the deadenylation enzymes responsible for the rate-limiting step in eukaryotic mRNA degradation, Caf1 and Ccr4, are subunits of the Ccr4-Not complex. Another subunit of this complex is a RING E3 ligase, Not4. It is essential for cellular protein solubility and has been proposed to be involved in co-translational quality control. An open question has been whether this role of Not4 resides strictly in the regulation of the deadenylation module of the Ccr4-Not complex. However, Not4 is important for proper assembly of the proteasome, and the Ccr4-Not complex may have multiple functional modules that participate in protein quality control in different ways. In this work we studied how the functions of the Caf1/Ccr4 and Not4 modules are connected. We concluded that Not4 plays a role in protein quality control independently of the Ccr4 deadenylase, and that it is involved in clearance of aberrant proteins at least in part via the proteasome. PMID:24465968

A Critical Role for Monocytes/Macrophages During Intestinal Inflammation-associated Lymphangiogenesis

PubMed Central

Becker, Felix; Kurmaeva, Elvira; Gavins, Felicity N. E.; Stevenson, Emily V.; Navratil, Aaron R.; Jin, Long; Tsunoda, Ikuo; Orr, A. Wayne; Alexander, Jonathan S.; Ostanin, Dmitry V.

2016-01-01

Background Inflammation-associated lymphangiogenesis (IAL) is frequently observed in inflammatory bowel diseases. IAL is believed to limit inflammation by enhancing fluid and immune cell clearance. Although monocytes/macrophages (MΦ) are known to contribute to intestinal pathology in inflammatory bowel disease, their role in intestinal IAL has never been studied mechanistically. We investigated contributions of monocytes/MΦ to the development of intestinal inflammation and IAL. Methods Because inflammatory monocytes express CC chemokine receptor 2 (CCR2), we used CCR2 diphtheria toxin receptor transgenic (CCR2.DTR) mice, in which monocytes can be depleted by diphtheria toxin injection, and CCR2−/− mice, which have reduced circulating monocytes. Acute or chronic colitis was induced by dextran sodium sulfate or adoptive transfer of CD4+CD45RBhigh T cells, respectively. Intestinal inflammation was assessed by flow cytometry, immunofluorescence, disease activity, and histopathology, whereas IAL was assessed by lymphatic vessel morphology and density. Results We demonstrated that intestinal MΦ expressed vascular endothelial growth factor-C/D. In acute colitis, monocyte-depleted mice were protected from intestinal injury and showed reduced IAL, which was reversed after transfer of wild-type monocytes into CCR2−/− mice. In chronic colitis, CCR2 deficiency did not attenuate inflammation but reduced IAL. Conclusions We propose a dual role of MΦ in (1) promoting acute inflammation and (2) contributing to IAL. Our data suggest that intestinal inflammation and IAL could occur independently, because IAL was reduced in the absence of monocytes/MΦ, even when inflammation was present. Future inflammatory bowel disease therapies might exploit promotion of IAL and suppression of MΦ independently, to restore lymphatic clearance and reduce inflammation. PMID:26950310

Development of operational models of receptor activation including constitutive receptor activity and their use to determine the efficacy of the chemokine CCL17 at the CC chemokine receptor CCR4

PubMed Central

Slack, RJ; Hall, DA

2012-01-01

BACKGROUND AND PURPOSE The operational model provides a key conceptual framework for the analysis of pharmacological data. However, this model does not include constitutive receptor activity, a frequent phenomenon in modern pharmacology, particularly in recombinant systems. Here, we developed extensions of the operational model which include constitutive activity and applied them to effects of agonists at the chemokine receptor CCR4. EXPERIMENTAL APPROACH The effects of agonists of CCR4 on [35S]GTPγS binding to recombinant cell membranes and on the filamentous (F-) actin content of human CD4+ CCR4+ T cells were determined. The basal [35S]GTPγS binding was changed by varying the GDP concentration whilst the basal F-actin contents of the higher expressing T cell populations were elevated, suggesting constitutive activity of CCR4. Both sets of data were analysed using the mathematical models. RESULTS The affinity of CCL17 (also known as TARC) derived from analysis of the T cell data (pKa= 9.61 ± 0.17) was consistent with radioligand binding experiments (9.50 ± 0.11) while that from the [35S]GTPγS binding experiments was lower (8.27 ± 0.09). Its intrinsic efficacy differed between the two systems (110 in T cells vs. 11). CONCLUSIONS AND IMPLICATIONS The presence of constitutive receptor activity allows the absolute intrinsic efficacy of agonists to be determined without a contribution from the signal transduction system. Intrinsic efficacy estimated in this way is consistent with Furchgott's definition of this property. CCL17 may have a higher intrinsic efficacy at CCR4 in human T cells than that expressed recombinantly in CHO cells. PMID:22335621

Investigation of Inhibition Mechanism of Chemokine Receptor CCR5 by Micro-second Molecular Dynamics Simulations.

PubMed

Salmas, Ramin Ekhteiari; Yurtsever, Mine; Durdagi, Serdar

2015-08-24

Chemokine receptor 5 (CCR5) belongs to G protein coupled receptors (GPCRs) and plays an important role in treatment of human immunodeficiency virus (HIV) infection since HIV uses CCR5 protein as a co-receptor. Recently, the crystal structure of CCR5-bound complex with an approved anti-retroviral drug (maroviroc) was resolved. During the crystallization procedure, amino acid residues (i.e., Cys224, Arg225, Asn226 and Glu227) at the third intra-cellular loop were replaced by the rubredoxin for stability reasons. In the current study, we aimed to understand the impact of the incorporated rubredoxin on the conformations of TM domains of the target protein. For this reason, rubredoxin was deleted from the crystal structure and the missing amino acids were engineered. The resultant structure was subjected to long (μs) molecular dynamics (MD) simulations to shed light into the inhibitory mechanism. The derived model structure displayed a significant deviation in the cytoplasmic domain of TM5 and IC3 in the absence of rubredoxin. The principal component analyses (PCA) and MD trajectory analyses revealed important structural and dynamical differences at apo and holo forms of the CCR5.

CRISPR/Cas9 system and its applications in human hematopoietic cells.

PubMed

Hu, Xiaotang

2016-11-01

Since 2012, the CRISPR-Cas9 system has been quickly and successfully tested in a broad range of organisms and cells including hematopoietic cells. The application of CRISPR-Cas9 in human hematopoietic cells mainly involves the genes responsible for HIV infection, β-thalassemia and sickle cell disease (SCD). The successful disruption of CCR5 and CXCR4 genes in T cells by CRISPR-Cas9 promotes the prospect of the technology in the functional cure of HIV. More recently, eliminating CCR5 and CXCR4 in induced pluripotent stem cells (iPSCs) derived from patients and targeting the HIV genome have been successfully carried out in several laboratories. The outcome from these approaches bring us closer to the goal of eradicating HIV infection. For hemoglobinopathies the ability to produce iPSC-derived from patients with the correction of hemoglobin (HBB) mutations by CRISPR-Cas9 has been tested in a number of laboratories. These corrected iPSCs also show the potential to differentiate into mature erythrocytes expressing high-level and normal HBB. In light of the initial success of CRESPR-Cas9 in target mutated gene(s) in the iPSCs, a combination of genomic editing and autogenetic stem cell transplantation would be the best strategy for root treatment of the diseases, which could replace traditional allogeneic stem cell transplantation. Copyright © 2016 Elsevier Inc. All rights reserved.

Enriching the Housing Environment for Mice Enhances Their NK Cell Antitumor Immunity via Sympathetic Nerve-Dependent Regulation of NKG2D and CCR5.

PubMed

Song, Yanfang; Gan, Yu; Wang, Qing; Meng, Zihong; Li, Guohua; Shen, Yuling; Wu, Yufeng; Li, Peiying; Yao, Ming; Gu, Jianren; Tu, Hong

2017-04-01

Mice housed in an enriched environment display a tumor-resistant phenotype due to eustress stimulation. However, the mechanisms underlying enriched environment-induced protection against cancers remain largely unexplained. In this study, we observed a significant antitumor effect induced by enriched environment in murine pancreatic cancer and lung cancer models. This effect remained intact in T/B lymphocyte-deficient Rag1 -/- mice, but was nearly eliminated in natural killer (NK) cell-deficient Beige mice or in antibody-mediated NK-cell-depleted mice, suggesting a predominant role of NK cells in enriched environment-induced tumor inhibition. Exposure to enriched environment enhanced NK-cell activity against tumors and promoted tumoral infiltration of NK cells. Enriched environment increased the expression levels of CCR5 and NKG2D (KLRK1) in NK cells; blocking their function effectively blunted the enriched environment-induced enhancement of tumoral infiltration and cytotoxic activity of NK cells. Moreover, blockade of β-adrenergic signaling or chemical sympathectomy abolished the effects of enriched environment on NK cells and attenuated the antitumor effect of enriched environment. Taken together, our results provide new insight into the mechanism by which eustress exerts a beneficial effect against cancer. Cancer Res; 77(7); 1611-22. ©2017 AACR . ©2017 American Association for Cancer Research.

Heme oxygenase-1 induction alters chemokine regulation and ameliorates human immunodeficiency virus-type-1 infection in lipopolysaccharide-stimulated macrophages

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhou, Zhao-Hua; Kumari, Namita; Nekhai, Sergei

2013-06-07

Highlights: •Lipopolysaccharide stimulation of heme oxygenase-1 (HO-1) ameliorated HIV-1 infection of primary human macrophages. •The partial protection by HO-1 against HIV infection was associated with induction of chemokines such as MIP1α and MIP1β. •This mechanism explains lipopolysaccharide-stimulated HO-1-mediated inhibition of HIV-1 infection of macrophages. -- Abstract: We have elucidated a putative mechanism for the host resistance against HIV-1 infection of primary human monocyte-derived macrophages (MDM) stimulated with lipopolysaccharide (LPS). We show that LPS-activated MDM both inhibited HIV-1 entry into the cells and were refractory to post-entry productive viral replication. LPS-treated cells were virtually negative for mature virions as revealed bymore » transmission electron microscopy. LPS activation of MDM markedly enhanced the expression of heme oxygenase-1 (HO-1), a potent inducible cytoprotective enzyme. Increased HO-1 expression was accompanied by elevated production of macrophage inflammatory chemokines (MIP1α and MIP1β) by LPS-activated MDM, significantly decreased surface chemokine receptor-5 (CCR-5) expression, and substantially reduced virus replication. Treatment of cells with HO-1 inhibitor SnPP IX (tin protoporphyrin IX) attenuated the LPS-mediated responses, HIV-1 replication and secretion of MIP1α, MIP1β, and LD78β chemokines with little change in surface CCR-5 expression. These results identify a novel role for HO-1 in the modulation of host immune response against HIV infection of MDM.« less

Simian Immunodeficiency Virus Targeting of CXCR3+ CD4+ T Cells in Secondary Lymphoid Organs Is Associated with Robust CXCL10 Expression in Monocyte/Macrophage Subsets.

