Association of TLR variants with susceptibility to Plasmodium vivax malaria and parasitemia in the Amazon region of Brazil

PubMed Central

Ramasawmy, Rajendranath; Ibiapina, Hiochelson Najibe Santos; Sampaio, Vanderson Souza; Xábregas, Lilyane Amorim; Brasil, Larissa Wanderley; Tarragô, Andréa Monteiro; Almeida, Anne Cristine Gomes; Kuehn, Andrea; Vitor-Silva, Sheila; Melo, Gisely Cardoso; Siqueira, André Machado; Monteiro, Wuelton Marcelo

2017-01-01

Background Plasmodium vivax malaria (Pv-malaria) is still considered a neglected disease despite an alarming number of individuals being infected annually. Malaria pathogenesis occurs with the onset of the vector-parasite-host interaction through the binding of pathogen-associated molecular patterns (PAMPs) and receptors of innate immunity, such as toll-like receptors (TLRs). The triggering of the signaling cascade produces an elevated inflammatory response. Genetic polymorphisms in TLRs are involved in susceptibility or resistance to infection, and the identification of genes involved with Pv-malaria response is important to elucidate the pathogenesis of the disease and may contribute to the formulation of control and elimination tools. Methodology/Principal findings A retrospective case-control study was conducted in an intense transmission area of Pv-malaria in the state of Amazonas, Brazil. Genetic polymorphisms (SNPs) in different TLRs, TIRAP, and CD14 were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis in 325 patients infected with P. vivax and 274 healthy individuals without malaria history in the prior 12 months from the same endemic area. Parasite load was determined by qPCR. Simple and multiple logistic/linear regressions were performed to investigate association between the polymorphisms and the occurrence of Pv-malaria and parasitemia. The C/T (TLR5 R392StopCodon) and T/T (TLR9 -1486C/T) genotypes appear to be risk factors for infection by P. vivax (TLR5: C/C vs. C/T [OR: 2.116, 95% CI: 1.054–4.452, p = 0.031]; TLR9: C/C vs. T/T [OR: 1.919, 95% CI: 1.159–3.177, p = 0.010]; respectively). Fever (COEF = 7599.46, 95% CI = 3063.80–12135.12, p = 0.001) and the C/C genotype of TLR9 -1237C/T (COEF = 17006.63, 95% CI = 3472.83–30540.44, p = 0.014) were independently associated with increased parasitemia in patients with Pv-malaria. Conclusions Variants of TLRs may predispose individuals to infection

Association of TLR variants with susceptibility to Plasmodium vivax malaria and parasitemia in the Amazon region of Brazil.

PubMed

Costa, Allyson Guimarães; Ramasawmy, Rajendranath; Ibiapina, Hiochelson Najibe Santos; Sampaio, Vanderson Souza; Xábregas, Lilyane Amorim; Brasil, Larissa Wanderley; Tarragô, Andréa Monteiro; Almeida, Anne Cristine Gomes; Kuehn, Andrea; Vitor-Silva, Sheila; Melo, Gisely Cardoso; Siqueira, André Machado; Monteiro, Wuelton Marcelo; Lacerda, Marcus Vinicius Guimarães; Malheiro, Adriana

2017-01-01

Plasmodium vivax malaria (Pv-malaria) is still considered a neglected disease despite an alarming number of individuals being infected annually. Malaria pathogenesis occurs with the onset of the vector-parasite-host interaction through the binding of pathogen-associated molecular patterns (PAMPs) and receptors of innate immunity, such as toll-like receptors (TLRs). The triggering of the signaling cascade produces an elevated inflammatory response. Genetic polymorphisms in TLRs are involved in susceptibility or resistance to infection, and the identification of genes involved with Pv-malaria response is important to elucidate the pathogenesis of the disease and may contribute to the formulation of control and elimination tools. A retrospective case-control study was conducted in an intense transmission area of Pv-malaria in the state of Amazonas, Brazil. Genetic polymorphisms (SNPs) in different TLRs, TIRAP, and CD14 were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis in 325 patients infected with P. vivax and 274 healthy individuals without malaria history in the prior 12 months from the same endemic area. Parasite load was determined by qPCR. Simple and multiple logistic/linear regressions were performed to investigate association between the polymorphisms and the occurrence of Pv-malaria and parasitemia. The C/T (TLR5 R392StopCodon) and T/T (TLR9 -1486C/T) genotypes appear to be risk factors for infection by P. vivax (TLR5: C/C vs. C/T [OR: 2.116, 95% CI: 1.054-4.452, p = 0.031]; TLR9: C/C vs. T/T [OR: 1.919, 95% CI: 1.159-3.177, p = 0.010]; respectively). Fever (COEF = 7599.46, 95% CI = 3063.80-12135.12, p = 0.001) and the C/C genotype of TLR9 -1237C/T (COEF = 17006.63, 95% CI = 3472.83-30540.44, p = 0.014) were independently associated with increased parasitemia in patients with Pv-malaria. Variants of TLRs may predispose individuals to infection by P. vivax. The TLR5 R392StopCodon and TLR9 -1486C/T variants

Structure-Activity Relationship in TLR4 Mutations: Atomistic Molecular Dynamics Simulations and Residue Interaction Network Analysis

NASA Astrophysics Data System (ADS)

Anwar, Muhammad Ayaz; Choi, Sangdun

2017-03-01

Toll-like receptor 4 (TLR4), a vital innate immune receptor present on cell surfaces, initiates a signaling cascade during danger and bacterial intrusion. TLR4 needs to form a stable hexamer complex, which is necessary to dimerize the cytoplasmic domain. However, D299G and T399I polymorphism may abrogate the stability of the complex, leading to compromised TLR4 signaling. Crystallography provides valuable insights into the structural aspects of the TLR4 ectodomain; however, the dynamic behavior of polymorphic TLR4 is still unclear. Here, we employed molecular dynamics simulations (MDS), as well as principal component and residue network analyses, to decipher the structural aspects and signaling propagation associated with mutations in TLR4. The mutated complexes were less cohesive, displayed local and global variation in the secondary structure, and anomalous decay in rotational correlation function. Principal component analysis indicated that the mutated complexes also exhibited distinct low-frequency motions, which may be correlated to the differential behaviors of these TLR4 variants. Moreover, residue interaction networks (RIN) revealed that the mutated TLR4/myeloid differentiation factor (MD) 2 complex may perpetuate abnormal signaling pathways. Cumulatively, the MDS and RIN analyses elucidated the mutant-specific conformational alterations, which may help in deciphering the mechanism of loss-of-function mutations.

Budesonide increases TLR4 and TLR2 expression in Treg lymphocytes of allergic asthmatics.

PubMed

Pace, Elisabetta; Di Sano, Caterina; Ferraro, Maria; Bruno, Andreina; Caputo, Valentina; Gallina, Salvatore; Gjomarkaj, Mark

2015-06-01

Reduced innate immunity responses as well as reduced T regulatory activities characterise bronchial asthma. In this study the effect of budesonide on the expression of TLR4 and TLR2 in T regulatory lymphocyte sub-population was assessed. TLR4 and TLR2 expression in total peripheral blood mononuclear cells (PBMC), in CD4+/CD25+ and in CD4+/CD25- was evaluated, by flow cytometric analysis, in mild intermittent asthmatics (n = 14) and in controls (n = 11). The in vitro effects of budesonide in modulating: TLR4 and TLR2 expression in controls and in asthmatics; IL-10 expression and cytokine release (IL-6 and TNF-α selected by a multiplex assay) in asthmatics were also explored. TLR4 and TLR2 were reduced in total PBMC from asthmatics in comparison to PBMC from controls. CD4+CD25+ cells expressed at higher extent TLR2 and TLR4 in comparison to CD4+CD25- cells. Budesonide was able to increase the expression of TLR4, TLR2 and IL-10 in CD4+/CD25 highly+ cells from asthmatics. TLR4 ligand, LPS induced Foxp3 expression. Budesonide was also able to reduce the release of IL-6 and TNF-α by PBMC of asthmatics. Budesonide potentiates the activity of Treg by increasing TLR4, TLR2 and IL-10 expression. This event is associated to the decreased release of IL-6 and TNF-α in PBMC treated with budesonide. These findings shed light on new mechanisms by which corticosteroids, drugs widely used for the clinical management of bronchial asthma, control T lymphocyte activation. Copyright © 2015 Elsevier Ltd. All rights reserved.

Polymorphisms in the TLR4 and TLR5 gene are significantly associated with inflammatory bowel disease in German shepherd dogs.

PubMed

Kathrani, Aarti; House, Arthur; Catchpole, Brian; Murphy, Angela; German, Alex; Werling, Dirk; Allenspach, Karin

2010-12-23

Inflammatory bowel disease (IBD) is considered to be the most common cause of vomiting and diarrhoea in dogs, and the German shepherd dog (GSD) is particularly susceptible. The exact aetiology of IBD is unknown, however associations have been identified between specific single-nucleotide polymorphisms (SNPs) in Toll-like receptors (TLRs) and human IBD. However, to date, no genetic studies have been undertaken in canine IBD. The aim of this study was to investigate whether polymorphisms in canine TLR 2, 4 and 5 genes are associated with IBD in GSDs. Mutational analysis of TLR2, TLR4 and TLR5 was performed in 10 unrelated GSDs with IBD. Four non-synonymous SNPs (T23C, G1039A, A1571T and G1807A) were identified in the TLR4 gene, and three non-synonymous SNPs (G22A, C100T and T1844C) were identified in the TLR5 gene. The non-synonymous SNPs identified in TLR4 and TLR5 were evaluated further in a case-control study using a SNaPSHOT multiplex reaction. Sequencing information from 55 unrelated GSDs with IBD were compared to a control group consisting of 61 unrelated GSDs. The G22A SNP in TLR5 was significantly associated with IBD in GSDs, whereas the remaining two SNPs were found to be significantly protective for IBD. Furthermore, the two SNPs in TLR4 (A1571T and G1807A) were in complete linkage disequilibrium, and were also significantly associated with IBD. The TLR5 risk haplotype (ACC) without the two associated TLR4 SNP alleles was significantly associated with IBD, however the presence of the two TLR4 SNP risk alleles without the TLR5 risk haplotype was not statistically associated with IBD. Our study suggests that the three TLR5 SNPs and two TLR4 SNPs; A1571T and G1807A could play a role in the pathogenesis of IBD in GSDs. Further studies are required to confirm the functional importance of these polymorphisms in the pathogenesis of this disease.

Impaired Innate Immunity in Tlr4 −/− Mice but Preserved CD8+ T Cell Responses against Trypanosoma cruzi in Tlr4-, Tlr2-, Tlr9- or Myd88-Deficient Mice

PubMed Central

Tzelepis, Fanny; Klezewsky, Weberton; da Silva, Raquel N.; Neves, Fabieni S.; Cavalcanti, Gisele S.; Boscardin, Silvia; Nunes, Marise P.; Santiago, Marcelo F.; Nóbrega, Alberto; Rodrigues, Maurício M.; Bellio, Maria

2010-01-01

The murine model of T. cruzi infection has provided compelling evidence that development of host resistance against intracellular protozoans critically depends on the activation of members of the Toll-like receptor (TLR) family via the MyD88 adaptor molecule. However, the possibility that TLR/MyD88 signaling pathways also control the induction of immunoprotective CD8+ T cell-mediated effector functions has not been investigated to date. We addressed this question by measuring the frequencies of IFN-γ secreting CD8+ T cells specific for H-2Kb-restricted immunodominant peptides as well as the in vivo Ag-specific cytotoxic response in infected animals that are deficient either in TLR2, TLR4, TLR9 or MyD88 signaling pathways. Strikingly, we found that T. cruzi-infected Tlr2−/−, Tlr4−/−, Tlr9−/ − or Myd88−/− mice generated both specific cytotoxic responses and IFN-γ secreting CD8+ T cells at levels comparable to WT mice, although the frequency of IFN-γ+CD4+ cells was diminished in infected Myd88−/− mice. We also analyzed the efficiency of TLR4-driven immune responses against T. cruzi using TLR4-deficient mice on the C57BL genetic background (B6 and B10). Our studies demonstrated that TLR4 signaling is required for optimal production of IFN-γ, TNF-α and nitric oxide (NO) in the spleen of infected animals and, as a consequence, Tlr4−/− mice display higher parasitemia levels. Collectively, our results indicate that TLR4, as well as previously shown for TLR2, TLR9 and MyD88, contributes to the innate immune response and, consequently, resistance in the acute phase of infection, although each of these pathways is not individually essential for the generation of class I-restricted responses against T. cruzi. PMID:20442858

Association between variations in the TLR4 gene and incident type 2 diabetes is modified by the ratio of total cholesterol to HDL-cholesterol

PubMed Central

Kolz, Melanie; Baumert, Jens; Müller, Martina; Khuseyinova, Natalie; Klopp, Norman; Thorand, Barbara; Meisinger, Christine; Herder, Christian; Koenig, Wolfgang; Illig, Thomas

2008-01-01

Background Toll-like receptor 4 (TLR4), the signaling receptor for lipopolysaccharides, is an important member of the innate immunity system. Since several studies have suggested that type 2 diabetes might be associated with changes in the innate immune response, we sought to investigate the association between genetic variants in the TLR4 gene and incident type 2 diabetes. Methods A case-cohort study was conducted in initially healthy, middle-aged subjects from the MONICA/KORA Augsburg studies including 498 individuals with incident type 2 diabetes and 1,569 non-cases. Seven SNPs were systematically selected in the TLR4 gene and haplotypes were reconstructed. Results The effect of TLR4 SNPs on incident type 2 diabetes was modified by the ratio of total cholesterol to high-density lipoprotein cholesterol (TC/HDL-C). In men, four out of seven TLR4 variants showed significant interaction with TC/HDL-C after correction for multiple testing (p < 0.01). The influence of the minor alleles of those variants on the incidence of type 2 diabetes was observed particularly for male patients with high values of TC/HDL-C. Consistent with these findings, haplotype-based analyses also revealed that the effect of two haplotypes on incident type 2 diabetes was modified by TC/HDL-C in men (p < 10-3). However, none of the investigated variants or haplotypes was associated with type 2 diabetes in main effect models without assessment of effect modifications. Conclusion We conclude that minor alleles of several TLR4 variants, although not directly associated with type 2 diabetes might increase the risk for type 2 diabetes in subjects with high TC/HDL-C. Additionally, our results confirm previous studies reporting sex-related dissimilarities in the development of type 2 diabetes. PMID:18298826

Polymorphisms in the Tlr4 and Tlr5 Gene Are Significantly Associated with Inflammatory Bowel Disease in German Shepherd Dogs

PubMed Central

Kathrani, Aarti; House, Arthur; Catchpole, Brian; Murphy, Angela; German, Alex; Werling, Dirk; Allenspach, Karin

2010-01-01

Inflammatory bowel disease (IBD) is considered to be the most common cause of vomiting and diarrhoea in dogs, and the German shepherd dog (GSD) is particularly susceptible. The exact aetiology of IBD is unknown, however associations have been identified between specific single-nucleotide polymorphisms (SNPs) in Toll-like receptors (TLRs) and human IBD. However, to date, no genetic studies have been undertaken in canine IBD. The aim of this study was to investigate whether polymorphisms in canine TLR 2, 4 and 5 genes are associated with IBD in GSDs. Mutational analysis of TLR2, TLR4 and TLR5 was performed in 10 unrelated GSDs with IBD. Four non-synonymous SNPs (T23C, G1039A, A1571T and G1807A) were identified in the TLR4 gene, and three non-synonymous SNPs (G22A, C100T and T1844C) were identified in the TLR5 gene. The non-synonymous SNPs identified in TLR4 and TLR5 were evaluated further in a case-control study using a SNaPSHOT multiplex reaction. Sequencing information from 55 unrelated GSDs with IBD were compared to a control group consisting of 61 unrelated GSDs. The G22A SNP in TLR5 was significantly associated with IBD in GSDs, whereas the remaining two SNPs were found to be significantly protective for IBD. Furthermore, the two SNPs in TLR4 (A1571T and G1807A) were in complete linkage disequilibrium, and were also significantly associated with IBD. The TLR5 risk haplotype (ACC) without the two associated TLR4 SNP alleles was significantly associated with IBD, however the presence of the two TLR4 SNP risk alleles without the TLR5 risk haplotype was not statistically associated with IBD. Our study suggests that the three TLR5 SNPs and two TLR4 SNPs; A1571T and G1807A could play a role in the pathogenesis of IBD in GSDs. Further studies are required to confirm the functional importance of these polymorphisms in the pathogenesis of this disease. PMID:21203467

Accumulation of stimulants of Toll-like receptor (TLR)-2 and TLR4 in meat products stored at 5 °C.