PubMed

Fujino, Masayuki; Sato, Hirotaka; Okamura, Tomotaka; Uda, Akihiko; Takeda, Satoshi; Ahmed, Nursarat; Shichino, Shigeyuki; Shiino, Teiichiro; Saito, Yohei; Watanabe, Satoru; Sugimoto, Chie; Kuroda, Marcelo J; Ato, Manabu; Nagai, Yoshiyuki; Izumo, Shuji; Matsushima, Kouji; Miyazawa, Masaaki; Ansari, Aftab A; Villinger, Francois; Mori, Kazuyasu

2017-07-01

Glycosylation of Env defines pathogenic properties of simian immunodeficiency virus (SIV). We previously demonstrated that pathogenic SIVmac239 and a live-attenuated, quintuple deglycosylated Env mutant (Δ5G) virus target CD4 + T cells residing in different tissues during acute infection. SIVmac239 and Δ5G preferentially infected distinct CD4 + T cells in secondary lymphoid organs (SLOs) and within the lamina propria of the small intestine, respectively (C. Sugimoto et al., J Virol 86:9323-9336, 2012, https://doi.org/10.1128/JVI.00948-12). Here, we studied the host responses relevant to SIV targeting of CXCR3 + CCR5 + CD4 + T cells in SLOs. Genome-wide transcriptome analyses revealed that Th1-polarized inflammatory responses, defined by expression of CXCR3 chemokines, were distinctly induced in the SIVmac239-infected animals. Consistent with robust expression of CXCL10, CXCR3 + T cells were depleted from blood in the SIVmac239-infected animals. We also discovered that elevation of CXCL10 expression in blood and SLOs was secondary to the induction of CD14 + CD16 + monocytes and MAC387 + macrophages, respectively. Since the significantly higher levels of SIV infection in SLOs occurred with a massive accumulation of infiltrated MAC387 + macrophages, T cells, dendritic cells (DCs), and residential macrophages near high endothelial venules, the results highlight critical roles of innate/inflammatory responses in SIVmac239 infection. Restricted infection in SLOs by Δ5G also suggests that glycosylation of Env modulates innate/inflammatory responses elicited by cells of monocyte/macrophage/DC lineages. IMPORTANCE We previously demonstrated that a pathogenic SIVmac239 virus and a live-attenuated, deglycosylated mutant Δ5G virus infected distinct CD4 + T cell subsets in SLOs and the small intestine, respectively (C. Sugimoto et al., J Virol 86:9323-9336, 2012, https://doi.org/10.1128/JVI.00948-12). Accordingly, infections with SIVmac239, but not with Δ5G, deplete CXCR3 + CCR5 + CD4 + T (Th1) cells during the primary infection, thereby compromising the cellular immune response. Thus, we hypothesized that distinct host responses are elicited by the infections with two different viruses. We found that SIVmac239 induced distinctly higher levels of inflammatory Th1 responses than Δ5G. In particular, SIVmac239 infection elicited robust expression of CXCL10, a chemokine for CXCR3 + cells, in CD14 + CD16 + monocytes and MAC387 + macrophages recently infiltrated in SLOs. In contrast, Δ5G infection elicited only modest inflammatory responses. These results suggest that the glycosylation of Env modulates the inflammatory/Th1 responses through the monocyte/macrophage subsets and elicits marked differences in SIV infection and clinical outcomes. Copyright © 2017 American Society for Microbiology.

Simian Immunodeficiency Virus Targeting of CXCR3+ CD4+ T Cells in Secondary Lymphoid Organs Is Associated with Robust CXCL10 Expression in Monocyte/Macrophage Subsets

PubMed Central

Fujino, Masayuki; Sato, Hirotaka; Okamura, Tomotaka; Uda, Akihiko; Takeda, Satoshi; Ahmed, Nursarat; Shichino, Shigeyuki; Shiino, Teiichiro; Saito, Yohei; Watanabe, Satoru; Sugimoto, Chie; Kuroda, Marcelo J.; Ato, Manabu; Nagai, Yoshiyuki; Izumo, Shuji; Matsushima, Kouji; Miyazawa, Masaaki; Ansari, Aftab A.; Villinger, Francois

2017-01-01

ABSTRACT Glycosylation of Env defines pathogenic properties of simian immunodeficiency virus (SIV). We previously demonstrated that pathogenic SIVmac239 and a live-attenuated, quintuple deglycosylated Env mutant (Δ5G) virus target CD4+ T cells residing in different tissues during acute infection. SIVmac239 and Δ5G preferentially infected distinct CD4+ T cells in secondary lymphoid organs (SLOs) and within the lamina propria of the small intestine, respectively (C. Sugimoto et al., J Virol 86:9323–9336, 2012, https://doi.org/10.1128/JVI.00948-12). Here, we studied the host responses relevant to SIV targeting of CXCR3+ CCR5+ CD4+ T cells in SLOs. Genome-wide transcriptome analyses revealed that Th1-polarized inflammatory responses, defined by expression of CXCR3 chemokines, were distinctly induced in the SIVmac239-infected animals. Consistent with robust expression of CXCL10, CXCR3+ T cells were depleted from blood in the SIVmac239-infected animals. We also discovered that elevation of CXCL10 expression in blood and SLOs was secondary to the induction of CD14+ CD16+ monocytes and MAC387+ macrophages, respectively. Since the significantly higher levels of SIV infection in SLOs occurred with a massive accumulation of infiltrated MAC387+ macrophages, T cells, dendritic cells (DCs), and residential macrophages near high endothelial venules, the results highlight critical roles of innate/inflammatory responses in SIVmac239 infection. Restricted infection in SLOs by Δ5G also suggests that glycosylation of Env modulates innate/inflammatory responses elicited by cells of monocyte/macrophage/DC lineages. IMPORTANCE We previously demonstrated that a pathogenic SIVmac239 virus and a live-attenuated, deglycosylated mutant Δ5G virus infected distinct CD4+ T cell subsets in SLOs and the small intestine, respectively (C. Sugimoto et al., J Virol 86:9323–9336, 2012, https://doi.org/10.1128/JVI.00948-12). Accordingly, infections with SIVmac239, but not with Δ5G, deplete CXCR3+ CCR5+ CD4+ T (Th1) cells during the primary infection, thereby compromising the cellular immune response. Thus, we hypothesized that distinct host responses are elicited by the infections with two different viruses. We found that SIVmac239 induced distinctly higher levels of inflammatory Th1 responses than Δ5G. In particular, SIVmac239 infection elicited robust expression of CXCL10, a chemokine for CXCR3+ cells, in CD14+ CD16+ monocytes and MAC387+ macrophages recently infiltrated in SLOs. In contrast, Δ5G infection elicited only modest inflammatory responses. These results suggest that the glycosylation of Env modulates the inflammatory/Th1 responses through the monocyte/macrophage subsets and elicits marked differences in SIV infection and clinical outcomes. PMID:28424283

Eotaxin Increases Monolayer Permeability of Human Coronary Artery Endothelial Cells

PubMed Central

Jamaluddin, Md Saha; Wang, Xinwen; Wang, Hao; Rafael, Cubas; Yao, Qizhi; Chen, Changyi

2009-01-01

Objective The objective of this study was to determine the effects and molecular mechanisms of eotaxin, a newly discovered chemokine (CCL11), on endothelial permeability in the human coronary artery endothelial cells (HCAECs). Methods and Results Cells were treated with eotaxin, and the monolayer permeability was studied by using a costar transwell system with a Texas-Red-labeled dextran tracer. Eotaxin significantly increased monolayer permeability in a concentration-dependent manner. In addition, eotaxin treatment significantly decreased the mRNA and protein levels of endothelial junction molecules including zonula occludens-1 (ZO-1), occludin and claudin-1 in a concentration-dependent manner as determined by real time RT-PCR and Western blot analysis, respectively. Increased oxidative stress was observed in eotaxin-treated HCAECs by analysis of cellular glutathione levels. Furthermore, eotaxin treatment substantially activated the phosphorylation of MAPK p38. HCAECs expressed CCR3. Consequently, antioxidants (ginkgolide B and MnTBAP), specific p38 inhibitor SB203580 and anti-CCR3 antibody effectively blocked the eotaxin-induced permeability increase in HCAECs. Eotaxin also increased phosphorylation of Stat3 and nuclear translocation of NF-κB in HCAECs. Conclusions Eotaxin increases vascular permeability through CCR3, the down regulation of tight junction proteins, increase of oxidative stress and activation of MAPK p38, Stat3 and NF-kB pathways in HCAECs. PMID:19778943

HIV-1 Pathogenesis: The Virus

PubMed Central

Swanstrom, Ronald; Coffin, John

2012-01-01

Transmission of HIV-1 results in the establishment of a new infection, typically starting from a single virus particle. That virion replicates to generate viremia and persistent infection in all of the lymphoid tissue in the body. HIV-1 preferentially infects T cells with high levels of CD4 and those subsets of T cells that express CCR5, particularly memory T cells. Most of the replicating virus is in the lymphoid tissue, yet most of samples studied are from blood. For the most part the tissue and blood viruses represent a well-mixed population. With the onset of immunodeficiency, the virus evolves to infect new cell types. The tropism switch involves switching from using CCR5 to CXCR4 and corresponds to an expansion of infected cells to include naïve CD4+ T cells. Similarly, the virus evolves the ability to enter cells with low levels of CD4 on the surface and this potentiates the ability to infect macrophages, although the scope of sites where infection of macrophages occurs and the link to pathogenesis is only partly known and is clear only for infection of the central nervous system. A model linking viral evolution to these two pathways has been proposed. Finally, other disease states related to immunodeficiency may be the result of viral infection of additional tissues, although the evidence for a direct role for the virus is less strong. Advancing immunodeficiency creates an environment in which viral evolution results in viral variants that can target new cell types to generate yet another class of opportunistic infections (i.e., HIV-1 with altered tropism). PMID:23143844

Elevated levels of 2-arachidonoylglycerol promote atherogenesis in ApoE-/- mice.

PubMed

Jehle, Julian; Schöne, Benedikt; Bagheri, Sayeh; Avraamidou, Elina; Danisch, Melina; Frank, Imke; Pfeifer, Philipp; Bindila, Laura; Lutz, Beat; Lütjohann, Dieter; Zimmer, Andreas; Nickenig, Georg

2018-01-01

The endocannabinoid (eCB) 2-arachidonoylglycerol (2-AG) is a known modulator of inflammation and ligand to both, pro-inflammatory cannabinoid receptor 1 (CB1) and anti-inflammatory CB2. While the role of both receptors in atherogenesis has been studied extensively, the significance of 2-AG for atherogenesis is less well characterized. The impact of 2-AG on atherogenesis was studied in two treatment groups of ApoE-/- mice. One group received the monoacylglycerol lipase (MAGL)-inhibitor JZL184 [5 mg/kg i.p.], which impairs 2-AG degradation and thus causes elevated 2-AG levels, the other group received vehicle for four weeks. Simultaneously, both groups were fed a high-cholesterol diet. The atherosclerotic plaque burden was assessed in frozen sections through the aortic sinus following oil red O staining and infiltrating macrophages were detected by immunofluorescence targeting CD68. In vitro, the effect of 2-AG on B6MCL macrophage migration was assessed by Boyden chamber experiments. Transcription of adhesion molecules and chemokine receptors in macrophages was assessed by qPCR. As expected, application of the MAGL-inhibitor JZL184 resulted in a significant increase in 2-AG levels in vascular tissue (98.2 ± 16.1 nmol/g vs. 27.3 ± 4.5 nmol/g; n = 14-16; p < 0.001). ApoE-/- mice with elevated 2-AG levels displayed a significantly increased plaque burden compared to vehicle treated controls (0.44 ± 0.03 vs. 0.31 ± 0.04; n = 14; p = 0.0117). This was accompanied by a significant increase in infiltrating macrophages within the atherosclerotic vessel wall (0.33 ± 0.02 vs. 0.27 ± 0.01; n = 13-14; p = 0.0076). While there was no alteration to the white blood counts of JZL184-treated animals, 2-AG enhanced macrophage migration in vitro by 1.8 ± 0.2 -fold (n = 4-6; p = 0.0393) compared to vehicle, which was completely abolished by co-administration of either CB1- or CB2-receptor-antagonists. qPCR analyses of 2-AG-stimulated macrophages showed an enhanced transcription of the chemokine CCL5 (1.59 ± 0.23 -fold; n = 5-6; p = 0.0589) and its corresponding receptors CCR1 (2.04 ± 0.46 -fold; n = 10-11; p = 0.0472) and CCR5 (2.45 ± 0.62 -fold; n = 5-6; p = 0.0554). Taken together, elevated 2-AG levels appear to promote atherogenesis in vivo. Our data suggest that 2-AG promotes macrophage migration, possibly by the CCL5-CCR5/CCR1 axis, and thereby contributes to vascular inflammation. Thus, decreasing vascular 2-AG levels might represent a promising therapeutic strategy in patients suffering from atherosclerosis and coronary heart disease.

Generation of genetic constructs that simultaneously express several shRNAs.

PubMed

Kretova, Olga V; Alembekov, Ildar R; Tchurikov, Nickolai A

2012-05-01

RNAi has potential as an antiviral gene therapy strategy. Cassette constructs simultaneously expressing several siRNAs could prove to be the most efficient technique in developing gene therapy approaches for highly mutable viruses such as HIV-1. Here we describe a rapid and cost-saving protocol to generate cassettes that simultaneously express three siRNAs for repression of HIV-1 and CCR5 transcripts. siRNA biological activity was tested in a non-viral system, and exhibited both efficiency and specificity. Our results suggest this protocol can be used to rapidly generate cassette constructs for antiviral gene therapy applications.

Type III interferon is a critical regulator of innate antifungal immunity.

PubMed

Espinosa, Vanessa; Dutta, Orchi; McElrath, Constance; Du, Peicheng; Chang, Yun-Juan; Cicciarelli, Bryan; Pitler, Amy; Whitehead, Ian; Obar, Joshua J; Durbin, Joan E; Kotenko, Sergei V; Rivera, Amariliz

2017-10-06

Type III interferons (IFN-λs) are the most recently found members of the IFN cytokine family and engage IFNLR1 and IL10R2 receptor subunits to activate innate responses against viruses. We have identified IFN-λs as critical instructors of antifungal neutrophil responses. Using Aspergillus fumigatus ( Af ) as a model to study antifungal immune responses, we found that depletion of CCR2 + monocytes compromised the ability of neutrophils to control invasive fungal growth. Using an unbiased approach, we identified type I and III IFNs as critical regulators of the interplay between monocytes and neutrophils responding to Af We found that CCR2 + monocytes are an important early source of type I IFNs that prime optimal expression of IFN-λ. Type III IFNs act directly on neutrophils to activate their antifungal response, and mice with neutrophil-specific deletion of IFNLR1 succumb to invasive aspergillosis. Dysfunctional neutrophil responses in CCR2-depleted mice were rescued by adoptive transfer of pulmonary CCR2 + monocytes or by exogenous administration of IFN-α and IFN-λ. Thus, CCR2 + monocytes promote optimal activation of antifungal neutrophils by initiating a coordinated IFN response. We have identified type III IFNs as critical regulators of neutrophil activation and type I IFNs as early stimulators of IFN-λ expression. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

CXCL4/Platelet Factor 4 is an agonist of CCR1 and drives human monocyte migration.

PubMed

Fox, James M; Kausar, Fahima; Day, Amy; Osborne, Michael; Hussain, Khansa; Mueller, Anja; Lin, Jessica; Tsuchiya, Tomoko; Kanegasaki, Shiro; Pease, James E

2018-06-21

Activated platelets release micromolar concentrations of the chemokine CXCL4/Platelet Factor-4. Deposition of CXCL4 onto the vascular endothelium is involved in atherosclerosis, facilitating monocyte arrest and recruitment by an as yet, unidentified receptor. Here, we demonstrate that CXCL4 drives chemotaxis of the monocytic cell line THP-1. Migration and intracellular calcium responses induced by CXCL4 were pertussis toxin-sensitive, implicating a GPCR in signal transduction. Cell treatment with chondroitinase ABC ablated migration, suggesting that cis presentation of CXCL4 by cell surface glycosaminoglycans to a GPCR is required. Although CXCR3 has been previously described as a CXCL4 receptor, THP-1 cells were unresponsive to CXCR3 ligands and CXCL4-induced migration was insensitive to a CXCR3 antagonist, suggesting that an alternative receptor is involved. Interrogating CC-class chemokine receptor transfectants, we unexpectedly found that CXCL4 could induce the migration of CCR1-expressing cells and also induce CCR1 endocytosis. Extending our findings to primary human monocytes, we observed that CXCL4 induced CCR1 endocytosis and could induce monocyte chemotaxis in a CCR1 antagonist-sensitive manner. Collectively, our data identify CCR1 as a previously elusive monocyte CXCL4 receptor and suggest that CCR1 may play a role in inflammation where the release of CXCL4 is implicated.

Prenatally diagnosed de novo apparently balanced complex chromosome rearrangements: Two new cases and review of the literature

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ruiz, C.; Grubs, R.E.; Jewett, T.

Complex chromosome rearrangements (CCR) are rare structural rearrangements. Currently six cases of prenatally diagnosed balanced de novo CCR have been described. We present two new cases of prenatally ascertained balanced de novo CCR. In the first case, an amniocentesis revealed a balanced de novo three-way CCR involving chromosomes 5,6, and 11 with a pericentric inversion of chromosome 5 [four breaks]. In the second case a balanced de novo rearrangement was identified by amniocentesis which involved a reciprocal translocation between chromosomes 3 and 8 and a CCR involving chromosomes 6,7, and 18 [six breaks]. The use of whole chromosome painting helpedmore » elucidate the nature of these rearrangements. A review of the postnatally ascertained cases suggests that most patients have congenital anomalies, minor anomalies, and/or developmental delay/mental retardation. In addition, there appears to be a relationship between the number of chromosome breaks and the extent of phenotypic effects. The paucity of information regarding prenatally diagnosed CCR and the bias of ascertainment of postnatal CCR cases poses a problem in counseling families. 38 refs., 3 figs., 4 tabs.« less

Therapeutic Targeting of CC Ligand 21 or CC Chemokine Receptor 7 Abrogates Pulmonary Fibrosis Induced by the Adoptive Transfer of Human Pulmonary Fibroblasts to Immunodeficient Mice

PubMed Central

Pierce, Elizabeth M.; Carpenter, Kristin; Jakubzick, Claudia; Kunkel, Steven L.; Flaherty, Kevin R.; Martinez, Fernando J.; Hogaboam, Cory M.

2007-01-01

Idiopathic interstitial pneumonias (IIPs) are a collection of pulmonary fibrotic diseases of unknown etiopathogenesis. CC chemokine receptor 7 (CCR7) is expressed in IIP biopsies and primary fibroblast lines, but its role in pulmonary fibrosis was not previously examined. To study the in vivo role of CCR7 in a novel model of pulmonary fibrosis, 1.0 × 106 primary fibroblasts grown from idiopathic pulmonary fibrosis/usual interstitial pneumonia, nonspecific interstitial pneumonia, or histologically normal biopsies were injected intravenously into C.B-17 severe combined immunodeficiency (SCID)/beige (bg) mice. At days 35 and 63 after idiopathic pulmonary fibrosis/usual interstitial pneumonia fibroblast injection, patchy interstitial fibrosis and increased hydroxyproline were present in the lungs of immunodeficient mice. Adoptively transferred nonspecific interstitial pneumonia fibroblasts caused a more diffuse interstitial fibrosis and increased hydroxyproline levels at both times, but injected normal human fibroblasts did not induce interstitial remodeling changes in C.B-17SCID/bg mice. Systemic therapeutic immunoneutralization of either human CCR7 or CC ligand 21, its ligand, significantly attenuated the pulmonary fibrosis in groups of C.B-17SCID/bg mice that received either type of IIP fibroblasts. Thus, the present study demonstrates that pulmonary fibrosis is initiated by the intravenous introduction of primary human fibroblast lines into immunodeficient mice, and this fibrotic response is dependent on the interaction between CC ligand 21 and CCR7. PMID:17392156

Association of chemokine receptor gene (CCR2-CCR5) haplotypes with acquisition and control of HIV-1 infection in Zambians

PubMed Central

2011-01-01

Background Polymorphisms in chemokine (C-C motif) receptors 2 and 5 genes (CCR2 and CCR5) have been associated with HIV-1 infection and disease progression. We investigated the impact of CCR2-CCR5 haplotypes on HIV-1 viral load (VL) and heterosexual transmission in an African cohort. Between 1995 and 2006, cohabiting Zambian couples discordant for HIV-1 (index seropositive and HIV-1 exposed seronegative {HESN}) were monitored prospectively to determine the role of host genetic factors in HIV-1 control and heterosexual transmission. Genotyping for eight CCR2 and CCR5 variants resolved nine previously recognized haplotypes. By regression and survival analytic techniques, controlling for non-genetic factors, we estimated the effects of these haplotypic variants on a) index partner VL, b) seroconverter VL, c) HIV-1 transmission by index partners, d) HIV-1 acquisition by HESN partners. Results Among 567 couples, 240 virologically linked transmission events had occurred through 2006. HHF*2 homozygosity was associated with significantly lower VL in seroconverters (mean beta = -0.58, log10 P = 0.027) and the HHD/HHE diplotype was associated with significantly higher VL in the seroconverters (mean beta = 0.54, log10 P = 0.014) adjusted for age and gender in multivariable model. HHD/HHE was associated with more rapid acquisition of infection by the HESNs (HR = 2.0, 95% CI = 1.20-3.43, P = 0.008), after adjustments for index partner VL and the presence of genital ulcer or inflammation in either partner in Cox multivariable models. The HHD/HHE effect was stronger in exposed females (HR = 2.1, 95% CI = 1.14-3.95, P = 0.018). Conclusions Among Zambian discordant couples, HIV-1 coreceptor gene haplotypes and diplotypes appear to modulate HIV-1 VL in seroconverters and alter the rate of HIV-1 acquisition by HESNs. These associations replicate or resemble findings reported in other African and European populations. PMID:21429204

CCL3L1 copy number, CCR5 genotype and susceptibility to tuberculosis.

PubMed

Carpenter, Danielle; Taype, Carmen; Goulding, Jon; Levin, Mike; Eley, Brian; Anderson, Suzanne; Shaw, Marie-Anne; Armour, John A L

2014-01-09

Tuberculosis is a major infectious disease and functional studies have provided evidence that both the chemokine MIP-1α and its receptor CCR5 play a role in susceptibility to TB. Thus by measuring copy number variation of CCL3L1, one of the genes that encode MIP-1α, and genotyping a functional promoter polymorphism -2459A > G in CCR5 (rs1799987) we investigate the influence of MIP-1α and CCR5, independently and combined, in susceptibility to clinically active TB in three populations, a Peruvian population (n = 1132), a !Xhosa population (n = 605) and a South African Coloured population (n = 221). The three populations include patients with clinically diagnosed pulmonary TB, as well as other, less prevalent forms of extrapulmonary TB. Copy number of CCL3L1 was measured using the paralogue ratio test and exhibited ranges between 0-6 copies per diploid genome (pdg) in Peru, between 0-12 pdg in !Xhosa samples and between 0-10 pdg in South African Coloured samples. The CCR5 promoter polymorphism was observed to differ significantly in allele frequency between populations (*A; Peru f = 0.67, !Xhosa f = 0.38, Coloured f = 0.48). The case-control association studies performed however find, surprisingly, no evidence for an influence of variation in genes coding for MIP-1α or CCR5 individually or together in susceptibility to clinically active TB in these populations.

CCL2 and CCR2 variants are associated with skeletal muscle strength and change in strength with resistance training.