PubMed

Erridge, Clett

2011-03-01

Recent evidence suggests that exposure to stimulants of the innate immune receptors Toll-like receptor (TLR)-2 and TLR4 may contribute to the development of atherosclerosis and insulin resistance. We showed recently that common foodsuffs can contain TLR-stimulants, and that the greatest concentrations were present in meat-based products. Using a recently developed quantitative bioassay, we here examined the kinetics of accumulation of TLR2- and TLR4-stimulants in a variety of meat products held at 5 °C in air or under a modified atmosphere for up to 8 d. Meat content of TLR-stimulants increased with time in each meat examined and was paralleled by growth of pseudomonads and Enterobacteriaceae, suggesting that bacterial lipopeptides and lipopolysaccharides are the likely sources of TLR2- and TLR4-stimulants, respectively. TLR-stimulants reached the highest levels (approximately 80 μg lipopeptide-equivalents per gramme and approximately 7 μg lipopolysaccharide-equivalents per gram) in meat that was minced rather than intact, and when stored in air rather than under a modified atmosphere. TLR2- and TLR4-stimulants in meat products cooked for 1 h retained approximately 20% and approximately 40% of their bioactivity, respectively. In summary, storage conditions and microbial flora critically regulate the kinetics of TLR2- and TLR4-stimulant accumulation in meat products and these may retain biological activity after cooking. The novel assays presented in this work could be used to predict the potential of foodstuffs to promote inflammatory signaling in human subjects, which may be deleterious to health. These assays may also be used to monitor the historical microbial flora in food products after cooking or other forms of food processing may have rendered the original microflora nonviable.

Polymorphisms in genes TLR1, 2 and 4 are associated with differential cytokine and chemokine serum production in patients with leprosy.

PubMed

Santana, Nadja de Lima; Rêgo, Jamile Leão; Oliveira, Joyce Moura; Almeida, Lucas Frederico de; Braz, Marcos; Machado, Lídia Maria Medeiros; Machado, Paulo Roberto Lima; Castellucci, Léa Cristina

2017-04-01

Leprosy or hansen's disease is a spectral disease whose clinical forms mostly depends on host's immune and genetic factors. Different Toll-like receptors (TLR) variants have been described associated with leprosy, but with some lack of replication across different populations. To evaluate the role of polymorphisms in genes TLR1, TLR2 and TLR4 and susceptibility to leprosy in a genetic case control study; to verify the association between genotypes of these markers and the immunological profile in the serum of patients with leprosy. Pre-designed TaqMan® assays were used to genotype markers at TLR1 (rs4833095, rs5743551), TLR2 (rs7656411, rs3804099) and TLR4 (rs1927914, rs1927911). A panel of cytokines and chemokines was accessed by enzime-linked immunosorbent assay (ELISA) test in the serum of a subgroup of patients with and without leprosy reactions. Our results show an association between the T allele of rs3804099 at the TLR2 gene and increased risk for leprosy per se [Odds ratio (OR) = 1.296, p = 0,022]. In addition, evaluating the association between different genotypes of the TLR1, 2 and 4 markers and cytokine/chemokine serological levels, IL-17 appears as an immunological marker regulated by the polymorphism of the three TLR genes evaluated, whereas different TLR1 genotypes were associated with differential production of IL-12p40 and MCP-1(CCL2). Furthermore, other relevant serum markers such as CXCL-10 and IL-6 seemed to be regulated by TLR2 variants and IL-1β was related to TLR4 genotypes. All together our data points that the tested TLR markers may have a regulatory role in the immunity against Mycobacterium leprae, by driving the host's production of key cytokines and chemokines involved in the pathogenesis of this disease.

Analysis of NOD1, NOD2, TLR1, TLR2, TLR4, TLR5, TLR6 and TLR9 genes in anal furunculosis of German shepherd dogs.

PubMed

House, A K; Binns, M M; Gregory, S P; Catchpole, B

2009-03-01

Anal furunculosis (AF) primarily affects German shepherd dogs (GSD) and is characterised by inflammation and ulceration of the perianal tissues with development of cutaneous sinuses or rectocutaneous fistulae. Investigation of pattern recognition receptor (PRR) function has suggested that defective responses might occur in AF-affected GSD. The aim of the current study was to investigate whether canine PRR genes are involved in determining susceptibility to AF in this breed. Chromosomal location and coding sequences for NOD1, NOD2, TLR1, TLR2, TLR4, TLR5, TLR6 and TLR9 were determined and microsatellite markers identified for each gene. Microsatellite genotyping of 100 control GSD and 47 AF-affected GSD showed restricted allelic variation for AHT H91 (associated with TLR5) and REN216 NO5 (associated with both TLR1 and TLR6) compared with non-GSD dogs. Genotyping of single nucleotide polymorphisms identified in canine TLR1, TLR5, TLR6 and NOD2 genes failed to show any significant associations between PRR polymorphisms and AF. The highly restricted PRR genotypes seen in GSD are likely to have resulted from selective breeding and might influence innate immune responses in this breed.

A Protective Hsp70-TLR4 Pathway in Lethal Oxidant Lung Injury

PubMed Central

Zhang, Yi; Zhang, Xuchen; Shan, Peiying; Hunt, Clayton R.; Pandita, Tej K.; Lee, Patty J.

2013-01-01

Administering high levels of inspired oxygen, or hyperoxia, is commonly used as a life-sustaining measure in critically ill patients. However, prolonged exposures can exacerbate respiratory failure. Our previous study showed that toll-like receptor 4 (TLR4) confers protection against hyperoxia-induced lung injury and mortality. Hsp70 has potent cytoprotective properties and has been described as a TLR4 ligand in cell lines. We sought to elucidate the relationship between TLR4 and Hsp70 in hyperoxia-induced lung injury in vitro and in vivo and to define the signaling mechanisms involved. Wild type, TLR4−/− and Trif−/− (a TLR4 adapter protein) murine lung endothelial cells (MLEC) were exposed to hyperoxia. We found markedly elevated levels of intracellular and secreted Hsp70 from mice lung and MLEC after hyperoxia. We confirmed that Hsp70 and TLR4 co-immunoprecipitate in lung tissue and MLEC. Hsp70-mediated NFκB activation appears to depend upon TLR4. In the absence of TLR4, Hsp70 loses its protective effects in endothelial cells. Furthermore, these protective properties of Hsp70 are TLR4 adapter Trif-dependent, MyD88-independent. Hsp70-deficient mice have increased mortality during hyperoxia and lung-targeted adenoviral delivery of Hsp70 effectively rescues both Hsp70-deficient and wild type mice. Our studies are the first to define an Hsp70-TLR4-Trif cytoprotective axis in the lung and endothelial cells. This pathway is a potential therapeutic target against a range of oxidant-induced lung injuries. PMID:23817427

Role of the Toll Like receptor (TLR) radical cycle in chronic inflammation: possible treatments targeting the TLR4 pathway.

PubMed

Lucas, Kurt; Maes, Michael

2013-08-01

Activation of the Toll-like receptor 4 (TLR4) complex, a receptor of the innate immune system, may underpin the pathophysiology of many human diseases, including asthma, cardiovascular disorder, diabetes, obesity, metabolic syndrome, autoimmune disorders, neuroinflammatory disorders, schizophrenia, bipolar disorder, autism, clinical depression, chronic fatigue syndrome, alcohol abuse, and toluene inhalation. TLRs are pattern recognition receptors that recognize damage-associated molecular patterns and pathogen-associated molecular patterns, including lipopolysaccharide (LPS) from gram-negative bacteria. Here we focus on the environmental factors, which are known to trigger TLR4, e.g., ozone, atmosphere particulate matter, long-lived reactive oxygen intermediate, pentachlorophenol, ionizing radiation, and toluene. Activation of the TLR4 pathways may cause chronic inflammation and increased production of reactive oxygen and nitrogen species (ROS/RNS) and oxidative and nitrosative stress and therefore TLR-related diseases. This implies that drugs or substances that modify these pathways may prevent or improve the abovementioned diseases. Here we review some of the most promising drugs and agents that have the potential to attenuate TLR-mediated inflammation, e.g., anti-LPS strategies that aim to neutralize LPS (synthetic anti-LPS peptides and recombinant factor C) and TLR4/MyD88 antagonists, including eritoran, CyP, EM-163, epigallocatechin-3-gallate, 6-shogaol, cinnamon extract, N-acetylcysteine, melatonin, and molecular hydrogen. The authors posit that activation of the TLR radical (ROS/RNS) cycle is a common pathway underpinning many "civilization" disorders and that targeting the TLR radical cycle may be an effective method to treat many inflammatory disorders.

Single-nucleotide polymorphisms in Toll-like receptor (TLR)-2, TLR4 and heat shock protein 70 genes and susceptibility to scrub typhus.

PubMed

Janardhanan, Jeshina; Joseph Martin, Sherry; Astrup, Elisabeth; Veeramanikandan, R; Aukrust, Pål; Abraham, Ooriapadickal C; Varghese, George M

2013-11-01

Scrub typhus is a highly prevalent bacterial infection in India and South Asia that is caused by Orientia tsutsugamushi. The innate immune response to infections is modulated by Toll-like receptors (TLRs) and heat shock proteins (HSPs). This study was done to assess the prevalence and possible association of TLR and HSP polymorphisms in scrub typhus. TLR4 Asp299Gly, TLR4 Thr399Ile, TLR2 Arg753Gln and HSP70-2 A1267G are single-nucleotide polymorphisms (SNPs) that may modulate their activities, and these SNPs were assessed in 137 scrub typhus patients and 134 controls by PCR restriction fragment length polymorphism. We found that the two TLR4 mutations, TLR4 D299G and TLR4T399I, were present in 19.5% and 22% of the study population, respectively, and was in significant linkage disequilibrium with a D' of 0.8. The TLR2 mutation was found to be rare, whereas the HSP A>G mutation was very common (77.5%). Compared with the controls, the prevalence of heterozygous genotype of the TLR4D299G SNP, but not any of the other SNPs, was significantly higher among scrub typhus patients. Further studies using a larger sample size and more candidate genes may better enable in determining the role of these associations in susceptibility and severity of scrub typhus.

Genetic alterations within TLR genes in development of Toxoplasma gondii infection among Polish pregnant women.

PubMed

Wujcicka, Wioletta; Wilczyński, Jan; Nowakowska, Dorota

2017-09-01

The research was conducted to evaluate the role of genotypes, haplotypes and multiple-SNP variants in the range of TLR2, TLR4 and TLR9 single nucleotide polymorphisms (SNPs) in the development of Toxoplasma gondii infection among Polish pregnant women. The study was performed for 116 Polish pregnant women, including 51 patients infected with T. gondii, and 65 age-matched control pregnant individuals. Genotypes in TLR2 2258 G>A, TLR4 896 A>G, TLR4 1196 C>T and TLR9 2848 G>A SNPs were estimated by self-designed, nested PCR-RFLP assays. Randomly selected PCR products, representative for distinct genotypes in the studied polymorphisms, were confirmed by sequencing. All the genotypes were calculated for Hardy-Weinberg (H-W) equilibrium and TLR4 variants were tested for linkage disequilibrium. Relationships were assessed between alleles, genotypes, haplotypes or multiple-SNP variants in TLR polymorphisms and the occurrence of T. gondii infection in pregnant women, using a logistic regression model. All the analyzed genotypes preserved the H-W equilibrium among the studied groups of patients (P>0.050). Similar distribution of distinct alleles and individual genotypes in TLR SNPs, as well as of haplotypes in TLR4 polymorphisms, were observed in T. gondii infected and control uninfected pregnant women. However, the GACG multiple-SNP variant, within the range of all the four studied polymorphisms, was correlated with a decreased risk of the parasitic infection (OR 0.52, 95% CI 0.28-0.97; P≤0.050). The polymorphisms, located within TLR2, TLR4 and TLR9 genes, may be involved together in occurrence of T. gondii infection among Polish pregnant women. Copyright © 2017 Medical University of Bialystok. Published by Elsevier B.V. All rights reserved.

Polymorphisms in genes TLR1, 2 and 4 are associated with differential cytokine and chemokine serum production in patients with leprosy

PubMed Central

Santana, Nadja de Lima; Rêgo, Jamile Leão; Oliveira, Joyce Moura; de Almeida, Lucas Frederico; Braz, Marcos; Machado, Lídia Maria Medeiros; Machado, Paulo Roberto Lima; Castellucci, Léa Cristina

2017-01-01

BACKGROUND Leprosy or hansen’s disease is a spectral disease whose clinical forms mostly depends on host’s immune and genetic factors. Different Toll-like receptors (TLR) variants have been described associated with leprosy, but with some lack of replication across different populations. OBJECTIVES To evaluate the role of polymorphisms in genes TLR1, TLR2 and TLR4 and susceptibility to leprosy in a genetic case control study; to verify the association between genotypes of these markers and the immunological profile in the serum of patients with leprosy. METHODS Pre-designed TaqMan® assays were used to genotype markers at TLR1 (rs4833095, rs5743551), TLR2 (rs7656411, rs3804099) and TLR4 (rs1927914, rs1927911). A panel of cytokines and chemokines was accessed by enzime-linked immunosorbent assay (ELISA) test in the serum of a subgroup of patients with and without leprosy reactions. FINDINGS Our results show an association between the T allele of rs3804099 at the TLR2 gene and increased risk for leprosy per se [Odds ratio (OR) = 1.296, p = 0,022]. In addition, evaluating the association between different genotypes of the TLR1, 2 and 4 markers and cytokine/chemokine serological levels, IL-17 appears as an immunological marker regulated by the polymorphism of the three TLR genes evaluated, whereas different TLR1 genotypes were associated with differential production of IL-12p40 and MCP-1(CCL2). Furthermore, other relevant serum markers such as CXCL-10 and IL-6 seemed to be regulated by TLR2 variants and IL-1β was related to TLR4 genotypes. MAIN CONCLUSIONS All together our data points that the tested TLR markers may have a regulatory role in the immunity against Mycobacterium leprae, by driving the host’s production of key cytokines and chemokines involved in the pathogenesis of this disease. PMID:28327786

Mechanisms of Toll-like receptor 4 endocytosis reveal a common immune-evasion strategy used by pathogenic and commensal bacteria

PubMed Central

Tan, Yunhao; Zanoni, Ivan; Cullen, Thomas W.; Goodman, Andrew L.; Kagan, Jonathan C.

2015-01-01

Microbe-induced receptor trafficking has emerged as an essential means to promote innate immune signal transduction. Upon detection of bacterial lipopolysaccharides (LPS), CD14 induces an inflammatory endocytosis pathway that delivers Toll-like Receptor 4 (TLR4) to endosomes. Although several regulators of CD14-dependent TLR4 endocytosis have been identified, the cargo selection mechanism during this process remains unknown. We reveal that, in contrast to classic cytosolic interactions that promoted the endocytosis of transmembrane receptors, TLR4 was selected as cargo for inflammatory endocytosis entirely through extracellular interactions. Mechanistically, the extracellular protein MD-2 bound to and dimerized TLR4 in order to promote this endocytic event. Our analysis of LPS variants from human pathogens and gut commensals revealed a common mechanism by which bacteria prevent inflammatory endocytosis. We suggest that evasion of CD14-dependent endocytosis is an attribute that transcends the concept of pathogenesis, and may be a fundamental feature of bacteria that inhabit eukaryotic hosts. PMID:26546281

Atopic dermatitis: Interaction between genetic variants of GSTP1, TNF, TLR2, and TLR4 and air pollution in early life.

PubMed

Hüls, Anke; Klümper, Claudia; MacIntyre, Elaina A; Brauer, Michael; Melén, Erik; Bauer, Mario; Berdel, Dietrich; Bergström, Anna; Brunekreef, Bert; Chan-Yeung, Moira; Fuertes, Elaine; Gehring, Ulrike; Gref, Anna; Heinrich, Joachim; Standl, Marie; Lehmann, Irina; Kerkhof, Marjan; Koppelman, Gerard H; Kozyrskyj, Anita L; Pershagen, Göran; Carlsten, Christopher; Krämer, Ursula; Schikowski, Tamara

2018-04-06

Associations between traffic-related air pollution (TRAP) and childhood atopic dermatitis (AD) remain inconsistent, possibly due to unexplored gene-environment interactions. The aim of this study was to examine whether a potential effect of TRAP on AD prevalence in children is modified by selected single nucleotide polymorphisms (SNPs) related to oxidative stress and inflammation. Doctor-diagnosed AD up to age 2 years and at 7-8 years, as well as AD symptoms up to age 2 years, was assessed using parental-reported questionnaires in six birth cohorts (N = 5685). Associations of nitrogen dioxide (NO 2 ) estimated at the home address of each child at birth and nine SNPs within the GSTP1, TNF, TLR2, or TLR4 genes with AD were examined. Weighted genetic risk scores (GRS) were calculated from the above SNPs and used to estimate combined marginal genetic effects of oxidative stress and inflammation on AD and its interaction with TRAP. GRS was associated with childhood AD and modified the association between NO 2 and doctor-diagnosed AD up to the age of 2 years (P(interaction) = .029). This interaction was mainly driven by a higher susceptibility to air pollution in TNF rs1800629 minor allele (A) carriers. TRAP was not associated with the prevalence of AD in the general population. The marginal genetic association of a weighted GRS from GSTP1, TNF, TLR2, and TLR4SNPs and its interaction with air pollution supports the role of oxidative stress and inflammation in AD. © 2018 EAACI and John Wiley and Sons A/S. Published by John Wiley and Sons Ltd.

Profibrotic Effect of Interleukin-18 in HK-2 Cells Is Dependent on Stimulation of the Toll-like Receptor 4 (TLR4) Promoter and Increased TLR4 Expression*

PubMed Central

Meldrum, Kirstan K.; Zhang, Hongji; Hile, Karen L.; Moldower, Lyle L.; Dong, Zizheng; Meldrum, Daniel R.

2012-01-01

IL-18 is an important mediator of obstruction-induced renal fibrosis and tubular epithelial cell injury independent of TGF-β1 activity. We sought to determine whether the profibrotic effect of IL-18 is mediated through Toll-like receptor 4 (TLR4). Male C57BL6 wild type and mice transgenic for human IL-18-binding protein were subjected to left unilateral ureteral obstruction versus sham operation. The kidneys were harvested 1 week postoperatively and analyzed for IL-18 production and TLR4 expression. In a separate arm, renal tubular epithelial cells (HK-2) were directly stimulated with IL-18 in the presence or absence of a TLR4 agonist, TLR4 antagonist, or TLR4 siRNA knockdown. Cell lysates were analyzed for TLR4, α-smooth muscle actin, and E-cadherin expression. TLR4 promotor activity, as well as AP-1 activation and the effect of AP-1 knockdown on TLR4 expression, was evaluated in HK-2 cells in response to IL-18 stimulation. The results demonstrate that IL-18 induces TLR4 expression during unilateral ureteral obstruction and induces TLR4 expression in HK-2 cells via AP-1 activation. Inhibition of TLR4 or knockdown of TLR4 gene expression in turn prevents IL-18-induced profibrotic changes in HK-2 cells. These results suggest that IL-18 induces profibrotic changes in tubular epithelial cells via increased TLR4 expression/signaling. PMID:23027874

Colitis-associated variant of TLR2 causes impaired mucosal repair due to TFF3 deficiency

PubMed Central

Podolsky, Daniel K.; Gerken, Guido; Eyking, Annette; Cario, Elke

2009-01-01

Background & aims Goblet cells (GC) facilitate mucosal protection and epithelial barrier repair, yet the innate immune mechanisms that selectively drive GC functions have not been defined. The aim of this study was to determine whether TLR2 and modulation of GC-derived TFF3 are functionally linked in the intestine. Methods GC modulation was assessed using qRT-PCR, western blotting and confocal microscopy. DSS colitis was induced in wild-type, TFF3−/− and TLR2−/− mice. Recombinant TLR2 ligand or TFF3 peptide were orally administered after DSS termination. Caco-2 overexpressing full-length TLR2 or mutant TLR2-R753Q were tested for TFF3 synthesis and functional-related effects in a wounding-assay. Results Data from in-vitro (Ls174T) and ex-vivo models of murine and human GC reveal that TLR2 activation selectively induces synthesis of TFF3. In-vivo studies using TFF3−/− or TLR2−/− mice demonstrate the ability for oral treatment with a TLR2 agonist to confer anti-apoptotic protection of the intestinal mucosa against inflammatory stress-induced damage through TFF3. Recombinant TFF3 rescues TLR2-deficient mice from increased morbidity and mortality during acute colonic injury. Severe ulcerative colitis has recently been found to be associated with the R753Q polymorphism of the TLR2 gene. The relevance of the observed functional effect of TLR2 in regulating GC is confirmed by the finding that the UC-associated TLR2-R753Q variant is functionally deficient in the ability to induce TFF3 synthesis, thus leading to impaired wound healing. Conclusions These data demonstrate a novel function of TLR2 in intestinal GC that links products of commensal bacteria to innate immune protection of the host via TFF3. PMID:19303021

Colitis-associated variant of TLR2 causes impaired mucosal repair because of TFF3 deficiency.

PubMed

Podolsky, Daniel K; Gerken, Guido; Eyking, Annette; Cario, Elke

2009-07-01

Goblet cells (GC) facilitate mucosal protection and epithelial barrier repair, yet the innate immune mechanisms that selectively drive GC functions have not been defined. The aim of this study was to determine whether Toll-like receptor (TLR) 2 and modulation of GC-derived trefoil factor (TFF) 3 are functionally linked in the intestine. GC modulation was assessed using quantitative real-time polymerase chain reaction analysis (qRT-PCR), Western blotting, and confocal microscopy. Dextran sulfate sodium (DSS) colitis was induced in wild-type, TFF3(-/-), and TLR2(-/-) mice. Recombinant TLR2 ligand or TFF3 peptide were orally administered after DSS termination. Caco-2 cells overexpressing full-length TLR2 or mutant TLR2-R753Q were tested for TFF3 synthesis and functional-related effects in a wounding assay. Data from in vitro (Ls174T) and ex vivo models of murine and human GC reveal that TLR2 activation selectively induces synthesis of TFF3. In vivo studies using TFF3(-/-) or TLR2(-/-) mice demonstrate the ability for oral treatment with a TLR2 agonist to confer antiapoptotic protection of the intestinal mucosa against inflammatory stress-induced damage through TFF3. Recombinant TFF3 rescues TLR2-deficient mice from increased morbidity and mortality during acute colonic injury. Severe ulcerative colitis (UC) has recently been found to be associated with the R753Q polymorphism of the TLR2 gene. The relevance of the observed functional effect of TLR2 in regulating GC is confirmed by the finding that the UC-associated TLR2-R753Q variant is functionally deficient in the ability to induce TFF3 synthesis, thus leading to impaired wound healing. These data demonstrate a novel function of TLR2 in intestinal GC that links products of commensal bacteria to innate immune protection of the host via TFF3.

Stimulants of Toll-like receptor (TLR)-2 and TLR-4 are abundant in certain minimally-processed vegetables.

PubMed

Erridge, Clett

2011-06-01

Stimulants of the innate immune receptors Toll-like receptor (TLR)-2 and TLR4 have been shown to promote insulin resistance and atherosclerosis in animal models of these diseases. As minimally processed vegetables (MPV) can contain a relatively large bacterial load compared to other foodstuffs, we aimed to quantify the abundance of stimulants of TLR2 and TLR4 in MPV using a transfection-based bioassay calibrated with Escherichia coli LPS and the synthetic lipopeptide Pam(3)CSK(4). Of 5 classes of MPV and 3 classes of related vegetable products considered to be likely to contain a high microbial load, diced onion and bean sprouts contained the highest levels of stimulants of TLR2 (up to 18.5 μg Pam(3)CSK(4)-equivalents per g) and TLR4 (up to 11.4 μg LPS-equivalents per g). By contrast, the majority of fresh whole vegetables examined reproducibly contained minimal or undetectable levels of TLR2- or TLR4-stimulants. The accumulation of TLR-stimulants in MPVs correlated well with growth of enterobacterial spoilage organisms. In conclusion, the modern trend towards eating minimally processed vegetables rather than whole foods is likely to be associated with increased oral exposure to stimulants of TLR2 and TLR4. Copyright © 2011 Elsevier Ltd. All rights reserved.

Molecular characterization and expression profile of partial TLR4 gene in association to mastitis in crossbred cattle.

PubMed

Panigrahi, Manjit; Sharma, Arjava; Bhushan, Bharat

2014-01-01

Crossbred cattle are more prone to mastitis in comparison to indigenous cattle. Toll-like receptor 4 (TLR4) recognizes pathogen ligands, for example, lipopolysaccharide (LPS) endotoxin from Escherichia coli and mediates signaling to initiate innate and adaptive immune responses. Mutations in TLR4 can compromise the host immune response to certain pathogens, so it may be a potential candidate for marker assisted selection to enhance mastitis resistance in dairy cattle. Hence, in this study role of bovine TLR4 gene in mastitis resistance was investigated by association as well as expression profiling analysis in crossbred cattle. The animals were divided into mastitis affected and unaffected groups on the basis of history of animals and California Mastitis Test (CMT). PCR-SSCP and Sequence analysis revealed three genotypes of coreceptor binding region 1 (CRBR1) fragment of TLR4 gene namely AA, AB, and BB in both groups of cattle. The logistic regression model did not show any significant effect of these genotypes on the occurrence of clinical mastitis. Moreover, in vitro challenge of peripheral blood mononuclear cells (PBMCs) with LPS failed to show any association of the genotypes with TLR4 gene expression. In a nutshell, in the present study enough evidence was not found for association of the SNP variants of CRBR1 fragment of TLR4 gene with mastitis susceptibility in crossbred cattle.