PubMed

Harmon, Brennan T; Orkunoglu-Suer, E Funda; Adham, Kasra; Larkin, Justin S; Gordish-Dressman, Heather; Clarkson, Priscilla M; Thompson, Paul D; Angelopoulos, Theodore J; Gordon, Paul M; Moyna, Niall M; Pescatello, Linda S; Visich, Paul S; Zoeller, Robert F; Hubal, Monica J; Tosi, Laura L; Hoffman, Eric P; Devaney, Joseph M

2010-12-01

Baseline muscle size and muscle adaptation to exercise are traits with high variability across individuals. Recent research has implicated several chemokines and their receptors in the pathogenesis of many conditions that are influenced by inflammatory processes, including muscle damage and repair. One specific chemokine, chemokine (C-C motif) ligand 2 (CCL2), is expressed by macrophages and muscle satellite cells, increases expression dramatically following muscle damage, and increases expression further with repeated bouts of exercise, suggesting that CCL2 plays a key role in muscle adaptation. The present study hypothesizes that genetic variations in CCL2 and its receptor (CCR2) may help explain muscle trait variability. College-aged subjects [n = 874, Functional Single-Nucleotide Polymorphisms Associated With Muscle Size and Strength (FAMUSS) cohort] underwent a 12-wk supervised strength-training program for the upper arm muscles. Muscle size (via MR imaging) and elbow flexion strength (1 repetition maximum and isometric) measurements were taken before and after training. The study participants were then genotyped for 11 genetic variants in CCL2 and five variants in CCR2. Variants in the CCL2 and CCR2 genes show strong associations with several pretraining muscle strength traits, indicating that inflammatory genes in skeletal muscle contribute to the polygenic system that determines muscle phenotypes. These associations extend across both sexes, and several of these genetic variants have been shown to influence gene regulation.

The conserved RNA recognition motif and C3H1 domain of the Not4 ubiquitin ligase regulate in vivo ligase function.

PubMed

Chen, Hongfeng; Sirupangi, Tirupataiah; Wu, Zhao-Hui; Johnson, Daniel L; Laribee, R Nicholas

2018-05-25

The Ccr4-Not complex controls RNA polymerase II (Pol II) dependent gene expression and proteasome function. The Not4 ubiquitin ligase is a Ccr4-Not subunit that has both a RING domain and a conserved RNA recognition motif and C3H1 domain (referred to as the RRM-C domain) with unknown function. We demonstrate that while individual Not4 RING or RRM-C mutants fail to replicate the proteasomal defects found in Not4 deficient cells, mutation of both exhibits a Not4 loss of function phenotype. Transcriptome analysis revealed that the Not4 RRM-C affects a specific subset of Pol II-regulated genes, including those involved in transcription elongation, cyclin-dependent kinase regulated nutrient responses, and ribosomal biogenesis. The Not4 RING, RRM-C, or RING/RRM-C mutations cause a generalized increase in Pol II binding at a subset of these genes, yet their impact on gene expression does not always correlate with Pol II recruitment which suggests Not4 regulates their expression through additional mechanisms. Intriguingly, we find that while the Not4 RRM-C is dispensable for Ccr4-Not association with RNA Pol II, the Not4 RING domain is required for these interactions. Collectively, these data elucidate previously unknown roles for the conserved Not4 RRM-C and RING domains in regulating Ccr4-Not dependent functions in vivo.

PD-1HIGH Follicular CD4 T Helper Cell Subsets Residing in Lymph Node Germinal Centers Correlate with B Cell Maturation and IgG Production in Rhesus Macaques

PubMed Central

Xu, Huanbin; Wang, Xiaolei; Lackner, Andrew A.; Veazey, Ronald S.

2014-01-01

CD4+ T follicular helper (TFH) cells guide development and maturation of B cells and are crucial for effective antibody responses. Here we found rhesus macaque TFH cells, defined as CXCR5+CD4 T cells, contain two major populations: PD-1INT and PD-1HIGH cells. Of these, PD-1HIGHCD4+ T cells highly co-express ICOS but little CCR7, and reside in lymph node germinal centers (GCs), but not in blood. These cells secrete IL-21 and express transcriptional factor Bcl-6 at higher levels than CXCR5+PD-1INTCD4+ T cells. In addition, the frequency of PD-1HIGHCD4+ T cells is low in lymph nodes of newborns, but increases with age. Levels of PD-1HIGHCD4+ T cells correlate with mature B cells in lymph nodes, and PD-1 blockade in PD-1HIGHCD4+ T and B cell co-cultures significantly inhibits IgG production. In summary, PD-1HIGHCD4+ T cells residing in GC represent a specific TFH subset that contributes to maturation of B cells and IgG production. PMID:24678309

ADMA induces monocyte adhesion via activation of chemokine receptors in cultured THP-1 cells.

PubMed

Chen, Meifang; Li, Yuanjian; Yang, Tianlun; Wang, Yongjin; Bai, Yongping; Xie, Xiumei

2008-08-01

Asymmetric dimethylarginine (ADMA), an endogenous NOS inhibitor, is also an important inflammatory factor contributing to the development of atherosclerosis (AS). The present study was to test the effect of ADMA on angiotensin (Ang) II-induced monocytic adhesion. Human monocytoid cells (THP-1) or isolated peripheral blood monocyte cells (PBMCs) were incubated with Ang II (10(-6)M) or exogenous ADMA (30 microM) for 4 or 24h in the absence or presence of losartan or antioxidant PDTC. In cultured THP-1 cells, Ang II (10(-6)M) for 24h elevated the level of ADMA in the medium, upregulated the protein expression of protein arginine methyltransferase (PRMT) and decreased the activity of dimethylarginine dimethylaminohydrolase (DDAH). Both of Ang II and ADMA increased monocytic adhesion to human umbilical vein endothelial cells (HUVECs), elevated the levels of monocyte chemoattractant protein (MCP)-1, interleukin (IL)-8 and tumor necrosis factor (TNF)-alpha and upregulated CCR(2) and CXCR(2) mRNA expression, concomitantly with increase in reactive oxygen species (ROS) generation and activation of nuclear factor (NF)-kappaB. Pretreatment with losartan (10 microM) or PDTC (10 microM) abolished the effects mediated by Ang II or ADMA. In isolated PBMCs from healthy individuals, ADMA upregulated the expression of CXCR(2) mRNA, which was attenuated by losartan (10 microM), however, ADMA had no effect on surface protein expression of CCR(2). The present results suggest that ADMA may be involved in monocytic adhesion induced by Ang II via activation of chemokine receptors by ROS/NF-kappaB pathway.

International phase 3 study of azacitidine vs conventional care regimens in older patients with newly diagnosed AML with >30% blasts

PubMed Central

Seymour, John F.; Butrym, Aleksandra; Wierzbowska, Agnieszka; Selleslag, Dominik; Jang, Jun Ho; Kumar, Rajat; Cavenagh, James; Schuh, Andre C.; Candoni, Anna; Récher, Christian; Sandhu, Irwindeep; Bernal del Castillo, Teresa; Al-Ali, Haifa Kathrin; Martinelli, Giovanni; Falantes, Jose; Noppeney, Richard; Stone, Richard M.; Minden, Mark D.; McIntyre, Heidi; Songer, Steve; Lucy, Lela M.; Beach, C. L.; Döhner, Hartmut

2015-01-01

This multicenter, randomized, open-label, phase 3 trial evaluated azacitidine efficacy and safety vs conventional care regimens (CCRs) in 488 patients age ≥65 years with newly diagnosed acute myeloid leukemia (AML) with >30% bone marrow blasts. Before randomization, a CCR (standard induction chemotherapy, low-dose ara-c, or supportive care only) was preselected for each patient. Patients then were assigned 1:1 to azacitidine (n = 241) or CCR (n = 247). Patients assigned to CCR received their preselected treatment. Median overall survival (OS) was increased with azacitidine vs CCR: 10.4 months (95% confidence interval [CI], 8.0-12.7 months) vs 6.5 months (95% CI, 5.0-8.6 months), respectively (hazard ratio [HR] was 0.85; 95% CI, 0.69-1.03; stratified log-rank P = .1009). One-year survival rates with azacitidine and CCR were 46.5% and 34.2%, respectively (difference, 12.3%; 95% CI, 3.5%-21.0%). A prespecified analysis censoring patients who received AML treatment after discontinuing study drug showed median OS with azacitidine vs CCR was 12.1 months (95% CI, 9.2-14.2 months) vs 6.9 months (95% CI, 5.1-9.6 months; HR, 0.76; 95% CI, 0.60-0.96; stratified log-rank P = .0190). Univariate analysis showed favorable trends for azacitidine compared with CCR across all subgroups defined by baseline demographic and disease features. Adverse events were consistent with the well-established safety profile of azacitidine. Azacitidine may be an important treatment option for this difficult-to-treat AML population. This trial was registered at www.clinicaltrials.gov as #NCT01074047. PMID:25987659

A Linear Epitope in the N-Terminal Domain of CCR5 and Its Interaction with Antibody

PubMed Central

Chain, Benny; Arnold, Jack; Akthar, Samia; Brandt, Michael; Davis, David; Noursadeghi, Mahdad; Lapp, Thabo; Ji, Changhua; Sankuratri, Surya; Zhang, Yanjing; Govada, Lata; Saridakis, Emmanuel; Chayen, Naomi

2015-01-01

The CCR5 receptor plays a role in several key physiological and pathological processes and is an important therapeutic target. Inhibition of the CCR5 axis by passive or active immunisation offers one very selective strategy for intervention. In this study we define a new linear epitope within the extracellular domain of CCR5 recognised by two independently produced monoclonal antibodies. A short peptide encoding the linear epitope can induce antibodies which recognise the intact receptor when administered colinear with a tetanus toxoid helper T cell epitope. The monoclonal antibody RoAb 13 is shown to bind to both cells and peptide with moderate to high affinity (6x10^8 and 1.2x107 M-1 respectively), and binding to the peptide is enhanced by sulfation of tyrosines at positions 10 and 14. RoAb13, which has previously been shown to block HIV infection, also blocks migration of monocytes in response to CCR5 binding chemokines and to inflammatory macrophage conditioned medium. A Fab fragment of RoAb13 has been crystallised and a structure of the antibody is reported to 2.1 angstrom resolution. PMID:26030924

Visualizing High-Efficiency HIV Transfer | Center for Cancer Research

Cancer.gov

The Human Immunodeficiency Virus (HIV), the causative agent of Acquired Immunodeficiency Syndrome (AIDS), infects and eventually kills CD4 receptor-expressing T cells, which are critical for proper immune system function. The gp120 protein on the surface of HIV particles is known to bind CD4 and a co-receptor, either CCR5 or CXCR4, leading to fusion of the virus and T cell

Chemokine CCR3 ligands-binding peptides derived from a random phage-epitope library.

PubMed

Houimel, Mehdi; Mazzucchelli, Luca

2013-01-01

Eosinophils are major effectors cells implicated in a number of chronic inflammatory diseases in humans, particularly bronchial asthma and allergic rhinitis. The human chemokine receptor C-C receptor 3 (hCCR3) provides a mechanism for the recruitment of eosinophils into tissue and thus has recently become an attractive biological target for therapeutic intervention. In order to develop peptides antagonists of hCCR3-hCCL11 (human eotaxin) interactions, a random bacteriophage hexapeptide library was used to map structural features of hCCR3 by determining the epitopes of neutralizing anti-hCCR3 mAb 7B11. This mAb t is selective for hCCR3 and exhibit potent antagonist activity in receptor binding and functional assays. After three rounds of biopanning, four mAb7B11-binding peptides were identified from a 6-mer linear peptide library. The phage bearing the peptides showed specific binding to immobilized mAb 7B11 with over 94% of phages bound being competitively inhibited by free synthetic peptides. In FACScan analysis all selected phage peptides were able to strongly inhibit the binding of mAb 7B11 to hCCR3-transfected preB-300-19 murine cells. Furthermore, synthetic peptides of the corresponding phage epitopes were effective in blocking the antibody-hCCR3 interactions and to inhibit the binding of hCCL11 to hCCR3 transfectants. Chemically synthesized peptides CKGERF, FERKGK, SSMKVK and RHVSSQ, effectively competed for (125)I-hCCL11 binding to hCCR3 with IC(50) ranging from 3.5 to 9.7μM. Calcium release and chemotaxis of hCCR3 transfectants or human eosinophils were inhibited by all peptides in a dose-dependent manner. Furthermore, they showed inhibitory effects on chemotaxis of human eosinophils induced by hCCL11, hCCL5, hCCL7, hCCL8, and hCCL24. Specificities of all selected peptides were assessed with hCXCR1, hCXCR2, hCXCR3, and hCCR5 receptors. Peptides CKGERF and FERKGK showed inhibitory effects on eosinophil chemotaxis in a murine model of mCCL11-induced peritoneal eosinophilia. The development of peptides inhibiting the interactions between hCCR3 and its chemokine ligands will facilitate the development of small peptides antagonists with the hope of ameliorating chronic inflammatory diseases in humans. Copyright © 2012 Elsevier B.V. All rights reserved.

CCR4+T cell recruitment to the skin in mycosis fungoides: potential contributions by thymic stromal lymphopoietin and interleukin-16.

PubMed

Tuzova, Marina; Richmond, Jillian; Wolpowitz, Deon; Curiel-Lewandrowski, Clara; Chaney, Keri; Kupper, Thomas; Cruikshank, William

2015-02-01

Mycosis fungoides (MF) is characterized by skin accumulation of CCR4+CCR7- effector memory T cells; however the mechanism for their recruitment is not clearly identified. Thymic Stromal Lymphopoietin (TSLP) is a keratinocyte-derived cytokine that triggers Th2 immunity and is associated with T cell recruitment to the skin in atopic dermatitis. Interleukin-16 (IL-16) is a chemoattractant and growth factor for CD4+T cells. We hypothesized that TSLP and IL-16 could contribute to recruitment of malignant T cells in MF. We found elevated TSLP and IL-16 in very early stage patients' plasma and skin biopsies, prior to elevation in CCL22. Both TSLP and IL-16 induced migratory responses of CCR4+TSLPR+CD4+CCR7-CD31+cells, characteristic of malignant T cells in the skin. Co-stimulation also resulted in significant proliferative responses. We conclude that TSLP and IL-16, expressed at early stages of disease, function to recruit malignant T cells to the skin and contribute to their enhanced proliferation.

The discovery of tropane-derived CCR5 receptor antagonists.

PubMed

Armour, Duncan R; de Groot, Marcel J; Price, David A; Stammen, Blanda L C; Wood, Anthony; Perros, Manos; Burt, Catherine

2006-04-01

The development of compound 1, a piperidine-based CCR5 receptor antagonist with Type I CYP2D6 inhibition, into the tropane-derived analogue 5, is described. This compound, which is devoid of CYP2D6 liabilities, is a highly potent ligand for the CCR5 receptor and has broad-spectrum activity against a range of clinically relevant HIV isolates. The identification of human ether a-go-go-related gene channel inhibition within this series is described and the potential for QTc interval prolongation discussed. Furthermore, structure activity relationship (SAR) around the piperidine moiety is also described.

Digital gene expression profiling analysis and its application in the identification of genes associated with improved response to neoadjuvant chemotherapy in breast cancer.

PubMed

Liu, Xiaozhen; Jin, Gan; Qian, Jiacheng; Yang, Hongjian; Tang, Hongchao; Meng, Xuli; Li, Yongfeng

2018-04-23

This study aimed to screen sensitive biomarkers for the efficacy evaluation of neoadjuvant chemotherapy in breast cancer. In this study, Illumina digital gene expression sequencing technology was applied and differentially expressed genes (DEGs) between patients presenting pathological complete response (pCR) and non-pathological complete response (NpCR) were identified. Further, gene ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis were then performed. The genes in significant enriched pathways were finally quantified by quantitative real-time PCR (qRT-PCR) to confirm that they were differentially expressed. Additionally, GSE23988 from Gene Expression Omnibus database was used as the validation dataset to confirm the DEGs. After removing the low-quality reads, 715 DEGs were finally detected. After mapping to KEGG pathways, 10 DEGs belonging to the ubiquitin proteasome pathway (HECTD3, PSMB10, UBD, UBE2C, and UBE2S) and cytokine-cytokine receptor interactions (CCL2, CCR1, CXCL10, CXCL11, and IL2RG) were selected for further analysis. These 10 genes were finally quantified by qRT-PCR to confirm that they were differentially expressed (the log 2 fold changes of selected genes were - 5.34, 7.81, 6.88, 5.74, 3.11, 19.58, 8.73, 8.88, 7.42, and 34.61 for HECTD3, PSMB10, UBD, UBE2C, UBE2S, CCL2, CCR1, CXCL10, CXCL11, and IL2RG, respectively). Moreover, 53 common genes were confirmed by the validation dataset, including downregulated UBE2C and UBE2S. Our results suggested that these 10 genes belonging to these two pathways might be useful as sensitive biomarkers for the efficacy evaluation of neoadjuvant chemotherapy in breast cancer.

Creatinine reabsorption by the aged kidney.

PubMed

Musso, Carlos G; Michelángelo, Hernán; Vilas, Manuel; Reynaldi, Juliana; Martinez, Bernardo; Algranati, Luis; Macías Núñez, Juan F

2009-01-01

The handling of renal creatinine in human beings has classically been described as the result of two particular physiological processes: glomerular filtration and proximal tubular secretion. However, there are particular physiological situations in which tubular creatinine reabsorption has been documented, such as in the case of healthy newborns and premature babies. We performed a prospective study in order to evaluate if there is tubular creatinine reabsorption in healthy elderly people. We studied prospectively nine healthy volunteers, four of them young (20-33 years old) and the remaining five, old (65-73 years old). Since creatinine is secreted in the proximal tubules, and its secretion can be completely blocked by cimetidine administration, a creatinine clearance with cimetidine reliably represents the glomerular filtration rate. Therefore, if the ratio creatinine clearance (Ccr)/creatinine clearance with cimetidine (CcrWC) is higher than one, this would indicate net creatinine secretion, whereas a ratio lower than one would indicate a net renal creatinine tubular reabsorption; a ratio equal to one indicates creatinine filtration. Finally, the Ccr, CcrWC, and Ccr/CcrWC ratios were compared between the young and old group. Mann-Whitney and Wilcoxon tests were used. As expected, creatinine clearance in the elderly was significantly lower than in the young [Ccr: 74.4 ml/min (47.9-100.9) (old) vs. 153.8 ml/min (108.3-199.2) (young), p = 0.014]. Similarly, the creatinine clearance with cimetidine (CcrWC) was significantly lower in the elderly compared to the young [CcrWC: 81.8 ml/min (69.2-94.5) (old) vs. 122.5 ml/min (82.6-162.4) (young), p = 0.028]. The ratio of Ccr/CcrWC was 0.9 in the elderly vs. 1.26 in the young (p = 0.014), indicating net creatinine reabsorption in the elderly and net creatinine secretion in the young. Our findings indicate that there seems to be a net reabsorption of creatinine in the renal tubules of healthy old persons.

Induction of Simian AIDS in Infant Rhesus Macaques Infected with CCR5- or CXCR4-Utilizing Simian-Human Immunodeficiency Viruses Is Associated with Distinct Lesions of the Thymus

PubMed Central

Reyes, R. A.; Canfield, Don R.; Esser, Ursula; Adamson, Lourdes A.; Brown, Charles R.; Cheng-Mayer, Cecilia; Gardner, Murray B.; Harouse, Janet M.; Luciw, Paul A.

2004-01-01

Newborn rhesus macaques were infected with two chimeric simian-human immunodeficiency virus (SHIV) strains which contain unique human immunodeficiency virus type 1 (HIV-1) env genes and exhibit distinct phenotypes. Infection with either the CCR5-specific SHIVSF162P3 or the CXCR4-utilizing SHIVSF33A resulted in clinical manifestations consistent with simian AIDS. Most prominent in this study was the detection of severe thymic involution in all SHIVSF33A-infected infants, which is very similar to HIV-1-induced thymic dysfunction in children who exhibit a rapid pattern of disease progression. In contrast, SHIVSF162P3 induced only a minor disruption in thymic morphology. Consistent with the distribution of the coreceptors CXCR4 and CCR5 within the thymus, the expression of SHIVSF162P3 was restricted to the thymic medulla, whereas SHIVSF33A was preferentially detected in the cortex. This dichotomy of tissue tropism is similar to the differential tropism of HIV-1 isolates observed in the reconstituted human thymus in SCID-hu mice. Accordingly, our results show that the SHIV-monkey model can be used for the molecular dissection of cell and tissue tropisms controlled by the HIV-1 env gene and for the analysis of mechanisms of viral immunopathogenesis in AIDS. Furthermore, these findings could help explain the rapid progression of disease observed in some HIV-1-infected children. PMID:14747577

Physiological, physical and behavioural changes in dogs (Canis familiaris) when kennelled: testing the validity of stress parameters.

PubMed

Part, C E; Kiddie, J L; Hayes, W A; Mills, D S; Neville, R F; Morton, D B; Collins, L M

2014-06-22

Domestic dogs (Canis familiaris) housed in kennelling establishments are considered at risk of suffering poor welfare. Previous research supporting this hypothesis has typically used cortisol:creatinine ratios (C/Cr) to measure acute and chronic stress in kennelled dogs. However, the value of C/Cr as a welfare indicator has been questioned. This study aimed to test the validity of a range of physiological, physical and behavioural welfare indicators and to establish baseline values reflecting good dog welfare. Measurements were taken from 29 privately-owned dogs (14 males, 15 females), ranging in age and breed, in their own home and in a boarding kennel environment, following a within-subjects, counterbalanced design. Pairwise comparisons revealed that C/Cr and vanillylmandelic acid:creatinine ratios (VMA/Cr) were higher in the kennel than home environment (P=0.003; P=0.01, respectively) and were not associated with differences in movement/exercise between environments. Dogs' surface temperature was lower in kennels (P=0.001) and was not associated with ambient temperature. No association with age, or effects of kennel establishment, kennelling experience, sex or source were found. Dogs were generally more active in kennels, but showed considerable individual variability. C/Cr and 5-HIAA:creatinine ratios (5-HIAA/Cr) were negatively correlated with lip licking in kennels. Baseline values for each parameter are presented. The emotional valence of responses was ambiguous and no definitive evidence was found to suggest that dogs were negatively stressed by kennelling. It was concluded that C/Cr and, particularly, VMA/Cr and surface temperature provide robust indicators of psychological arousal in dogs, while spontaneous behaviour might be better used to facilitate interpretation of physiological and physical data on an individual level. Copyright © 2014 Elsevier Inc. All rights reserved.

The skin, a novel niche for recirculating B cells1

PubMed Central

Geherin, Skye A.; Fintushel, Sarah R.; Lee, Michael H.; Wilson, R. Paul; Patel, Reema T.; Alt, Carsten; Young, Alan J.; Hay, John B.; Debes, Gudrun F.