S100A9 Interaction with TLR4 Promotes Tumor Growth

PubMed Central

Källberg, Eva; Vogl, Thomas; Liberg, David; Olsson, Anders; Björk, Per; Wikström, Pernilla; Bergh, Anders; Roth, Johannes; Ivars, Fredrik; Leanderson, Tomas

2012-01-01

By breeding TRAMP mice with S100A9 knock-out (S100A9−/−) animals and scoring the appearance of palpable tumors we observed a delayed tumor growth in animals devoid of S100A9 expression. CD11b+ S100A9 expressing cells were not observed in normal prostate tissue from control C57BL/6 mice but were readily detected in TRAMP prostate tumors. Also, S100A9 expression was observed in association with CD68+ macrophages in biopsies from human prostate tumors. Delayed growth of TRAMP tumors was also observed in mice lacking the S100A9 ligand TLR4. In the EL-4 lymphoma model tumor growth inhibition was observed in S100A9−/− and TLR4−/−, but not in RAGE−/− animals lacking an alternative S100A9 receptor. When expression of immune-regulating genes was analyzed using RT-PCR the only common change observed in mice lacking S100A9 and TLR4 was a down-regulation of TGFβ expression in splenic CD11b+ cells. Lastly, treatment of mice with a small molecule (ABR-215050) that inhibits S100A9 binding to TLR4 inhibited EL4 tumor growth. Thus, S100A9 and TLR4 appear to be involved in promoting tumor growth in two different tumor models and pharmacological inhibition of S100A9-TLR4 interactions is a novel and promising target for anti-tumor therapies. PMID:22470535

Distinct Dictation of Japanese Encephalitis Virus-Induced Neuroinflammation and Lethality via Triggering TLR3 and TLR4 Signal Pathways

PubMed Central

Uyangaa, Erdenebelig; Kim, Seong Bum; Kim, Jin Hyoung; Kim, Bum Seok; Kim, Koanhoi; Eo, Seong Kug

2014-01-01

Japanese encephalitis (JE) is major emerging neurologic disease caused by JE virus. To date, the impact of TLR molecules on JE progression has not been addressed. Here, we determined whether each TLR modulates JE, using several TLR-deficient mouse strains (TLR2, TLR3, TLR4, TLR7, TLR9). Surprisingly, among the tested TLR-deficient mice there were contrasting results in TLR3−/− and TLR4−/− mice, i.e. TLR3−/− mice were highly susceptible to JE, whereas TLR4−/− mice showed enhanced resistance to JE. TLR3 ablation induced severe CNS inflammation characterized by early infiltration of inflammatory CD11b+Ly-6Chigh monocytes along with profoundly increased viral burden, proinflammatory cytokine/chemokine expression as well as BBB permeability. In contrast, TLR4−/− mice showed mild CNS inflammation manifested by reduced viral burden, leukocyte infiltration and proinflammatory cytokine expression. Interestingly, TLR4 ablation provided potent in vivo systemic type I IFN innate response, as well as ex vivo type I IFN production associated with strong induction of antiviral PRRs (RIG-I, MDA5), transcription factors (IRF-3, IRF-7), and IFN-dependent (PKR, Oas1, Mx) and independent ISGs (ISG49, ISG54, ISG56) by alternative activation of IRF3 and NF-κB in myeloid-derived DCs and macrophages, as compared to TLR3−/− myeloid-derived cells which were more permissive to viral replication through impaired type I IFN innate response. TLR4 ablation also appeared to mount an enhanced type I IFN innate and humoral, CD4+ and CD8+ T cell responses, which were mediated by altered immune cell populations (increased number of plasmacytoid DCs and NK cells, reduced CD11b+Ly-6Chigh monocytes) and CD4+Foxp3+ Treg number in lymphoid tissue. Thus, potent type I IFN innate and adaptive immune responses in the absence of TLR4 were closely coupled with reduced JE lethality. Collectively, these results suggest that a balanced triggering of TLR signal array by viral components

Lauric Acid Accelerates Glycolytic Muscle Fiber Formation through TLR4 Signaling.

PubMed

Wang, Leshan; Luo, Lv; Zhao, Weijie; Yang, Kelin; Shu, Gang; Wang, Songbo; Gao, Ping; Zhu, Xiaotong; Xi, Qianyun; Zhang, Yongliang; Jiang, Qingyan; Wang, Lina

2018-06-18

Lauric acid (LA), which is the primary fatty acid in coconut oil, was reported to have many metabolic benefits. TLR4 is a common receptor of lipopolysaccharides and involved mainly in inflammation responses. Here, we focused on the effects of LA on skeletal muscle fiber types and metabolism. We found that 200 μM LA treatment in C2C12 or dietary supplementation of 1% LA increased MHCIIb protein expression and the proportion of type IIb muscle fibers from 0.452 ± 0.0165 to 0.572 ± 0.0153, increasing the mRNA expression of genes involved in glycolysis, such as HK2 and LDH2 (from 1.00 ± 0.110 to 1.35 ± 0.0843 and from 1.00 ± 0.123 to 1.71 ± 0.302 in vivo, respectively), decreasing the catalytic activity of lactate dehydrogenase (LDH), and transforming lactic acid to pyruvic acid. Furthermore, LA activated TLR4 signaling, and TLR4 knockdown reversed the effect of LA on muscle fiber type and glycolysis. Thus, we inferred that LA promoted glycolytic fiber formation through TLR4 signaling.

TLR4 deficiency promotes autophagy during cigarette smoke-induced pulmonary emphysema.

PubMed

An, Chang Hyeok; Wang, Xiao Mei; Lam, Hilaire C; Ifedigbo, Emeka; Washko, George R; Ryter, Stefan W; Choi, Augustine M K

2012-11-01

Toll-like receptors (TLRs) exert important nonimmune functions in lung homeostasis. TLR4 deficiency promotes pulmonary emphysema. We examined the role of TLR4 in regulating cigarette smoke (CS)-induced autophagy, apoptosis, and emphysema. Lung tissue was obtained from chronic obstructive lung disease (COPD) patients. C3H/HeJ (Tlr4-mutated) mice and C57BL/10ScNJ (Tlr4-deficient) mice and their respective control strains were exposed to chronic CS or air. Human or mouse epithelial cells (wild-type, Tlr4-knockdown, and Tlr4-deficient) were exposed to CS-extract (CSE). Samples were analyzed for TLR4 expression, and for autophagic or apoptotic proteins by Western blot analysis or confocal imaging. Chronic obstructive lung disease lung tissues and human pulmonary epithelial cells exposed to CSE displayed increased TLR4 expression, and increased autophagic [microtubule-associated protein-1 light-chain-3B (LC3B)] and apoptotic (cleaved caspase-3) markers. Beas-2B cells transfected with TLR4 siRNA displayed increased expression of LC3B relative to control cells, basally and after exposure to CSE. The basal and CSE-inducible expression of LC3B and cleaved caspase-3 were elevated in pulmonary alveolar type II cells from Tlr4-deficient mice. Wild-type mice subjected to chronic CS-exposure displayed airspace enlargement;, however, the Tlr4-mutated or Tlr4-deficient mice exhibited a marked increase in airspace relative to wild-type mice after CS-exposure. The Tlr4-mutated or Tlr4-deficient mice showed higher levels of LC3B under basal conditions and after CS exposure. The expression of cleaved caspase-3 was markedly increased in Tlr4-deficient mice exposed to CS. We describe a protective regulatory function of TLR4 against emphysematous changes of the lung in response to CS.

TLR4 deficiency promotes autophagy during cigarette smoke-induced pulmonary emphysema

PubMed Central

An, Chang Hyeok; Wang, Xiao Mei; Lam, Hilaire C.; Ifedigbo, Emeka; Washko, George R.; Ryter, Stefan W.

2012-01-01

Toll-like receptors (TLRs) exert important nonimmune functions in lung homeostasis. TLR4 deficiency promotes pulmonary emphysema. We examined the role of TLR4 in regulating cigarette smoke (CS)-induced autophagy, apoptosis, and emphysema. Lung tissue was obtained from chronic obstructive lung disease (COPD) patients. C3H/HeJ (Tlr4-mutated) mice and C57BL/10ScNJ (Tlr4-deficient) mice and their respective control strains were exposed to chronic CS or air. Human or mouse epithelial cells (wild-type, Tlr4-knockdown, and Tlr4-deficient) were exposed to CS-extract (CSE). Samples were analyzed for TLR4 expression, and for autophagic or apoptotic proteins by Western blot analysis or confocal imaging. Chronic obstructive lung disease lung tissues and human pulmonary epithelial cells exposed to CSE displayed increased TLR4 expression, and increased autophagic [microtubule-associated protein-1 light-chain-3B (LC3B)] and apoptotic (cleaved caspase-3) markers. Beas-2B cells transfected with TLR4 siRNA displayed increased expression of LC3B relative to control cells, basally and after exposure to CSE. The basal and CSE-inducible expression of LC3B and cleaved caspase-3 were elevated in pulmonary alveolar type II cells from Tlr4-deficient mice. Wild-type mice subjected to chronic CS-exposure displayed airspace enlargement;, however, the Tlr4-mutated or Tlr4-deficient mice exhibited a marked increase in airspace relative to wild-type mice after CS-exposure. The Tlr4-mutated or Tlr4-deficient mice showed higher levels of LC3B under basal conditions and after CS exposure. The expression of cleaved caspase-3 was markedly increased in Tlr4-deficient mice exposed to CS. We describe a protective regulatory function of TLR4 against emphysematous changes of the lung in response to CS. PMID:22983353

TLR4 Asp299Gly polymorphism may be protective against chronic periodontitis.

PubMed

Sellers, R M; Payne, J B; Yu, F; LeVan, T D; Walker, C; Mikuls, T R

2016-04-01

logistic regression examined associations between the SNPs and periodontitis or ABHL. SNP interactions with smoking and P. gingivalis were analyzed. A significant, negative interaction was observed between the TLR4 SNP and the presence of P. gingivalis (p = 0.045) with respect to periodontitis. The TLR4 minor variant was also associated with less ABHL: 16.8% of individuals with low ABHL, 9.0% with moderate ABHL and 11.2% with high ABHL had the minor allele [p = 0.029; odds ratio = 0.58 (95% confidence interval: 0.36-0.95)]. The interaction between the TLR4 SNP and smoking was not significant with respect to periodontitis or ABHL. The CD14 SNP was not associated with periodontitis or ABHL. The TLR4 Asp299Gly SNP significantly interacted with P. gingivalis in conferring a decreased risk of periodontitis and may be protective against ABHL, a feature of periodontitis. Agents blocking TLR4 signaling, a strategy currently under investigation for the treatment of other inflammatory conditions, may warrant investigation in the context of periodontitis related to the presence of P. gingivalis. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Meat and fiber intake and interaction with pattern recognition receptors (TLR1, TLR2, TLR4, and TLR10) in relation to colorectal cancer in a Danish prospective, case-cohort study.

PubMed

Kopp, Tine Iskov; Vogel, Ulla; Tjonneland, Anne; Andersen, Vibeke

2018-03-01

Meat and dietary fiber are associated with increased and decreased risk of colorectal cancer (CRC), respectively. Toll-like receptors (TLRs) regulate the intestinal immune response in a complex interplay between the mucosal epithelium and the microbiota and may therefore be important modulators of diet-induced CRC together with other inflammatory mediators. Our aim was to investigate the association between functional TLR polymorphisms and risk of CRC and the interaction with dietary factors. Additionally, interactions with previously studied polymorphisms in IL10, IL1B, PTGS2, and NFKB1 were assessed in order to examine possible biological pathways in meat-induced CRC. A nested case-cohort study of 897 CRC cases and 1689 randomly selected participants from the Danish prospective "Diet, Cancer and Health" study encompassing 57,053 persons was performed using Cox proportional hazard models and the likelihood ratio test. We found associations between polymorphisms in TLR2 (P = 0.018) and TLR4 (P = 0.044) and risk of CRC per se, interactions between intake of red and processed meat (10 g/d) and polymorphisms in TLR1 (P-interaction = 0.032) and TLR10 (P-interaction = 0.026 and 0.036), and intake of cereals (50 g/d) and TLR4 (P-interaction = 0.044) in relation to risk of CRC. Intake of red and processed meat also interacted with combinations of polymorphisms in TLR1 and TLR10 and polymorphisms in NFKB1, IL10, IL1B, and PTGS2 (P-interaction; TLR1/rs4833095 × PTGS2/rs20417 = 0.021, TLR10/rs11096955 × IL10/rs3024505 = 0.047, TLR10/rs11096955 × PTGS2/rs20417 = 0.017, TLR10/rs4129009 × NFKB1/rs28362491 = 0.027, TLR10/rs4129009 × IL1B/rs4848306 = 0.020, TLR10/rs4129009 × IL1B/rs1143623 = 0.021, TLR10/rs4129009 × PTGS2/rs20417 = 0.027), whereas intake of dietary fiber (10 g/d) interacted with combinations of polymorphisms in TLR4, IL10, and PTGS2 (P-interaction; TLR4/rs1554973 × IL10/rs3024505 = 0.0012, TLR4/rs1554973 × PTGS2/rs20417 = 0.0041, TLR4/rs1554973 × PTGS

Benzenediamine analog FC-99 inhibits TLR2 and TLR4 signaling in peritoneal macrophage in vitro.

PubMed

Yang, Liu; Dou, Huan; Song, Yuxian; Hou, Yayi

2016-01-01

Inflammatory bowel disease (IBD) is an inflammatory disorder, characterized by abnormally increased expression of Toll-like receptors TLR2 and TLR4 in the colon and increased pro-inflammatory cytokine production by macrophages. In the present study, we explored the effect of FC-99, a novel benzenediamine analog, on dextran sulfate sodium (DSS)-induced mouse colitis and investigated its potential mechanism. The results revealed that FC-99 improved the colon morphology and the clinical parameters in DSS-induced mouse colitis. FC-99 inhibited the increase of DSS-induced T helper cells (Th) 1 and Th17 and enhanced the number of regulatory T cells (Treg) in mesenteric lymph nodes (MLN), but had no effect on Th2 cells. FC-99 also suppressed the DSS-induced secretion of interleukin (IL)-1β, IL-6, and the tumor necrosis factor (TNF)-α in the colon and hindered the infiltration of macrophages into colon lamina propria. Flow cytometric analysis also confirmed that FC-99 reduced CD11b(+)F4/80(+) colon macrophages, and down-regulated TNF-α level in situ. Moreover, FC-99 inhibited concentration-dependently the expression of TNF-α and IL-6 in vitro from mouse peritoneal macrophages, which were induced by TLR ligands: PamCSK4 and peptidoglycan (PGN, TLR2 ligand) as well as LPS (TLR4 ligand). Of note, FC-99 also suppressed the activation of TLR2 and TLR4 signaling pathways and the downstream nuclear factor-κB (NF-κB) in the DSS-induced mouse colitis. FC-99 improved the condition of DSS-induced mouse colitis by inhibiting the activation of TLR2 and TLR4 signaling pathways in macrophage. These results suggest that FC-99 may be developed as a new therapeutic drug for IBD. Copyright © 2015 Elsevier Inc. All rights reserved.

Differential Alteration in Intestinal Epithelial Cell Expression of Toll-Like Receptor 3 (TLR3) and TLR4 in Inflammatory Bowel Disease

PubMed Central

Cario, Elke; Podolsky, Daniel K.

2000-01-01

Initiation and perpetuation of the inflammatory intestinal responses in inflammatory bowel disease (IBD) may result from an exaggerated host defense reaction of the intestinal epithelium to endogenous lumenal bacterial flora. Intestinal epithelial cell lines constitutively express several functional Toll-like receptors (TLRs) which appear to be key regulators of the innate response system. The aim of this study was to characterize the expression pattern of TLR2, TLR3, TLR4, and TLR5 in primary intestinal epithelial cells from patients with IBD. Small intestinal and colonic biopsy specimens were collected from patients with IBD (Crohn's disease [CD], ulcerative colitis [UC]) and controls. Non-IBD specimens were assessed by immunofluorescence histochemistry using polyclonal antibodies specific for TLR2, TLR3, TLR4, and TLR5. Primary intestinal epithelial cells (IEC) of normal mucosa constitutively expressed TLR3 and TLR5, while TLR2 and TLR4 were only barely detectable. In active IBD, the expression of TLR3 and TLR4 was differentially modulated in the intestinal epithelium. TLR3 was significantly downregulated in IEC in active CD but not in UC. In contrast, TLR4 was strongly upregulated in both UC and CD. TLR2 and TLR5 expression remained unchanged in IBD. These data suggest that IBD may be associated with distinctive changes in selective TLR expression in the intestinal epithelium, implying that alterations in the innate response system may contribute to the pathogenesis of these disorders. PMID:11083826

TLR4 and TLR9 signals stimulate protective immunity against blood-stage Plasmodium yoelii infection in mice.