2012-01-01

B cells infiltrate the skin in many chronic inflammatory diseases caused by autoimmunity or infection. Despite potential contribution to disease, skin-associated B cells remain poorly characterized. Using an ovine model of granulomatous skin inflammation, we demonstrate that B cells increase in the skin and skin-draining afferent lymph during inflammation. Surprisingly, skin B cells are a heterogeneous population that is distinct from lymph node B cells, with more large lymphocytes as well as B-1-like B cells that co-express high levels IgM and CD11b. Skin B cells have increased MHCII, CD1, and CD80/86 expression compared with lymph node B cells, suggesting that they are well-suited for T cell activation at the site of inflammation. Furthermore, we show that skin accumulation of B cells and antibody-secreting cells during inflammation increases local antibody titers, which could augment host defense and autoimmunity. While skin B cells express typical skin homing receptors such as E-selectin ligand and alpha-4 and beta-1 integrins, they are unresponsive to ligands for chemokine receptors associated with T cell homing into skin. Instead, skin B cells migrate toward the cutaneously expressed CCR6 ligand CCL20. Our data support a model in which B cells use CCR6-CCL20 to recirculate through the skin, fulfilling a novel role in skin immunity and inflammation. PMID:22561151

Host genetic determinants of HIV pathogenesis: an immunologic perspective.

PubMed

Hunt, Peter W; Carrington, Mary

2008-05-01

The purpose of this review is to highlight recent advances in our understanding of host genetic determinants of HIV pathogenesis and to provide a theoretical framework for interpreting these studies in the context of our evolving understanding of HIV immunopathogenesis. The first genome-wide association analysis of host determinants of HIV pathogenesis and other recent studies evaluating the interaction between killer cell immunoglobulin-like receptors and human leukocyte antigen alleles have implicated both adaptive and innate immune responses in the control of HIV replication. Furthermore, genetic variation associated with the expression of CCR5 and its ligand have been strongly associated with both decreased susceptibility to HIV infection and delayed clinical progression, independent of their effects on viral replication, suggesting a potential role for CCR5 inhibitors as immune-based therapies in HIV disease. Host factors associated with the control of HIV replication may help identify important targets for vaccine design, while those associated with delayed clinical progression provide targets for future immune-based therapies against HIV infection.

A highly reproducible quantitative viral outgrowth assay for the measurement of the replication-competent latent HIV-1 reservoir.

PubMed

Fun, Axel; Mok, Hoi Ping; Wills, Mark R; Lever, Andrew M

2017-02-24

Cure of Human Immunodeficiency Virus (HIV) infection remains elusive due to the persistence of HIV in a latent reservoir. Strategies to eradicate latent infection can only be evaluated with robust, sensitive and specific assays to quantitate reactivatable latent virus. We have taken the standard peripheral blood mononuclear cell (PBMC) based viral outgrowth methodology and from it created a logistically simpler and more highly reproducible assay to quantify replication-competent latent HIV in resting CD4 + T cells, both increasing accuracy and decreasing cost and labour. Purification of resting CD4 + T cells from whole PBMC is expedited and achieved in 3 hours, less than half the time of conventional protocols. Our indicator cell line, SupT1-CCR5 cells (a clonal cell line expressing CD4, CXCR4 and CCR5) provides a readily available standardised readout. Reproducibility compares favourably to other published assays but with reduced cost, labour and assay heterogeneity without compromising sensitivity.

[Companion diagnostics in the era of personalized medicine--chairmen's introductory remarks].

PubMed

Fukutsuka, Katsuhiro; Takubo, Takayuki

2014-04-01

Personalized medicine is a medical model that proposes the customization of treatment for individual patients. In this model, diagnostic tests are essential for selecting safer and more efficacious treatments. The term "companion diagnostics" has been used to describe these tests, whereby molecular assays that measure the levels of proteins or specific gene mutations are used to provide a specific therapy for an individual by stratifying the disease status, selecting the proper medication, and tailoring dosages. Examples of companion diagnostics in the field of cancer medicine for molecular targeted therapy include tests for the ALK-fusion gene in non-small cell lung cancer and expression of CCR4 in adult T-cell leukemia. For breast cancer, the expression of HER2 protein is evaluated by immunohistochemistry (IHC), and gene amplification of HER2 is tested by fluorescence in situ hybridization (FISH); both tests consist of pre-analysis, analysis, and post-analysis processes that require quality control to ensure the reliability of the results. This symposium includes: 1) future aspects of companion diagnostics addressing many of the problems that must be overcome, 2) companion diagnostics using FISH focusing on HER2 amplification and ALK alteration, 3) newly developed diagnostic tests using tumor specimens and cell-free DNA in serum, and 4) CCR4 expression detected by IHC and flow cytometry.

Association of Canine Osteosarcoma and Monocyte Phenotype and Chemotactic Function.

PubMed

Tuohy, J L; Lascelles, B D X; Griffith, E H; Fogle, J E

2016-07-01

Monocytes/macrophages are likely key cells in immune modulation in dogs with osteosarcoma (OSA). Increased peripheral monocyte counts are negatively correlated with shorter disease-free intervals in dogs with OSA. Understanding the monocyte/macrophage's modulatory role in dogs with OSA can direct further studies in immunotherapy development for OSA. That OSA evades the immune response by down-regulating monocyte chemokine receptor expression and migratory function, and suppresses host immune responses. Eighteen dogs with OSA that have not received definitive treatment and 14 healthy age-matched controls Clinical study-expression of peripheral blood monocyte cell surface receptors, monocyte mRNA expression and cytokine secretion, monocyte chemotaxis, and survival were compared between clinical dogs with OSA and healthy control dogs. Cell surface expression of multiple chemokine receptors is significantly down-regulated in peripheral blood monocytes of dogs with OSA. The percentage expression of CCR2 (median 58%, range 2-94%) and CXCR2 expression (median 54%, range 2-92%) was higher in control dogs compared to dogs with OSA (CCR2 median 29%, range 3-45%, P = 0.0006; CXCR2 median 23%, range 0.2-52%, P = 0.0007). Prostaglandin E2 (PGE2 ) (OSA, median 347.36 pg/mL, range 103.4-1268.5; control, 136.23 pg/mL, range 69.93-542.6, P = .04) and tumor necrosis factor-alpha (TNF-α) (P = .02) levels are increased in OSA monocyte culture supernatants compared to controls. Peripheral blood monocytes of dogs with OSA exhibit decreased chemotactic function when compared to control dogs (OSA, median 1.2 directed to random migration, range 0.8-1.25; control, 1.6, range of 0.9-1.8, P = .018). Dogs with OSA have decreased monocyte chemokine receptor expression and monocyte chemotaxis, potential mechanisms by which OSA might evade the immune response. Reversal of monocyte dysfunction using immunotherapy could improve survival in dogs with OSA. Copyright © 2016 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.

African Ancestry Influences CCR5 –2459G>A Genotype-Associated Virologic Success of Highly Active Antiretroviral Therapy

PubMed Central

Cheruvu, Vinay K.; Igo, Robert P.; Jurevic, Richard J.; Serre, David; Zimmerman, Peter A.; Rodriguez, Benigno; Mehlotra, Rajeev K.

2014-01-01

Introduction In a North American, HIV-positive, highly active antiretroviral therapy (HAART)-treated, adherent cohort of self-identified white and black patients, we previously observed that chemokine (C-C motif) receptor 5 (CCR5) –2459G>A genotype had a strong association with time to achieve virologic success (TVLS) in black but not in white patients. Methods Using 128 genome-wide ancestry informative markers, we performed a quantitative assessment of ancestry in these patients (n = 310) to determine (1) whether CCR5 –2459G>A genotype is still associated with TVLS of HAART when ancestry, not self-identified race, is considered and (2) whether this association is influenced by varying African ancestry. Results We found that the interaction between CCR5 –2459G>A genotype and African ancestry (≤0.125 vs. ≥0.425 and <0.71 vs. ≥0.71) was significantly associated with TVLS (GG compared with AA, P = 0.044 and 0.018, respectively). Furthermore, the association between CCR5 –2459G>A genotype and TVLS was stronger in patients with African ancestry ≥0.71 than in patients with African ancestry ≥0.452, in both Kaplan-Meier (log-rank P = 0.039 and 0.057, respectively, for AA, GA, and GG) and Cox proportional hazards regression (relative hazard for GG compared with AA 2.59 [95% CI, 1.27–5.22; P = 0.01] and 2.26 [95% CI, 1.18–4.32; P = 0.01], respectively) analyses. Conclusions We observed that the association between CCR5 –2459G>A genotype and TVLS of HAART increased with stronger African ancestry. Understanding the genomic mechanisms by which African ancestry influences this association is critical, and requires further studies. PMID:24714069

Correlations between polymorphisms in the uridine diphosphate-glucuronosyltransferase 1A and C-C motif chemokine receptor 5 genes and infection with the hepatitis B virus in three ethnic groups in China.

PubMed

Zhang, Chan; He, Yan; Shan, Ke-Ren; Tan, Kui; Zhang, Ting; Wang, Chan-Juan; Guan, Zhi-Zhong

2018-02-01

Objective To determine whether genetic polymorphisms in the uridine diphosphate-glucuronosyltransferase 1A ( UGT1A) and the C-C motif chemokine receptor 5 ( CCR5) genes are associated with hepatitis B virus (HBV) infection in Yi, Yao and Han ethnic groups in the Guizhou Province of China. Methods The study enrolled subjects with and without HBV infection. Whole blood was used for DNA genotyping using standard techniques. The study determined the frequencies of several polymorphic alleles ( UGT1A6 [rs2070959], UGT1A1 [rs8175347], CCR5-59029 [rs1799987] and CCR5Δ32 [rs333]) and then characterized their relationship with HBV infection. Results A total of 404 subjects were enrolled in the study: 138 from the Yao group, 101 from the Yi group and 165 from the Han group. There was a significant difference in the frequency of UGT1A1 rs8175347 polymorphisms among the three groups. The rates of 7TA carriers of UGT1A1 rs8175347 in all three groups were significantly higher than the other genotypes. Individuals with genotype AA of UGT1A6 rs2070959 in the Yi group had a higher risk for HBV infection than in the Yao and Han groups. The frequency of genotype GG in CCR5-59029 in the Yao group was significantly higher than in the Yi group. The genotypes of CCR5Δ32 were not associated with HBV infection. Conclusion These findings provide genetic and epidemiological evidence for an association of UGT1A and CCR5-59029 polymorphisms with HBV infection in Chinese Yi and Yao populations.

Inflammatory Monocytes Mediate Early and Organ-Specific Innate Defense During Systemic Candidiasis

PubMed Central

Ngo, Lisa Y.; Kasahara, Shinji; Kumasaka, Debra K.; Knoblaugh, Sue E.; Jhingran, Anupam; Hohl, Tobias M.

2014-01-01

Candida albicans is a commensal fungus that can cause systemic disease in patients with breaches in mucosal integrity, indwelling catheters, and defects in phagocyte function. Although circulating human and murine monocytes bind C. albicans and promote inflammation, it remains unclear whether C-C chemokine receptor 2 (CCR2)– and Ly6C-expressing inflammatory monocytes exert a protective or a deleterious function during systemic infection. During murine systemic candidiasis, interruption of CCR2-dependent inflammatory monocyte trafficking into infected kidneys impaired fungal clearance and decreased murine survival. Depletion of CCR2-expressing cells led to uncontrolled fungal growth in the kidneys and brain and demonstrated an essential antifungal role for inflammatory monocytes and their tissue-resident derivatives in the first 48 hours postinfection. Adoptive transfer of purified inflammatory monocytes in depleted hosts reversed the defect in fungal clearance to a substantial extent, indicating a compartmentally and temporally restricted protective function that can be transferred to enhance systemic innate antifungal immunity. PMID:23922372

Concurrent CCR7 Overexpression and RelB Knockdown in Immature Dendritic Cells Induces Immune Tolerance and Improves Skin-Graft Survival in a Murine Model.