PubMed

Zhang, Yanjun; Zhu, Xiaotong; Feng, Yonghui; Pang, Wei; Qi, Zanmei; Cui, Liwang; Cao, Yaming

2016-11-01

The mechanisms regulating the induction of protective immunity against blood-stage malaria remain unclear. Resistant DBA/2 mouse develops a higher Th1 response compared with a susceptible BALB/c strain during Plasmodium yoelii (Py) infection. It is known that the T helper cell response is initiated and polarized by dendritic cells (DCs) of the innate immune system, during which TLR4 and TLR9 are important receptors for the innate recognition of the malaria parasite and its products. We hypothesized that TLR4/9 may play critical roles in the induction of protective immunity against Py infection. We used TLR4/9 antagonists and agonists to study their effects on mouse resistance to Py infection. We found that the administration of an antagonist prior to infection aggravated disease outcomes, impaired DC functions and suppressed the pro-inflammatory response to Py infection in resistant DBA/2 mice. Treatment with the TLR4 agonist lipopolysaccharide (LPS) but not TLR9 agonist significantly improved the survival rate of susceptible Py-infected BALB/c mice. LPS administration promoted the activation and expansion of DCs and drove a Th1-biased response. Our data demonstrate the important roles of TLR4/9 signals in inducing resistance to malaria parasites and provide evidence for the rational use of TLR agonists to potentiate protective immunity against Plasmodium infection. Copyright © 2016 Elsevier Inc. All rights reserved.

Shock-induced neutrophil mediated priming for acute lung injury in mice: divergent effects of TLR-4 and TLR-4/FasL deficiency.

PubMed

Ayala, Alfred; Chung, Chun-Shiang; Lomas, Joanne L; Song, Grace Y; Doughty, Lesley A; Gregory, Stephen H; Cioffi, William G; LeBlanc, Brian W; Reichner, Jonathan; Simms, H Hank; Grutkoski, Patricia S

2002-12-01

Acute lung injury (ALI) leading to respiratory distress is a common sequela of shock/trauma, however, modeling this process in mice with a single shock or septic event is inconsistent. One explanation is that hemorrhage is often just a "priming insult," thus, secondary stimuli may be required to "trigger" ALI. To test this we carried out studies in which we assessed the capacity of hemorrhage alone or hemorrhage followed by septic challenge (CLP) to induce ALI. Lung edema, bronchoalveolar lavage interleukin (IL)-6, alveolar congestion, as well as lung IL-6, macrophage inflammatory protein (MIP)-2, and myeloperoxidase (MPO) activity were all increased in mice subjected to CLP at 24 but not 72 hours following hemorrhage. This was associated with a marked increase in the susceptibility of these mice to septic mortality. Peripheral blood neutrophils derived from 24 hours post-hemorrhage, but not Sham animals, exhibited an ex vivo decrease in apoptotic frequency and an increase in respiratory burst capacity, consistent with in vivo "priming." Subsequently, we observed that adoptive transfer of neutrophils from hemorrhaged but not sham-hemorrhage animals to neutropenic recipients reproduce ALI when subsequently septically challenged, implying that this priming was mediated by neutrophils. We also found marked general increases in lung IL-6, MIP-2, and MPO in mice deficient for toll-like receptor (TLR-4) or the combined lack of TLR-4/FasL. However, the TLR-4 defect markedly attenuated neutrophil influx into the lung while not altering the change in local cytokine/chemokine expression. Alternatively, the combined loss of FasL and TLR-4 did not inhibit the increase in MPO and exacerbated lung IL-6/MIP-2 levels even further.

NOD2 Modulates Serotonin Transporter and Interacts with TLR2 and TLR4 in Intestinal Epithelial Cells.

PubMed

Layunta, Elena; Latorre, Eva; Forcén, Raquel; Grasa, Laura; Castro, Marta; Arias, Maykel A; Alcalde, Ana I; Mesonero, José Emilio

2018-06-15

Serotonin (5-HT) is a chief modulator of intestinal activity. The effects of 5-HT depend on its extracellular availability, which is mainly controlled by serotonin transporter (SERT), expressed in enterocytes. On the other hand, innate immunity, mediated by Toll-like receptors (TLRs) and nucleotide oligomerization domain (NOD)-like receptors (NLRs), is known to control intestinal microbiota and maintain intestinal homeostasis. The dysregulation of the intestinal serotonergic system and innate immunity has been observed in inflammatory bowel diseases (IBD), the incidence of which has severely increased all over the world. The aim of the present study, therefore, was to analyze the effect of NOD2 on intestinal SERT activity and expression, as well as to study the crosstalk of NOD2 with TLR2 and TLR4. Intestinal epithelial cell line Caco-2/TC7 was used to analyze SERT activity and SERT, NOD2, TLR2 and TLR4 molecular expression by real-time PCR and western blotting. Moreover, intestinal tract (ileum and colon) from mice deficient in TLR2, TLR4 or TLR2/4 receptors was used to test the interdependence of NOD2 with these TLR receptors. NOD2 activation inhibits SERT activity in Caco-2/TC7 cells, mainly due to the decrement of SERT molecular expression, with RIP2/RICK being the intracellular pathway involved in this effect. This inhibitory effect on SERT would yield an increment of extracellular 5-HT availability. In this sense, 5-HT strongly inhibits NOD2 expression. In addition, NOD2 showed greater interdependence with TLR2 than with TLR4. Indeed, NOD2 expression significantly increased in both cells treated with TLR2 agonists and the intestinal tract of Tlr2-/- mice. It may be inferred from our data that NOD2 could play a role in intestinal pathophysiology not only through its inherent innate immune role but also due to its interaction with other receptors as TLR2 and the modulation of the intestinal serotonergic system decreasing SERT activity and expression. © 2018

Bovine TLR2 and TLR4 mediate Cryptosporidium parvum recognition in bovine intestinal epithelial cells.

PubMed

Yang, Zhengtao; Fu, Yunhe; Gong, Pengtao; Zheng, Jingtong; Liu, Li; Yu, Yuqiang; Li, Jianhua; Li, He; Yang, Ju; Zhang, Xichen

2015-08-01

Cryptosporidium parvum (C. parvum) is an intestinal parasite that causes diarrhea in neonatal calves. It results in significant morbidity of neonatal calves and economic losses for producers worldwide. Innate resistance against C. parvum is thought to depend on engagement of pattern recognition receptors. However, the role of innate responses to C. parvum has not been elucidated in bovine. The aim of this study was to evaluate the role of TLRs in host-cell responses during C. parvum infection of cultured bovine intestinal epithelial cells. The expressions of TLRs in bovine intestinal epithelial cells were detected by qRT-PCR. To determine which, if any, TLRs may play a role in the response of bovine intestinal epithelial cells to C. parvum, the cells were stimulated with C. parvum and the expression of TLRs were tested by qRT-PCR. The expression of NF-κB was detected by western blotting. Further analyses were carried out in bovine TLRs transfected HEK293 cells and by TLRs-DN transfected bovine intestinal epithelial cells. The results showed that bovine intestinal epithelial cells expressed all known TLRs. The expression of TLR2 and TLR4 were up-regulated when bovine intestinal epithelial cells were treated with C. parvum. Meanwhile, C. parvum induced IL-8 production in TLR2 or TLR4/MD-2 transfected HEK293 cells. Moreover, C. parvum induced NF-κB activation and cytokine expression in bovine intestinal epithelial cells. The induction of NF-κB activation and cytokine expression by C. parvum were reduced in TLR2-DN and TLR4-DN transfected cells. The results showed that bovine intestinal epithelial cells expressed all known TLRs, and bovine intestinal epithelial cells recognized and responded to C. parvum via TLR2 and TLR4. Copyright © 2015 Elsevier Ltd. All rights reserved.

Altered Expression of TLR2 and TLR4 on Peripheral CD14+ Blood Monocytes in Children with Urinary Tract Infection.

PubMed

Karananou, Panagiota; Fleva, Alexandra; Tramma, Despoina; Alataki, Anastasia; Pavlitou-Tsiontsi, Aikaterini; Emporiadou-Peticopoulou, Maria; Papadopoulou-Alataki, Efimia

2016-01-01

Urinary tract infection (UTI) is the second most common bacterial infection, after otitis media, in infants and children. The mechanisms of disease susceptibility and the role of immunity in the pathogenesis of UTI in children have been evaluated. In recent years, Toll-Like Receptors (TLRs) have been recognized as specific components of the innate immune system constituting important mediators in host immune recognition. The aim of the present study was to determine ΤLR2 and TLR4 expression during the acute phase of UTI in infants and children by measuring the CD14/TLR2 and CD14/TLR4 expression on monocytes. We also attempted to compare the TLRs expression with the immunological status of the patients to healthy children. The study group consisted of 60 children (36 females and 24 males) and the control group included 60 age-matched pediatric subjects (27 females and 33 males). In our study, no antibody deficiency was found either in the children with UTI or in healthy subjects. There might be a connection between low IgA, IgG, and IgG subclasses serum levels and UTI as there was a statistically significant difference between patients and healthy children. A higher expression of CD14/TLR2 was revealed in patients (90,07%) compared to controls (85,48%) as well as CD14/TLR4 in patients (90,53%) compared to controls (87,25%) (statistically significant difference, p < 0,05). The results of this study could provide new understanding of UTIs' pathogenesis in children.

Innate immune signaling by Toll-like receptor-4 (TLR4) shapes the inflammatory microenvironment in colitis-associated tumors

PubMed Central

Fukata, Masayuki; Hernandez, Yasmin; Conduah, Daisy; Cohen, Jason; Chen, Anli; Breglio, Keith; Goo, Tyralee; Hsu, David; Xu, Ruliang; Abreu, Maria T.

2009-01-01

Patients with ulcerative colitis are at increased risk for developing colorectal cancer. We have shown that TLR4 is over-expressed in human colitis-associated cancer (CAC) and that mice deficient in TLR4 are markedly protected against colitis-associated neoplasia. We wished to elucidate the specific contributions of TLR4 signaling by myeloid cells and colonic epithelial cells (CEC) in colitis-associated tumorigenesis. TLR4-deficient mice or wild-type littermates (WT) were transplanted with bone marrow (BM) cells: TLR4-/- BM→WT mice (TLR4-expressing CEC) and WT BM→TLR4-/- mice (TLR4-expressing myeloid cells). Colitis-associated neoplasia was induced by azoxymethane (AOM 7.3mg/kg) injection and two cycles of dextran sodium sulfate (DSS) treatment. The number and size of dysplastic lesions were greater in TLR4-/- BM→WT mice than in WT BM→TLR4-/- mice (P<0.005). Histologically, TLR4-/- BM→WT mice had greater numbers of mucosal neutrophils and macrophages compared to WT BM→TLR4-/- mice. The chemokines KC and CCL2, important in recruitment of neutrophils and macrophages, respectively, were induced in mice expressing TLR4 in CEC rather than the myeloid compartment. The lamina propria infiltrate of mice expressing TLR4 in CEC was characterized by macrophages expressing Cox-2. Moreover, mice expressing TLR4 in CEC rather than the myeloid compartment had increased production of amphiregulin and EGFR activation. These findings indicate that TLR4 signaling on CEC is necessary for recruitment and activation of Cox-2 expressing macrophages and increasing the number and size of dysplastic lesions. Our results implicate innate immune signaling on CEC as a key regulator of a tumor-promoting microenvironment. PMID:19229991

LPS enhances TLR4 expression and IFN‑γ production via the TLR4/IRAK/NF‑κB signaling pathway in rat pulmonary arterial smooth muscle cells.

PubMed

Wang, Pengyan; Han, Xuhui; Mo, Biwen; Huang, Guojin; Wang, Changming

2017-09-01

The aim of the present study was to investigate the role of the Toll‑like receptor (TLR)4 signaling pathway in cellular response to lipopolysaccharide (LPS) in rat pulmonary artery smooth muscle cells (PASMCs). Chronic obstructive pulmonary disease (COPD) rats were established with passive inhaling cigarette smoke plus injection of LPS. The TLR4 protein in lung tissues was determined with immunohistochemical staining and protein levels of the components of the TLR4 pathway in PASMCs were analyzed with western blotting. The production of interferon (IFN)‑γ upon LPS stimulation in PASMCs was measured with ELISA. TLR4 expression in lung tissue from COPD rats was increased obviously compared with that in normal group. LPS enhances TLR4 expression in rat PASMCs and induced production of IFN‑γ dramatically. LPS treatment resulted in increased phosphor‑interleukin‑1 receptor‑associated kinase (IRAK), IκB and IκB kinase, as well as the total protein of nuclear factor (NF)‑κB p65. TLR4 inhibitor TAK‑242, IRAK1/4 inhibitor and NF‑κB inhibitor Bay 117082 were capable of suppressing the effects of LPS. TLR4 signaling pathway is functional in PASMCs, and may be involved in the inflammatory response during the pathogenesis of COPD.

Contrasted evolutionary histories of two Toll-like receptors (Tlr4 and Tlr7) in wild rodents (MURINAE)

PubMed Central

2013-01-01

Background In vertebrates, it has been repeatedly demonstrated that genes encoding proteins involved in pathogen-recognition by adaptive immunity (e.g. MHC) are subject to intensive diversifying selection. On the other hand, the role and the type of selection processes shaping the evolution of innate-immunity genes are currently far less clear. In this study we analysed the natural variation and the evolutionary processes acting on two genes involved in the innate-immunity recognition of Microbe-Associated Molecular Patterns (MAMPs). Results We sequenced genes encoding Toll-like receptor 4 (Tlr4) and 7 (Tlr7), two of the key bacterial- and viral-sensing receptors of innate immunity, across 23 species within the subfamily Murinae. Although we have shown that the phylogeny of both Tlr genes is largely congruent with the phylogeny of rodents based on a comparably sized non-immune sequence dataset, we also identified several potentially important discrepancies. The sequence analyses revealed that major parts of both Tlrs are evolving under strong purifying selection, likely due to functional constraints. Yet, also several signatures of positive selection have been found in both genes, with more intense signal in the bacterial-sensing Tlr4 than in the viral-sensing Tlr7. 92% and 100% of sites evolving under positive selection in Tlr4 and Tlr7, respectively, were located in the extracellular domain. Directly in the Ligand-Binding Region (LBR) of TLR4 we identified two rapidly evolving amino acid residues and one site under positive selection, all three likely involved in species-specific recognition of lipopolysaccharide of gram-negative bacteria. In contrast, all putative sites of LBRTLR7 involved in the detection of viral nucleic acids were highly conserved across rodents. Interspecific differences in the predicted 3D-structure of the LBR of both Tlrs were not related to phylogenetic history, while analyses of protein charges clearly discriminated Rattini and Murini

Customized laboratory TLR4 and TLR2 detection method from peripheral human blood for early detection of doxorubicin-induced cardiotoxicity.

PubMed

Pop-Moldovan, A L; Trofenciuc, N-M; Dărăbanţiu, D A; Precup, C; Branea, H; Christodorescu, R; Puşchiţă, M

2017-05-01

Cancer treatments can have significant cardiovascular adverse effects that can cause cardiomyopathy and heart failure with reduced survival benefit and considerable decrease in the use of antineoplastic therapy. The purpose of this study is to assess the role of TLR2 and TLR4 gene expression as an early marker for the risk of doxorubicin-induced cardiomyopathy in correlation with early diastolic dysfunction in patients treated with doxorubicin. Our study included 25 consecutive patients who received treatment with doxorubicin for hematological malignancies (leukemia, lymphomas or multiple myeloma), aged 18-65 years, with a survival probability>6 months and with left ventricular ejection fraction>50%. Exclusion criteria consisted of the following: previous anthracycline therapy, previous radiotherapy, history of heart failure or chronic renal failure, atrial fibrillation, and pregnancy. In all patients, in fasting state, a blood sample was drawn for the assessment of TLR2 and TLR4 gene expression. Gene expression was assessed by quantitative reverse transcription PCR (qRT-PCR) using blood collection, RNA isolation, cDNA reverse transcription, qRT-PCR and quantification of the relative expression. At enrollment, all patients were evaluated clinically; an ECG and an echocardiography were performed. The average amount of gene expression units was 0.113 for TLR4 (range 0.059-0.753) and 0.218 for TLR2 (range 0.046-0.269). The mean mRNA extracted quantity was 113 571 ng/μl. As for the diastolic function parameters, criteria for diastolic dysfunction were present after 6 months in 16 patients (64%). In these patients, the mean values for TLR4 were 0.1198625 and for TLR2 were 0.16454 gene expression units. As for the diastolic function parameters, criteria for diastolic dysfunction were present after 6 months in 16 patients (64%). In these patients, the mean value for TLR2 was 0.30±0.19 and for TLR4 was 0.15±0.04. The corresponding values for the patients who did not

Respiratory syncytial virus prolifically infects N2a neuronal cells, leading to TLR4 and nucleolin protein modulations and RSV F protein co-localization with TLR4 and nucleolin.

PubMed

Yuan, Xiaoling; Hu, Tao; He, Hanwen; Qiu, Huan; Wu, Xuan; Chen, Jingxian; Wang, Minmin; Chen, Cheng; Huang, Shenghai

2018-02-10

Respiratory syncytial virus (RSV) infects the central nervous system, resulting in neurological symptoms. However, the precise underlying pathogenic mechanisms have not been elucidated. In the present study, the infectivity of RSV on N2a neuronal cells and the possible roles of Toll-like receptor 4 (TLR4) and nucleolin (C23) during RSV infection were investigated. We compared two experimental groups (infected and non-infected) and monitored the RSV viral titers in the culture supernatant by a viral plaque assay. We also inspected the morphology of the nucleus in infected N2a cells. We measured the level of RSV F protein and studied its co-localization with TLR4 and nucleolin using immunofluorescence assays and laser confocal microscopy. The potential interaction of RSV F protein with TLR4 and nucleolin was examined by coimmunoprecipitation. The expression changes of TLR4, nucleolin, TLR3 and TLR7 proteins in N2a cells and IL-6 and TNF-α in the culture supernatant were investigated by Western Blot analysis and ELISA assay. Changes in neuronal cell apoptosis status was examined by flow cytometry. The results demonstrated prolific RSV infection of N2a cells, which triggered a decrease of NeuN protein expression, coinciding with an increase of nuclear lesions, F protein expression, RSV viral titers, and late apoptotic levels of N2a cells. RSV infection induced co-localization of RSV F protein with TLR4 and nucleolin, which could potentially lead to a direct interaction. Furthermore, it was found that TLR4 and nucleolin levels increased early after infection and decreased subsequently, whereas TLR3 and TLR7 expression increased throughout RSV infection. The RSV Long strain can prolifically infect N2a neuronal cells, modulating the expression of TLR4 and nucleolin, as well as TLR3, TLR7 and their downstream inflammatory factors, and inducing the co-localization of the RSV F protein with TLR4 and nucleolin.

Discovery and Validation of a New Class of Small Molecule Toll-Like Receptor 4 (TLR4) Inhibitors

PubMed Central

Neal, Matthew D.; Jia, Hongpeng; Eyer, Benjamin; Good, Misty; Guerriero, Christopher J.; Sodhi, Chhinder P.; Afrazi, Amin; Prindle, Thomas; Ma, Congrong; Branca, Maria; Ozolek, John; Brodsky, Jeffrey L.; Wipf, Peter; Hackam, David J.

2013-01-01

Many inflammatory diseases may be linked to pathologically elevated signaling via the receptor for lipopolysaccharide (LPS), toll-like receptor 4 (TLR4). There has thus been great interest in the discovery of TLR4 inhibitors as potential anti-inflammatory agents. Recently, the structure of TLR4 bound to the inhibitor E5564 was solved, raising the possibility that novel TLR4 inhibitors that target the E5564-binding domain could be designed. We utilized a similarity search algorithm in conjunction with a limited screening approach of small molecule libraries to identify compounds that bind to the E5564 site and inhibit TLR4. Our lead compound, C34, is a 2-acetamidopyranoside (MW 389) with the formula C17H27NO9, which inhibited TLR4 in enterocytes and macrophages in vitro, and reduced systemic inflammation in mouse models of endotoxemia and necrotizing enterocolitis. Molecular docking of C34 to the hydrophobic internal pocket of the TLR4 co-receptor MD-2 demonstrated a tight fit, embedding the pyran ring deep inside the pocket. Strikingly, C34 inhibited LPS signaling ex-vivo in human ileum that was resected from infants with necrotizing enterocolitis. These findings identify C34 and the β-anomeric cyclohexyl analog C35 as novel leads for small molecule TLR4 inhibitors that have potential therapeutic benefit for TLR4-mediated inflammatory diseases. PMID:23776545

Toll-like receptor 4 (TLR4) deficient mice are protected from adipose tissue inflammation in aging.

PubMed

Ghosh, Amiya K; O'Brien, Martin; Mau, Theresa; Yung, Raymond

2017-09-07

Adipose tissue (AT) inflammation is a central mechanism for metabolic dysfunction in both diet-induced obesity and age-associated obesity. Studies in diet-induced obesity have characterized the role of Fetuin A (Fet A) in Free Fatty Acids (FFA)-mediated TLR4 activation and adipose tissue inflammation. However, the role of Fet A & TLR4 in aging-related adipose tissue inflammation is unknown. In the current study, analysis of epidymymal fat pads of C57/Bl6 male mice, we found that, in contrast to data from diet-induced obesity models, adipose tissue from aged mice have normal Fet A and TLR4 expression. Interestingly, aged TLR4-deficient mice have diminished adipose tissue inflammation compared to normal controls. We further demonstrated that reduced AT inflammation in old TLR4-deficient mice is linked to impaired ER stress, augmented autophagy activity, and diminished senescence phenomenon. Importantly, old TLR4-deficient mice have improved glucose tolerance compared to age-matched wild type mice, suggesting that the observed reduced AT inflammation in aged TLR4-deficient mice has important physiological consequences. Taken together, our present study establishes novel aspect of aging-associated AT inflammation that is distinct from diet-induced AT inflammation. Our results also provide strong evidence that TLR4 plays a significant role in promoting aging adipose tissue inflammation.

Toll-like receptor 4 (TLR4) deficient mice are protected from adipose tissue inflammation in aging

PubMed Central

Ghosh, Amiya K.; O'Brien, Martin; Mau, Theresa; Yung, Raymond

2017-01-01

Adipose tissue (AT) inflammation is a central mechanism for metabolic dysfunction in both diet-induced obesity and age-associated obesity. Studies in diet-induced obesity have characterized the role of Fetuin A (Fet A) in Free Fatty Acids (FFA)-mediated TLR4 activation and adipose tissue inflammation. However, the role of Fet A & TLR4 in aging-related adipose tissue inflammation is unknown. In the current study, analysis of epidymymal fat pads of C57/Bl6 male mice, we found that, in contrast to data from diet-induced obesity models, adipose tissue from aged mice have normal Fet A and TLR4 expression. Interestingly, aged TLR4-deficient mice have diminished adipose tissue inflammation compared to normal controls. We further demonstrated that reduced AT inflammation in old TLR4-deficient mice is linked to impaired ER stress, augmented autophagy activity, and diminished senescence phenomenon. Importantly, old TLR4-deficient mice have improved glucose tolerance compared to age-matched wild type mice, suggesting that the observed reduced AT inflammation in aged TLR4-deficient mice has important physiological consequences. Taken together, our present study establishes novel aspect of aging-associated AT inflammation that is distinct from diet-induced AT inflammation. Our results also provide strong evidence that TLR4 plays a significant role in promoting aging adipose tissue inflammation. PMID:28898202

Combating Drug Abuse by Targeting Toll-Like Receptor 4 (TLR4)

DTIC Science & Technology

2013-10-01

dosing with the TLR4 signaling inhibitor (+)- naltrexone ? If so, is Fig  3:  Study  was  conducted  as  previously  described  in...inhibited by systemic dosing with the TLR4 signaling inhibitor (+)- naltrexone ? If so, is morphine reinforcement inhibited by microinjecting LPS-RS into... naltrexone ; drug abuse; glial activation; therapeutic approach to treating drug abuse; opioids; cocaine 16. SECURITY CLASSIFICATION OF: 17

B cell TLR1/2, TLR4, TLR7 and TLR9 interact in induction of class switch DNA recombination: modulation by BCR and CD40, and relevance to T-independent antibody responses.

PubMed

Pone, Egest J; Lou, Zheng; Lam, Tonika; Greenberg, Milton L; Wang, Rui; Xu, Zhenming; Casali, Paolo

2015-02-01

Ig class switch DNA recombination (CSR) in B cells is crucial to the maturation of antibody responses. It requires IgH germline IH-CH transcription and expression of AID, both of which are induced by engagement of CD40 or dual engagement of a Toll-like receptor (TLR) and B cell receptor (BCR). Here, we have addressed cross-regulation between two different TLRs or between a TLR and CD40 in CSR induction by using a B cell stimulation system involving lipopolysaccharides (LPS). LPS-mediated long-term primary class-switched antibody responses and memory-like antibody responses in vivo and induced generation of class-switched B cells and plasma cells in vitro. Consistent with the requirement for dual TLR and BCR engagement in CSR induction, LPS, which engages TLR4 through its lipid A moiety, triggered cytosolic Ca2+ flux in B cells through its BCR-engaging polysaccharidic moiety. In the presence of BCR crosslinking, LPS synergized with a TLR1/2 ligand (Pam3CSK4) in CSR induction, but much less efficiently with a TLR7 (R-848) or TLR9 (CpG) ligand. In the absence of BCR crosslinking, R-848 and CpG, which per se induced marginal CSR, virtually abrogated CSR to IgG1, IgG2a, IgG2b, IgG3 and/or IgA, as induced by LPS or CD154 (CD40 ligand) plus IL-4, IFN-γ or TGF-β, and reduced secretion of class-switched Igs, without affecting B cell proliferation or IgM expression. The CSR inhibition by TLR9 was associated with the reduction in AID expression and/or IgH germline IH-S-CH transcription, and required co-stimulation of B cells by CpG with LPS or CD154. Unexpectedly, B cells also failed to undergo CSR or plasma cell differentiation when co-stimulated by LPS and CD154. Overall, by addressing the interaction of TLR1/2, TLR4, TLR7 and TLR9 in the induction of CSR and modulation of TLR-dependent CSR by BCR and CD40, our study suggests the complexity of how different stimuli cross-regulate an important B cell differentiation process and an important role of TLRs in inducing

B cell TLR1/2, TLR4, TLR7 and TLR9 interact in induction of class switch DNA recombination: modulation by BCR and CD40, and relevance to T-independent antibody responses

PubMed Central

Pone, Egest J.; Lou, Zheng; Lam, Tonika; Greenberg, Milton L.; Wang, Rui; Xu, Zhenming; Casali, Paolo

2015-01-01

Ig class switch DNA recombination (CSR) in B cells is crucial to the maturation of antibody responses. It requires IgH germline IH-CH transcription and expression of AID, both of which are induced by engagement of CD40 or dual engagement of a Toll-like receptor (TLR) and B cell receptor (BCR). Here, we have addressed cross-regulation between two different TLRs or between a TLR and CD40 in CSR induction by using a B cell stimulation system involving lipopolysaccharides (LPS). LPS mediated long-term primary class-switched antibody responses and memory-like antibody responses in vivo and induced generation of class-switched B cells and plasma cells in vitro. Consistent with the requirement for dual TLR and BCR engagement in CSR induction, LPS, which engages TLR4 through its lipid A moiety, triggered cytosolic Ca2+ flux in B cells through its BCR-engaging polysaccharidic moiety. In the presence of BCR crosslinking, LPS synergized with a TLR1/2 ligand (Pam3CSK4) in CSR induction, but much less efficiently with a TLR7 (R-848) or TLR9 (CpG) ligand. In the absence of BCR crosslinking, R-848 and CpG, which per se induced marginal CSR, virtually abrogated CSR to IgG1, IgG2a, IgG2b, IgG3 and/or IgA, as induced by LPS or CD154 (CD40 ligand) plus IL-4, IFN-γ or TGF-β, and reduced secretion of class-switched Igs, without affecting B cell proliferation or IgM expression. The CSR inhibition by TLR9 was associated with the reduction in AID expression and/or IgH germline IH-S-CH transcription, and required co-stimulation of B cells by CpG with LPS or CD154. Unexpectedly, B cells also failed to undergo CSR or plasma cell differentiation when co-stimulated by LPS and CD154. Overall, by addressing the interaction of TLR1/2, TLR4, TLR7 and TLR9 in the induction of CSR and modulation of TLR-dependent CSR by BCR and CD40, our study suggests the complexity of how different stimuli cross-regulate an important B cell differentiation process and an important role of TLRs in inducing

Ethanol consumption in mice lacking CD14, TLR2, TLR4, or MyD88

PubMed Central

Blednov, Yuri A.; Black, Mendy; Chernis, Julia; Da Costa, Adriana; Mayfield, Jody; Harris, R. Adron

2016-01-01

Background Molecular and behavioral studies support a role for innate immune proinflammatory pathways in mediating the effects of alcohol. Increased levels of Toll-like receptors (TLRs) have been observed in animal models of alcohol consumption and in human alcoholics, and many of these TLRs signal via the MyD88-dependent pathway. We hypothesized that this pathway is involved in alcohol drinking and examined some of its key signaling components. Methods Different ethanol drinking paradigms were studied in male and female control C57BL/6J mice vs. mice lacking CD14, TLR2, TLR4 (C57BL/10ScN), or MyD88. We studied continuous and intermittent access two-bottle choice (2BC) and one-bottle and 2BC drinking-in-the-dark (DID) tests as well as preference for saccharin, quinine, and NaCl. Results In the 2BC continuous access test, ethanol intake decreased in male TLR2 knockout (KO) mice, and we previously reported reduced 2BC drinking in male and female CD14 KO mice. In the intermittent access 2BC test, ethanol intake decreased in CD14 KO male and female mice, whereas drinking increased in MyD88 KO male mice. In the 2BC-DID test, ethanol drinking decreased in male and female mice lacking TLR2, whereas drinking increased in MyD88 KO male mice. In the one-bottle DID test, ethanol intake decreased in female TLR2 KO mice. TLR2 KO and CD14 KO mice did not differ in saccharin preference but showed reduced preference for NaCl. MyD88 KO mice showed a slight reduction in preference for saccharin. Conclusions Deletion of key components of the MyD88-dependent pathway produced differential effects on ethanol intake by decreasing (TLR2 KO and CD14 KO) or increasing (MyD88 KO) drinking, while deletion of TLR4 had no effect. Some of the drinking effects depended on the sex of the mice and/or the ethanol-drinking model. PMID:28146272

Functional variants in intercellular adhesion molecule-1 and toll-like receptor-4 genes are more frequent in children with febrile urinary tract infection with renal parenchymal involvement.