PubMed

Dong, Zhiwei; Chen, Yajie; Peng, Yuan; Wang, Fan; Yang, Zichen; Huang, Guangtao; Chen, Yu; Yuan, Zhiqiang; Cao, Tongtong; Peng, Yizhi

2017-01-01

Skin transplantation aims to cover skin defects but often fails due to immune rejection of the transplantated tissue. Immature dendritic cells (imDCs) induce immune tolerance but have a low migration rate. After stimulation, imDCs transform into mature DCs, which activate immune rejection. Thus, inducing imDC to obtain a high migration counteracts development of immune tolerance. We transfected imDCs with a recombinant adenovirus carrying the CCR7 gene (Ad-CCR7) and a small interfering RNA targeting RelB (RelB-siRNA) to concurrently overexpress CCR7 and downregulate RelB expression. Functionally, such cells showed a significantly enhanced migration rate in the chemotactic assay and decreased T-cell proliferation after lipopolysaccharide stimulation in mixed lymphocyte reactions. Cotransfected cells showed an increased ability to induce immune tolerance by upregulating T regulatory (Treg) cells and shifting the Th1/Th2 ratio. Cotransfection of Ad-CCR7 and RelB-siRNA endowed imDCs with resistance to apoptosis and cell death. CCR7 overexpression and RelB knockdown (KD) in imDCs improve skin-graft survival in a murine skin-transplantation model. Transfection with Ad-CCR7 and RelB KD in imDCs may be an effective approach inducing immune tolerance, thus being potentially valuable for inhibiting allograft rejection. © 2017 The Author(s). Published by S. Karger AG, Basel.

CCL3L1 copy number, CCR5 genotype and susceptibility to tuberculosis

PubMed Central

2014-01-01

Background Tuberculosis is a major infectious disease and functional studies have provided evidence that both the chemokine MIP-1α and its receptor CCR5 play a role in susceptibility to TB. Thus by measuring copy number variation of CCL3L1, one of the genes that encode MIP-1α, and genotyping a functional promoter polymorphism -2459A > G in CCR5 (rs1799987) we investigate the influence of MIP-1α and CCR5, independently and combined, in susceptibility to clinically active TB in three populations, a Peruvian population (n = 1132), a !Xhosa population (n = 605) and a South African Coloured population (n = 221). The three populations include patients with clinically diagnosed pulmonary TB, as well as other, less prevalent forms of extrapulmonary TB. Methods and results Copy number of CCL3L1 was measured using the paralogue ratio test and exhibited ranges between 0–6 copies per diploid genome (pdg) in Peru, between 0–12 pdg in !Xhosa samples and between 0–10 pdg in South African Coloured samples. The CCR5 promoter polymorphism was observed to differ significantly in allele frequency between populations (*A; Peru f = 0.67, !Xhosa f = 0.38, Coloured f = 0.48). Conclusions The case–control association studies performed however find, surprisingly, no evidence for an influence of variation in genes coding for MIP-1α or CCR5 individually or together in susceptibility to clinically active TB in these populations. PMID:24405814

Anti-MAdCAM Antibody Increases ß7+ T Cells and CCR9 Gene Expression in the Peripheral Blood of Patients With Crohn’s Disease

PubMed Central

Hassan-Zahraee, Mina; Banerjee, Anindita; Cheng, John B; Zhang, Weidong; Ahmad, Alaa; Page, Karen; von Schack, David; Zhang, Baohong; Martin, Steven W; Nayak, Satyaprakash; Reddy, Padma; Xi, Li; Neubert, Hendrik; Fernandez Ocana, Mireia; Gorelick, Ken; Clare, Robert; Vincent, Michael; Cataldi, Fabio; Hung, Kenneth

2018-01-01

Abstract Objective To define pharmacodynamic biomarkers in the peripheral blood of patients with Crohn’s disease [CD] after treatment with PF-00547659, an anti-human mucosal addressin cell adhesion molecule-1 [MAdCAM-1] monoclonal antibody. Methods In this Phase 2, randomised, double-blind, controlled study [OPERA], blood samples were analysed from patients with moderate to severe active CD who received placebo or 22.5 mg, 75 mg, or 225 mg of PF-00547659 subcutaneously at baseline and at Weeks 4 and 8, with follow-up at Week 12. Soluble MAdCAM [sMAdCAM] was measured by mass spectrometry, β7-expressing T cells by flow cytometry, and gene transcriptome by RNA sequencing. Results A slight increase in sMAdCAM was measured in the placebo group from baseline to Week 12 [6%], compared with significant decreases in all PF-00547659 groups [–87% to –98%]. A slight increase from baseline to Week 12 was observed in frequency and molecules of equivalent soluble fluorochrome for β7+ central memory T cells in the placebo group [4%], versus statistically significant increases in the active treatment groups [48% to 81%]. Similar trends were seen for β7+ effector memory T cells [placebo, 8%; PF-00547659, 84–138%] and β7+ naïve T cells [8%; 13–50%]. CCR9 gene expression had statistically significant up-regulation [p = 1.09e-06; false discovery rate < 0.1] with PF-00547659 treatment, and was associated with an increase in β7+ T cells. Conclusions Results of the OPERA study demonstrate positive pharmacology and dose-dependent changes in pharmacodynamic biomarker measurements in blood, including changes in cellular composition of lymphocytes and corresponding CCR9 gene expression changes. PMID:28961803

Anti-MAdCAM Antibody Increases ß7+ T Cells and CCR9 Gene Expression in the Peripheral Blood of Patients With Crohn's Disease.

PubMed

Hassan-Zahraee, Mina; Banerjee, Anindita; Cheng, John B; Zhang, Weidong; Ahmad, Alaa; Page, Karen; von Schack, David; Zhang, Baohong; Martin, Steven W; Nayak, Satyaprakash; Reddy, Padma; Xi, Li; Neubert, Hendrik; Fernandez Ocana, Mireia; Gorelick, Ken; Clare, Robert; Vincent, Michael; Cataldi, Fabio; Hung, Kenneth

2018-01-05

To define pharmacodynamic biomarkers in the peripheral blood of patients with Crohn's disease [CD] after treatment with PF-00547659, an anti-human mucosal addressin cell adhesion molecule-1 [MAdCAM-1] monoclonal antibody. In this Phase 2, randomised, double-blind, controlled study [OPERA], blood samples were analysed from patients with moderate to severe active CD who received placebo or 22.5 mg, 75 mg, or 225 mg of PF-00547659 subcutaneously at baseline and at Weeks 4 and 8, with follow-up at Week 12. Soluble MAdCAM [sMAdCAM] was measured by mass spectrometry, β7-expressing T cells by flow cytometry, and gene transcriptome by RNA sequencing. A slight increase in sMAdCAM was measured in the placebo group from baseline to Week 12 [6%], compared with significant decreases in all PF-00547659 groups [-87% to -98%]. A slight increase from baseline to Week 12 was observed in frequency and molecules of equivalent soluble fluorochrome for β7+ central memory T cells in the placebo group [4%], versus statistically significant increases in the active treatment groups [48% to 81%]. Similar trends were seen for β7+ effector memory T cells [placebo, 8%; PF-00547659, 84-138%] and β7+ naïve T cells [8%; 13-50%]. CCR9 gene expression had statistically significant up-regulation [p = 1.09e-06; false discovery rate < 0.1] with PF-00547659 treatment, and was associated with an increase in β7+ T cells. Results of the OPERA study demonstrate positive pharmacology and dose-dependent changes in pharmacodynamic biomarker measurements in blood, including changes in cellular composition of lymphocytes and corresponding CCR9 gene expression changes. Copyright © 2017 European Crohn’s and Colitis Organisation (ECCO). Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com

Prenatal exposure to ethanol stimulates hypothalamic CCR2 chemokine receptor system: Possible relation to increased density of orexigenic peptide neurons and ethanol drinking in adolescent offspring

PubMed Central

Chang, G.-Q.; Karatayev, O.; Leibowitz, S. F.

2015-01-01

Clinical and animal studies indicate that maternal consumption of ethanol during pregnancy increases alcohol drinking in the offspring. Possible underlying mechanisms may involve orexigenic peptides, which are stimulated by prenatal ethanol exposure and themselves promote drinking. Building on evidence that ethanol stimulates neuroimmune factors such as the chemokine CCL2 that in adult rats is shown to colocalize with the orexigenic peptide, melanin-concentrating hormone (MCH) in the lateral hypothalamus (LH), the present study sought to investigate the possibility that CCL2 or its receptor CCR2 in LH are stimulated by prenatal ethanol exposure, perhaps specifically within MCH neurons. Our paradigm of intraoral administration of ethanol to pregnant rats, at low-to-moderate doses (1 or 3 g/kg/day) during peak hypothalamic neurogenesis, caused in adolescent male offspring two-fold increase in drinking of and preference for ethanol and reinstatement of ethanol drinking in a two-bottle choice paradigm under an intermittent access schedule. This effect of prenatal ethanol exposure was associated with an increased expression of MCH and density of MCH+ neurons in LH of preadolescent offspring. Whereas CCL2+ cells at this age were low in density and unaffected by ethanol, CCR2+ cells were dense in LH and increased by prenatal ethanol, with a large percentage (83–87%) identified as neurons and found to colocalize MCH. Prenatal ethanol also stimulated the genesis of CCR2+ and MCH+ neurons in the embryo, which co-labeled the proliferation marker, BrdU. Ethanol also increased the genesis and density of neurons that co-expressed CCR2 and MCH in LH, with triple-labeled CCR2+/MCH+/BrdU+ neurons that were absent in control rats accounting for 35% of newly generated neurons in ethanol-exposed rats. With both the chemokine and MCH systems believed to promote ethanol consumption, this greater density of CCR2+/MCH+ neurons in the LH of preadolescent rats suggests that these systems function together in promoting alcohol drinking during adolescence. PMID:26365610

Saponins extracted from Dioscorea collettii rhizomes regulate the expression of urate transporters in chronic hyperuricemia rats.

PubMed

Zhu, Liran; Dong, Yifan; Na, Sha; Han, Ru; Wei, Chengyin; Chen, Guangliang

2017-09-01

The current study aimed to investigate whether the saponins, bioactive component of effects of D. collettii, could reduce the serum uric acid level in a hyperuricemic mouse via regulation of urate transporters. Chronic hyperuricemia model was established by combine administration of adenine (100mg/kg) and ethambutol (250mg/kg). In the model group, the serum uric acid (SUA), urine uric acid (UUA) volume, and 24-h UUA values increased significantly, while the uric acid clearance rate (CUr) and creatinine clearance rate (CCr) values decreased. Further, the model groups showed significantly lower expression of organic anion transporter 1 (OAT1) and organic anion transporter 3 (OAT3) and significantly higher expression of renal tubular urate transporter 1 (URAT1), glucose transporter 9 (GLUT9) and URAT1 mRNA than the normal control group. Saponins administration was found to have a dose-dependent effect, as evidenced by the increase in the 24-h UUA, CUr and CCr values; the decrease in SUA; the decrease in the renal expression of URAT1 mRNA and URAT1 and GLUT9 proteins; and the increase in the renal expression of the OAT1 and OAT3 proteins. The saponins extracted from D. collettii rhizomes had an obvious anti-hyperuricemic effect through downregulation of the URAT1 mRNA and the URAT1 and GLUT9 proteins and upregulation of the OAT1 and OAT3 proteins. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

CD4+ Memory Stem Cells Are Infected by HIV-1 in a Manner Regulated in Part by SAMHD1 Expression

PubMed Central

Tabler, Caroline O.; Lucera, Mark B.; Haqqani, Aiman A.; McDonald, David J.; Migueles, Stephen A.; Connors, Mark

2014-01-01

ABSTRACT CD4+ and CD8+ memory T cells with stem cell-like properties (TSCM cells) have been identified in mice, humans, and nonhuman primates and are being investigated for antitumor and antiviral vaccines and immunotherapies. Whether CD4+ TSCM cells are infected by human immunodeficiency virus (HIV) was investigated by using a combination HIV reporter virus system in vitro and by direct staining for HIV p24 antigen ex vivo. A proportion of TSCM cells were found to express the HIV coreceptors CCR5 and CXCR4 and were infected by HIV both in vitro and in vivo. Analysis of viral outcome following fusion using the combination reporter virus system revealed that TSCM cells can become productively or latently infected, although the vast majority of TSCM cells are abortively infected. Knockdown of the HIV restriction factor SAMHD1 using Vpx-containing simian immunodeficiency virus (SIV) virion-like particles enhanced the productive infection of TSCM cells, indicating that SAMHD1 contributes to abortive infection in these cells. These results demonstrate that CD4+ TSCM cells are targets for HIV infection, that they become productively or latently infected at low levels, and that SAMHD1 expression promotes abortive infection of this important memory cell subset. IMPORTANCE Here we demonstrate the susceptibility of CD4+ memory stem cells (TSCM cells) to infection by HIV in vitro and in vivo, provide an in-depth analysis of coreceptor expression, demonstrate the infection of naïve and memory CD4+ T cell subsets with both CCR5- and CXCR4-tropic HIV, and also perform outcome analysis to calculate the percentage of cells that are productively, latently, or abortively infected. Through these outcome studies, we determined that the vast majority of TSCM cells are abortively infected by HIV, and we demonstrate that knockdown of SAMHD1 significantly increases the frequency of infection of this CD4+ T cell subset, indicating that SAMHD1 is an active restriction factor in TSCM cells. PMID:24554663

Measurement of glomerular filtration rate, renal plasma flow, and endogenous creatinine clearance in cheetahs (Acinonyx jubatus jubatus).

PubMed

Holder, Erin Hall; Citino, Scott B; Businga, Nancy; Cartier, Leslie; Brown, Scott A

2004-06-01

Glomerular filtration rate (GFR), renal plasma flow (RPF), and the endogenous creatinine clearance (CCr) rate were determined in 13 captive cheetahs, Acinonyx jubatus jubatus (seven females and six males, 1.5-7.5 yr of age, x = 5.02 yr), during general anesthesia with Telazol and isoflurane by measuring the urinary clearances of inulin, para-aminohipppuric acid, and endogenous creatinine, respectively. Methods to determine GFR, RPF, and endogenous CCr in captive cheetahs were evaluated, and the relationship between GFR and CCr for this species was determined. The GFR and the RPF were stable during the procedure, with mean values of 1.59+/-0.17 ml/min/kg body weight and 5.12+/-1.15 ml/min/kg body weight, respectively. Although the mean value for CCr (1.47+/-0.20 ml/min/kg body weight) was significantly less than the corresponding value for GFR, the mean difference (0.11+/-0.02 ml/min/kg weight) between the two measurements was slight, and the values were highly correlated (R2 = 0.928; P < 0.0001). The measurement of CCr in cheetahs should provide a reliable estimate of GFR, facilitating the early detection of renal disease in this species.

A Distinct Inhibitory Function for miR-18a in Th17 Cell Differentiation.

PubMed

Montoya, Misty M; Maul, Julia; Singh, Priti B; Pua, Heather H; Dahlström, Frank; Wu, Nanyan; Huang, Xiaozhu; Ansel, K Mark; Baumjohann, Dirk

2017-07-15

Th17 cell responses orchestrate immunity against extracellular pathogens but also underlie autoimmune disease pathogenesis. In this study, we uncovered a distinct and critical role for miR-18a in limiting Th17 cell differentiation. miR-18a was the most dynamically upregulated microRNA of the miR-17-92 cluster in activated T cells. miR-18a deficiency enhanced CCR6 + RAR-related orphan receptor (ROR)γt + Th17 cell differentiation in vitro and increased the number of tissue Th17 cells expressing CCR6, RORγt, and IL-17A in airway inflammation models in vivo. Sequence-specific miR-18 inhibitors increased CCR6 and RORγt expression in mouse and human CD4 + T cells, revealing functional conservation. miR-18a directly targeted Smad4 , Hif1a , and Rora , all key transcription factors in the Th17 cell gene-expression program. These findings indicate that activating signals influence the outcome of Th cell differentiation via differential regulation of mature microRNAs within a common cluster. Copyright © 2017 by The American Association of Immunologists, Inc.

Prenatally diagnosed de novo complex chromosome rearrangements: Two new cases and review of the literature

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ruiz, C.; Grubs, R.E.; Jewett, T.

1994-09-01

Complex chromosome rearrangements (CCR) are rare structural rearrangements involving at least three chromosomes with three or more breakpoints. Although there have been numerous reports of individuals with CCR, most have been ascertained through the presence of multiple congenital anomalies, recurrent pregnancy loss, or infertility. Few cases have been ascertained prenatally. We present two new cases of prenatally ascertained CCR. In the first case, an amniocentesis revealed an apparently balanced de novo rearrangement in which chromosomes 5, 6 and 11 were involved in a three-way translocation: 46,XY,t(6;5)(5;11)(q23;p14.3;q15;p13). The pregnancy was unevenful. Recently, at the age of 9 months, a physical andmore » developmental evaluation were normal but, height, weight, and head circumference were below the 5th percentile. In the second case an amniocentesis revealed an unbalanced de novo rearrangement involving separate translocations and an interstitial deletion: 46,XY,del(6)(q25.3q27),t(3;8)(p13;q21.3),t(6;18)(p11.2;q11.2). A meconium plug was present at birth and at 6 months of age surgery for Hirschsprung`s disease was required. Currently, at 10 months of age, the patient has hypotonia and developmental delay. The paucity of information regarding prenatally diagnosed CCR poses a problem in counseling families. Of the four prenatally diagnosed balanced de novo CCR cases, three had abnormal outcomes. In a review of the literature, approximately 70% of the postnatally ascertained balanced de novo CCR cases were associated with congenital anomalies, growth retardation and/or mental retardation. More information regarding the outcome of prenatally ascertained balanced de novo CCR is required for accurate risk assessment.« less

Dogs infected with the blood trypomastigote form of Trypanosoma cruzi display an increase expression of cytokines and chemokines plus an intense cardiac parasitism during acute infection.

PubMed

de Souza, Sheler Martins; Vieira, Paula Melo de Abreu; Roatt, Bruno Mendes; Reis, Levi Eduardo Soares; da Silva Fonseca, Kátia; Nogueira, Nívia Carolina; Reis, Alexandre Barbosa; Tafuri, Washington Luiz; Carneiro, Cláudia Martins

2014-03-01

The recent increase in immigration of people from areas endemic for Chagas disease (Trypanosoma cruzi) to the United States and Europe has raised concerns about the transmission via blood transfusion and organ transplants in these countries. Infection by these pathways occurs through blood trypomastigotes (BT), and these forms of T. cruzi are completely distinct of metacyclic trypomastigotes (MT), released by triatomine vector, in relation to parasite-host interaction. Thus, research comparing infection with these different infective forms is important for explaining the potential impacts on the disease course. Here, we investigated tissue parasitism and relative mRNA expression of cytokines, chemokines, and chemokine receptors in the heart during acute infection by MT or BT forms in dogs. BT-infected dogs presented a higher cardiac parasitism, increased relative mRNA expression of pro-inflammatory and immunomodulatory cytokines and of the chemokines CCL3/MIP-1α, CCL5/RANTES, and the chemokine receptor CCR5 during the acute phase of infection, as compared to MT-infected dogs. These results suggest that infection with BT forms may lead to an increased immune response, as revealed by the cytokines ratio, but this kind of immune response was not able to control the cardiac parasitism. Infection with the MT form presented an increase in the relative mRNA expression of IL-12p40 as compared to that of IL-10 or TGF-β1. Correlation analysis showed increased relative mRNA expression of IFN-γ as well as IL-10, which may be an immunomodulatory response, as well as an increase in the correlation of CCL5/RANTES and its CCR5 receptor. Our findings revealed a difference between inoculum sources of T. cruzi, as vectorial or transfusional routes of T. cruzi infection may trigger distinct parasite-host interactions during the acute phase, which may influence immunopathological aspects of Chagas disease. Copyright © 2013 Elsevier Ltd. All rights reserved.

CCR5 adopts three homodimeric conformations that control cell surface delivery.

PubMed

Jin, Jun; Momboisse, Fanny; Boncompain, Gaelle; Koensgen, Florian; Zhou, Zhicheng; Cordeiro, Nelia; Arenzana-Seisdedos, Fernando; Perez, Franck; Lagane, Bernard; Kellenberger, Esther; Brelot, Anne

2018-05-08

Biophysical methods and x-ray crystallography have revealed that class A G protein-coupled receptors (GPCRs) can form homodimers. We combined computational approaches with receptor cross-linking, energy transfer, and a newly developed functional export assay to characterize the residues involved in the dimerization interfaces of the chemokine receptor CCR5, the major co-receptor for HIV-1 entry into cells. We provide evidence of three distinct CCR5 dimeric organizations, involving residues of transmembrane helix 5. Two dimeric states corresponded to unliganded receptors, whereas the binding of the inverse agonist maraviroc stabilized a third state. We found that CCR5 dimerization was required for targeting the receptor to the plasma membrane. These data suggest that dimerization contributes to the conformational diversity of inactive class A GPCRs and may provide new opportunities to investigate the cellular entry of HIV-1 and mechanisms for its inhibition. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Maraviroc: the evidence for its potential in the management of HIV

PubMed Central

Profit, Louise

2007-01-01

Introduction: New antiretroviral agents that are more convenient, better tolerated with fewer short- and long-term side effects, and that have novel resistance patterns are needed at all lines of therapy in patients infected with human immunodeficiency virus (HIV). Therefore, next generation products of current classes and alternative classes of antiretroviral agents are needed. The CC-chemokine receptor 5 (CCR5) antagonists are a novel class of antiretroviral agents that prevent the entry of HIV into host cells by blocking the CCR5 coreceptor. Within this class, maraviroc is the agent furthest along in development. Aims: The aim of this review is to evaluate the emerging evidence for the use of the CCR5 antagonist maraviroc in antiretroviral treatment-naïve and treatment-experienced patients with HIV-1 infection. Evidence review: Preliminary evidence from phase I/IIa short-term studies suggest that maraviroc monotherapy is effective at reducing HIV viral load, and is generally well tolerated. In-vitro evidence suggests that maraviroc will be effective in drug-naïve patients with CCR5-tropic virus, as well as in those with CCR5-tropic virus who have developed HIV resistance to existing antiretroviral regimens. However, it is not known how quickly resistance may develop to maraviroc in clinical practice. Clinical potential: Current evidence supports the continued development of maraviroc as a potentially useful, alternative treatment for the management of HIV infection. Maraviroc monotherapy has a high potency and long half-life, allowing single-pill dosing. Therefore, it is expected that maraviroc will have a beneficial effect on patient adherence and viral load in combination with other antiretroviral agents. Maraviroc is only effective against CCR5-tropic virus, which predominates throughout infection but is more common in patients at the early asymptomatic stage of infection. PMID:21221194