PubMed

Hussein, Almontaser; Saad, Khaled; Askar, Eman; Zahran, Asmaa M; Farghaly, Hekma; Metwalley, Kotb; Elderwy, Ahmad A

2018-02-01

We studied the functional polymorphisms of intercellular adhesion molecule-1 (ICAM-1) and toll-like receptor-4 (TLR-4) genes and risk of acute pyelonephritis (APN) in children attending Assiut University Children's Hospitals, Egypt, from 2011 to 2015. Urinary tract infections (UTIs) were diagnosed in 380 children: 98 had APN and 282 had lower UTIs. Four single-nucleotide polymorphisms in ICAM-1 and TLR-4 genes were genotyped in all subjects: ICAM-1 rs1799969 Gly241Arg, ICAM-1 rs5498 Glu469Lys, TLR-4 rs4896791 Thr399Ile and TLR-4 rs4896790 Asp299Gly. Patients with APN were significantly more likely to have AA genotype of the ICAM-1 rs5498 (1462 A/G) polymorphism (p = 0.04) than children with lower UTIs and the TLR-4 Asp299Gly GG genotype (p = 0.002) and G allele (p = 0.006) than healthy controls. The association with the ICAM-1 Glu469Lys (1462A/G) was less evident. The GG genotype was associated with a modest relative risk of 1.4 (p = 0.1) of developing APN, but was not an independent odds ratio, at 1.2 (p = 0.48). Functional variants in ICAM-1 and TLR-4 genes were increasingly common in children with febrile UTIs with renal parenchymal involvement, but the ICAM-1 Glu469Lys (1462A/G) association was less evident. TLR4 Asp299Gly might independently increase renal parenchymal infection rather than renal scarring. ©2017 Foundation Acta Paediatrica. Published by John Wiley & Sons Ltd.

TLR4 signaling in effector CD4+ T cells regulates TCR activation and experimental colitis in mice

PubMed Central

González-Navajas, José M.; Fine, Sean; Law, Jason; Datta, Sandip K.; Nguyen, Kim P.; Yu, Mandy; Corr, Maripat; Katakura, Kyoko; Eckman, Lars; Lee, Jongdae; Raz, Eyal

2010-01-01

TLRs sense various microbial products. Their function has been best characterized in DCs and macrophages, where they act as important mediators of innate immunity. TLR4 is also expressed on CD4+ T cells, but its physiological function on these cells remains unknown. Here, we have shown that TLR4 triggering on CD4+ T cells affects their phenotype and their ability to provoke intestinal inflammation. In a model of spontaneous colitis, Il10–/–Tlr4–/– mice displayed accelerated development of disease, with signs of overt colitis as early as 8 weeks of age, when compared with Il10–/– and Il10–/–Tlr9–/– mice, which did not develop colitis by 8 months. Similar results were obtained in a second model of colitis in which transfer of naive Il10–/–Tlr4–/– CD4+ T cells into Rag1–/– recipients sufficient for both IL-10 and TLR4 induced more aggressive colitis than the transfer of naive Il10–/– CD4+ T cells. Mechanistically, LPS stimulation of TLR4-bearing CD4+ T cells inhibited ERK1/2 activation upon subsequent TCR stimulation via the induction of MAPK phosphatase 3 (MKP-3). Our data therefore reveal a tonic inhibitory role for TLR4 signaling on subsequent TCR-dependent CD4+ T cell responses. PMID:20051628

[Expressions and significance of TLR2 and TLR4 in Kupffer cells of tree shrews chronically infected with hepatitis B virus].

PubMed

Ruan, Ping; Yang, Chun; Su, Jianjia; Ou, Chao; Cao, Ji; Luo, Chengpiao; Tang, Yanping; Qin, Hong; Sun, Wen; Li, Yuan

2014-07-01

To investigate the mRNA expression levels of Toll-like receptors 2 (TLR2) and TLR4 in Kupffer cells of tree shrews that were chronically infected with hepatitis B virus (HBV), and the effects of these receptors on the function of Kupffer cells. The tree shrews were divided into tree shrews proved with chronic HBV infection, tree shrews suspected with chronic HBV infection, and normal control tree shrews without hepatitis B vaccination. The samples of serum and liver biopsy were collected periodically, and the levels of HBV DNA in serum and liver tissues were detected by fluorescence-based quantitative real-time PCR (qRT-PCR). Meanwhile, Kupffer cells were isolated from the biopsied liver tissues, and then purified and primarily cultured. Afterwards, qRT-PCR was applied to detect the mRNA expression levels of TLR2, TLR4 and TNF-α in the Kupffer cells. Cell migration assay and lysosome-specific fluorescent probe were adopted to analyze the effects of TLR2 and TLR4 on the migration capacity of Kupffer cells and the quantity of lysosomes in these cells. The mRNA expression levels of TLR2 and TLR4 in tree shrews proved with chronic HBV infection were lower than those in the ones suspected with chronic HBV infection and normal controls without hepatitis B vaccination (P<0.05), and these expression levels were all negatively correlated with the level of HBV DNA in liver tissues of the animals (P<0.05), but were positively correlated with the number of migrated Kupffer cells, the density of lysosomes and the mRNA expression level of TNF-α (P<0.05). TLR2 and TLR4 in Kupffer cells may play important roles in the chronic process of hepatic pathological changes in tree shrews infected with HBV through their influence on the function of Kupffer cells.

Differential regulation of IL-23 production in M1 macrophages by TIR8/SIGIRR through TLR4- or TLR7/8-mediated signaling.

PubMed

Yamaguchi, Rui; Sakamoto, Arisa; Yamamoto, Takatoshi; Narahara, Shinji; Sugiuchi, Hiroyuki; Yamaguchi, Yasuo

2017-11-01

Cross-talks between toll-like receptors (TLRs) including various negative regulatory mechanisms are many unknown. We investigated the differential mechanism of IL-23 production in M1 macrophages by single immunoglobulin interleukin-1 receptor-related (SIGIRR) molecule through TLR4 or TLR7/8. IL-12p40 production by M1 macrophages pretreated with human neutrophil elastase (HNE) was synergistically enhanced IL-12p40, but not IL-23 production, after exposure to lipopolysaccharide (LPS). LPS (a TLR4 agonist) induced a slight increase of IL-23 production, while Resiquimod (a TLR7/8 agonist) significantly enhanced IL-23 production. Expression of SIGIRR protein, a negative regulator of TLR4, was higher in M1 macrophages than in monocytes. Interestingly, SIGIRR siRNA induced a slight increment of IL-23 production after exposure of macrophages to LPS, while IL-23 production in response to Resiquimod was significantly upregulated by SIGIRR siRNA. Silencing SIGIRR enhanced IRF4 protein level determined by western blotting or ELISA. IRF4 siRNA dramatically restored IL-23 production after exposure to Resiquimod in macrophages transfected with SIGIRR siRNA. In conclusion, production of IL-23 is differentially regulated in M1 macrophages by SIGIRR through TLR4- or TLR7/8-mediated signaling. SIGIRR is both a negative regulator of TLR4 and a positive regulator of TLR7/8. Copyright © 2017 Elsevier Ltd. All rights reserved.

May TLR4 Asp299Gly and IL17 His161Arg polymorphism be associated with progression of primary measles infection to subacute sclerosing panencephalitis?

PubMed

Karakas-Celik, Sevim; Piskin, Ibrahim Etem; Keni, Mehmet Fatih; Calık, Mustafa; Iscan, Akın; Dursun, Ahmet

2014-09-01

SSPE is a progressive neurological disorder of children. Only some of the children who are infected with measles virus develop SSPE, which supports individual variation. TLR-2 and TLR-4 play an important role in innate immunity by recognizing envelope proteins of MV. Another important cytokine that plays an important role in orchestrating innate immune function is IL-17. The purpose of our study is to elucidate whether the TLR2, TLR4, IL17F and IL17A gene polymorphisms are susceptibility genes for the development of SSPE. Using the PCR-RFLP methods, the single nucleotide polymorphisms of TLR2 (Arg753Gln, Arg677Trp, -194 to -174 del), TLR4 (Asp299Gly and Thr399Ile) IL17F (His161Arg, Glu126Gly) and IL17A were studied in 54 patients with SSPE and 81 healthy controls. For Asp299Gly polymorphism of the TLR4 gene we found that there were no control individuals who were homozygous carriers of the Gly/Gly genotype, and the risk for SSPE increased at approximately 4.7 fold for the heterozygous carriers of the Asp/Gly genotype (OR 4.727, 95%-CI 1.192-18.742; P=0.01), when compared to healthy controls. Also our findings demonstrate that homozygosity for the Arg161 variant of the IL17F His161Arg polymorphism is inversely associated with development of SSPE (OR 0.114 95%-CI 0.026-0.494; P<0.001). In conclusion, it is suggested that variation in susceptibility to SSPE disease may be in part due to variations in TLR4 and IL17 function resulting from polymorphisms of TLR4 Asp299Gly and IL17F His161Arg. Copyright © 2014 Elsevier B.V. All rights reserved.

TLR4 signaling augments B lymphocyte migration and overcomes the restriction that limits access to germinal center dark zones

PubMed Central

Hwang, Il-Young; Park, Chung; Harrison, Kathleen

2009-01-01

B lymphocyte–intrinsic Toll-like receptor (TLR) signals amplify humoral immunity and can exacerbate autoimmune diseases. We identify a new mechanism by which TLR signals may contribute to autoimmunity and chronic inflammation. We show that TLR4 signaling enhances B lymphocyte trafficking into lymph nodes (LNs), induces B lymphocyte clustering and interactions within LN follicles, leads to sustained in vivo B cell proliferation, overcomes the restriction that limits the access of nonantigen-activated B cells to germinal center dark zones, and enhances the generation of memory and plasma cells. Intravital microscopy and in vivo tracking studies of B cells transferred to recipient mice revealed that TLR4-activated, but not nonstimulated, B cells accumulated within the dark zones of preexisting germinal centers even when transferred with antigen-specific B cells. The TLR4-activated cells persist much better than nonstimulated cells, expanding both within the memory and plasma cell compartments. TLR-mediated activation of B cells may help to feed and stabilize the spontaneous and ectopic germinal centers that are so commonly found in autoimmune individuals and that accompany chronic inflammation. PMID:19917774

Role of TLR4 signaling in the nephrotoxicity of heme and heme proteins.

PubMed

Nath, Karl A; Belcher, John D; Nath, Meryl C; Grande, Joseph P; Croatt, Anthony J; Ackerman, Allan W; Katusic, Zvonimir S; Vercellotti, Gregory M

2018-05-01

Destabilized heme proteins release heme, and free heme is toxic. Heme is now recognized as an agonist for the Toll-like receptor-4 (TLR4) receptor. This study examined whether the TLR4 receptor mediates the nephrotoxicity of heme, specifically, the effects of heme on renal blood flow and inflammatory responses. We blocked TLR4 signaling by the specific antagonist TAK-242. Intravenous administration of heme to mice promptly reduced renal blood flow, an effect attenuated by TAK-242. In vitro, TAK-242 reduced heme-elicited activation of NF-κB and its downstream gene monocyte chemoattractant protein-1(MCP-1); in contrast, TAK-242 failed to reduce heme-induced activation of the anti-inflammatory transcription factor Nrf2 and its downstream gene heme oxygenase-1 (HO-1). TAK-242 did not reduce heme-induced renal MCP-1 upregulation in vivo. TAK-242 did not reduce dysfunction and histological injury in the glycerol model of heme protein-induced acute kidney injury (AKI), findings corroborated by studies in TLR4 +/+ and TLR4 -/- mice. We conclude that 1) acute heme-mediated renal vasoconstriction occurs through TLR4 signaling; 2) proinflammatory effects of heme in renal epithelial cells involve TLR4 signaling, whereas the anti-inflammatory effects of heme do not; 3) TLR4 signaling does not mediate the proinflammatory effects of heme in the kidney; and 4) major mechanisms underlying glycerol-induced, heme protein-mediated AKI do not involve TLR4 signaling. These findings in the glycerol model are in stark contrast with findings in virtually all other AKI models studied to date and emphasize the importance of TLR4-independent pathways of heme protein-mediated injury in this model. Finally, these studies urge caution when using observations derived in vitro to predict what occurs in vivo.

TLR4 plays a crucial role in MSC-induced inhibition of NK cell function

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

Lu, Ying; Liu, Jin; Liu, Yang

2015-08-21

Mesenchymal stem cells (MSC) are a kind of stromal cell within the tumor microenvironment. In our research, MSC derived from acute myeloid leukemia patients' bone marrow (AML-MSC) and lung cancer tissues (LC-MSC) as well as normal bone marrow-derived MSC (BM-MSC) cultured in conditioned medium of HeLa cells were found to have higher expressions of Toll-like receptor (TLR4) mRNA compared with BM-MSC. The sorted TLR4-positive MSC (TLR4+ MSC) differed in cytokine (interleukin-6, interleukin-8, and monocyte chemoattractant protein-1) secretion from those of unsorted MSC. MSC was reported to inhibit natural killer (NK) cell proliferation and function. In this research, we confirmed thatmore » TLR4+ MSC aggravate this suppression. Furthermore, when TLR4 in the sorted cells were stimulated by LPS or following blocked by antibody, the suppression on NK cell proliferation and cytotoxicity were more intensive or recovered respectively. Compared to unsorted MSC, NKG2D receptor expression on NK cells were also inhibited by TLR4+ MSC. These findings suggest that activation of TLR4 pathway is important for TLR4+ MSC and MSC to obstruct anti-tumor immunity by inhibiting NK cell function, which may provide a potential stroma-targeted tumor therapy. - Highlights: • TLR4+ MSC inhibit NK cell proliferation in vivo and in vitro. • TLR4+ MSC inhibit NKG2D expression on NK cells and NK cell cytotoxicity. • The distinguished cytokine expression of TLR4+ MSC may contribute to the inhibition on NK cell function.« less

[Changes of FoxP3, CD4(+)CD25(+) regulatory T cells, TLR2 and TLR9 in children with infectious mononucleosis].

PubMed

Wang, Qiang; Wang, Zuo-Feng; Cao, Mei; Wang, Zhi-Ying

2013-04-01

The aim of this study was to investigate the effects of TLR2, TLR9, CD4(+)CD25(+) regulatory T cells (Treg) and transcription factor FoxP3 in the pathogenesis of children with infectious mononucleosis (IM). Thirty-five acute IM patients admitted in our hospital from April 2010 to January 2011 were enrolled in this study. Thirty-five healthy subjects were taken as control. The thirty-five patients before treatment were considered as patients in acute stage, after treatment and without clinical symptom they were thought as patients in recovery stage. The expression levels of TLR2, TLR9 and FoxP3 mRNA were detected by real time PCR using SYBR Green I. The expression of T lymphocyte subset CD4(+)CD25(+) in peripheral blood mononuclear cells was detected by flow cytometry. The results showed that the relative levels of TLR2 mRNA (4.03 ± 0.56), TLR9 mRNA (8.88 ± 1.56) in peripheral blood mononuclear cells of IM patients in acute stage were significantly higher than those of the controls [TLR2 mRNA (2.22 ± 0.57), TLR9 mRNA (3.63 ± 1.30)] and IM patients in recovery stage [TLR2 mRNA (2.76 ± 0.83), TLR9 mRNA (5.34 ± 1.60)] (P < 0.01). The result of CD4(+)CD25(+) (2.38 ± 1.32%) and relative level of FoxP3 mRNA(2.82 ± 0.90) in peripheral blood mononuclear cells of IM patients in acute stage were lower than those of the control [CD4(+)CD25(+) (7.85 ± 1.97%), FoxP3 mRNA (4.65 ± 1.23) ] (P < 0.01). There was no significant difference in CD4(+)CD25(+) (6.81 ± 1.84%), FoxP3 mRNA(4.11 ± 1.37) levels between IM patients in recovery stage and the controls (P > 0.05). It is concluded that the expression of CD4(+)CD25(+)regulatory T cells is reduced, and its special transcription factor FoxP3 mRNA is down-regulated, but expression levels of TLR2 mRNA, TLR9 mRNA are up-regulated in IM patients of acute stage.

Whole genome sequencing of an African American family highlights toll like receptor 6 variants in Kawasaki disease susceptibility.

PubMed

Kim, Jihoon; Shimizu, Chisato; Kingsmore, Stephen F; Veeraraghavan, Narayanan; Levy, Eric; Ribeiro Dos Santos, Andre M; Yang, Hai; Flatley, Jay; Hoang, Long Truong; Hibberd, Martin L; Tremoulet, Adriana H; Harismendy, Olivier; Ohno-Machado, Lucila; Burns, Jane C

2017-01-01

Kawasaki disease (KD) is the most common acquired pediatric heart disease. We analyzed Whole Genome Sequences (WGS) from a 6-member African American family in which KD affected two of four children. We sought rare, potentially causative genotypes by sequentially applying the following WGS filters: sequence quality scores, inheritance model (recessive homozygous and compound heterozygous), predicted deleteriousness, allele frequency, genes in KD-associated pathways or with significant associations in published KD genome-wide association studies (GWAS), and with differential expression in KD blood transcriptomes. Biologically plausible genotypes were identified in twelve variants in six genes in the two affected children. The affected siblings were compound heterozygous for the rare variants p.Leu194Pro and p.Arg247Lys in Toll-like receptor 6 (TLR6), which affect TLR6 signaling. The affected children were also homozygous for three common, linked (r2 = 1) intronic single nucleotide variants (SNVs) in TLR6 (rs56245262, rs56083757 and rs7669329), that have previously shown association with KD in cohorts of European descent. Using transcriptome data from pre-treatment whole blood of KD subjects (n = 146), expression quantitative trait loci (eQTL) analyses were performed. Subjects homozygous for the intronic risk allele (A allele of TLR6 rs56245262) had differential expression of Interleukin-6 (IL-6) as a function of genotype (p = 0.0007) and a higher erythrocyte sedimentation rate at diagnosis. TLR6 plays an important role in pathogen-associated molecular pattern recognition, and sequence variations may affect binding affinities that in turn influence KD susceptibility. This integrative genomic approach illustrates how the analysis of WGS in multiplex families with a complex genetic disease allows examination of both the common disease-common variant and common disease-rare variant hypotheses.

Toll like receptor 4 (TLR4) mediates the stimulating activities of chitosan oligosaccharide on macrophages.

PubMed

Zhang, Pei; Liu, Weizhi; Peng, Yanfei; Han, Baoqin; Yang, Yan

2014-11-01

The in vivo and in vitro immunostimulating properties of chitosan oligosaccharide (COS) prepared by enzymatic hydrolysis of chitosan and the mechanisms mediating the effects were investigated. Our data showed that the highly active chitosanase isolated could hydrolyze chitosan to the polymerization degree of 3-8. The resulting COS was an efficient immunostimulator. COS markedly enhanced the proliferation and neutral red phagocytosis by RAW 264.7 macrophages. The production of nitric oxide (NO) and tumor necrosis factor alpha (TNF-α) by macrophages was significantly increased after incubation with COS. Oral administration of COS in mice could increase spleen index and serum immunoglobin G (IgG) contents. COS was labeled with FITC to study the pinocytosis by macrophages. Results showed that FITC-COS was phagocyted by macrophages and anti-murine TLR4 antibody completely blocked FITC-COS pinocytosis. RT-PCR indicated that COS treatment of macrophages significantly increased TLR4 and inducible nitric oxide synthase (iNOS) mRNA levels. When cells were pretreated with anti-murine TLR4 antibody, the effect of COS on TLR4 and iNOS mRNA induction was decreased. COS-induced NO secretion by macrophages was also markedly decreased by anti-murine TLR4 antibody pretreatment. In conclusion, the present study revealed that COS possesses potent immune-stimulating properties by activating TLR4 on macrophages. Copyright © 2014 Elsevier B.V. All rights reserved.

Elevated Muscle TLR4 Expression and Metabolic Endotoxemia in Human Aging

PubMed Central

Ghosh, Sangeeta; Lertwattanarak, Raweewan; Garduño, Jose de Jesus; Galeana, Joaquin Joya; Li, Jinqi; Zamarripa, Frank; Lancaster, Jack L.; Mohan, Sumathy; Hussey, Sophie

2015-01-01

Aging is associated with alterations in glucose metabolism and sarcopenia that jointly contribute to a higher risk of developing type 2 diabetes. Because aging is considered as a state of low-grade inflammation, in this study we examined whether older, healthy (lean, community-dwelling) participants have altered signaling flux through toll-like receptor 4 (TLR4), a key mediator of innate and adaptive immune responses. We also examined whether a 4-month aerobic exercise program would have an anti-inflammatory effect by reducing TLR4 expression and signaling. At baseline, muscle TLR4, nuclear factor κB p50 and nuclear factor κB p65 protein content, and c-Jun N-terminal kinase phosphorylation were significantly elevated in older versus young participants. The plasma concentration of the TLR4 agonist lipopolysaccharide and its binding protein also were significantly elevated in older participants, indicative of metabolic endotoxemia, which is a recently described phenomenon of increased plasma endotoxin level in metabolic disease. These alterations in older participants were accompanied by decreased insulin sensitivity, quadriceps muscle volume, and muscle strength. The exercise training program increased insulin sensitivity, without affecting quadriceps muscle volume or strength. Muscle TLR4, nuclear factor κB, and c-Jun N-terminal kinase, and plasma lipopolysaccharide and lipopolysaccharide binding protein were not changed by exercise. In conclusion, insulin resistance and sarcopenia of aging are associated with increased TLR4 expression/signaling, which may be secondary to metabolic endotoxemia. PMID:24846769

Purified monomeric ligand.MD-2 complexes reveal molecular and structural requirements for activation and antagonism of TLR4 by Gram-negative bacterial endotoxins.

PubMed

Gioannini, Theresa L; Teghanemt, Athmane; Zhang, DeSheng; Esparza, Gregory; Yu, Liping; Weiss, Jerrold

2014-08-01

A major focus of work in our laboratory concerns the molecular mechanisms and structural bases of Gram-negative bacterial endotoxin recognition by host (e.g., human) endotoxin-recognition proteins that mediate and/or regulate activation of Toll-like receptor (TLR) 4. Here, we review studies of wild-type and variant monomeric endotoxin.MD-2 complexes first produced and characterized in our laboratories. These purified complexes have provided unique experimental reagents, revealing both quantitative and qualitative determinants of TLR4 activation and antagonism. This review is dedicated to the memory of Dr. Theresa L. Gioannini (1949-2014) who played a central role in many of the studies and discoveries that are reviewed.

TLR2 and TLR4 expression in peripheral blood mononuclear cells of patients with chronic cystic echinococcosis and its relationship with IL-10.

PubMed

Shan, J-Y; Ji, W-Z; Li, H-T; Tuxun, T; Lin, R-Y; Wen, H

2011-12-01

This study aims at relating Toll-like receptors (TLR) and human systemic cytokines in patients with chronic cystic echinococcosis (CE). By real-time fluorescent quantitative reverse-transcription polymerase chain reaction, we measured the expression level of TLR2 and TLR4 mRNA in peripheral blood mononuclear cells (PBMCs), and using ELISA, we detected the cytokines IFN-γ, IL-12p70, IL-10, IL-4 and IL-17A from 34 chronic CE cases (four patients with biliary leakage; four patients with secondary location including three in lung and one in bone) and 22 healthy controls (HC). TLR2 and TLR4 mRNA expression were significantly higher in the CE group (P<0·05); levels of serum IL-10, IL-4 and IL-12p70 in patients with CE were significantly higher than those in controls (P<0·05). There were no differences in IFN-γ and IL-17A levels between the CE group and the HC group (P>0·05). In the patients with CE, positive correlations were noted between the expression of TLR2 and the serum level of IL-10, as well as between the expression of TLR4 and the serum level of IL-10. Our findings supported the hypothesis that during chronic CE infection, altered TLR expression might be involved in the cytokine modulation, which allowed the parasite to escape host immunosurveillance and promoted chronic infection. © 2011 Blackwell Publishing Ltd.

Elevated muscle TLR4 expression and metabolic endotoxemia in human aging.

PubMed

Ghosh, Sangeeta; Lertwattanarak, Raweewan; Garduño, Jose de Jesus; Galeana, Joaquin Joya; Li, Jinqi; Zamarripa, Frank; Lancaster, Jack L; Mohan, Sumathy; Hussey, Sophie; Musi, Nicolas

2015-02-01

Aging is associated with alterations in glucose metabolism and sarcopenia that jointly contribute to a higher risk of developing type 2 diabetes. Because aging is considered as a state of low-grade inflammation, in this study we examined whether older, healthy (lean, community-dwelling) participants have altered signaling flux through toll-like receptor 4 (TLR4), a key mediator of innate and adaptive immune responses. We also examined whether a 4-month aerobic exercise program would have an anti-inflammatory effect by reducing TLR4 expression and signaling. At baseline, muscle TLR4, nuclear factor κB p50 and nuclear factor κB p65 protein content, and c-Jun N-terminal kinase phosphorylation were significantly elevated in older versus young participants. The plasma concentration of the TLR4 agonist lipopolysaccharide and its binding protein also were significantly elevated in older participants, indicative of metabolic endotoxemia, which is a recently described phenomenon of increased plasma endotoxin level in metabolic disease. These alterations in older participants were accompanied by decreased insulin sensitivity, quadriceps muscle volume, and muscle strength. The exercise training program increased insulin sensitivity, without affecting quadriceps muscle volume or strength. Muscle TLR4, nuclear factor κB, and c-Jun N-terminal kinase, and plasma lipopolysaccharide and lipopolysaccharide binding protein were not changed by exercise. In conclusion, insulin resistance and sarcopenia of aging are associated with increased TLR4 expression/signaling, which may be secondary to metabolic endotoxemia. © The Author 2014. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Constitutive activation of epithelial TLR4 augments inflammatory responses to mucosal injury and drives colitis-associated tumorigenesis

PubMed Central

Fukata, Masayuki; Shang, Limin; Santaolalla, Rebeca; Sotolongo, John; Pastorini, Cristhine; España, Cecilia; Ungaro, Ryan; Harpaz, Noam; Cooper, Harry S.; Elson, Greg; Kosco-Vilbois, Marie; Zaias, Julia; Perez, Maria T.; Mayer, Lloyd; Vamadevan, Arunan S.; Lira, Sergio A.; Abreu, Maria T.

2010-01-01

Chronic intestinal inflammation culminates in cancer and a link to TLR4 has been suggested by our observation that TLR4 deficiency prevents colitis-associated neoplasia. In the current study, we address the effect of the aberrant activation of epithelial TLR4 on induction of colitis and colitis-associated tumor development. We take a translational approach to address the consequences of increased TLR signaling in the intestinal mucosa. Mice transgenic for a constitutively-active TLR4 under the intestine-specific villin promoter (villin-TLR4 mice) were treated with DSS for acute colitis and azoxymethane-dextran sulfate sodium. TLR4 expression was analyzed by immunohistochemistry in colonic tissue from patients with ulcerative colitis and ulcerative colitis associated cancer. The effect of an antagonist TLR4 Ab was tested in prevention of colitis-associated neoplasia in the AOM-DSS model. Villin-TLR4 mice were highly susceptible to both acute colitis and colitis-associated neoplasia. Villin-TLR4 mice had increased epithelial expression of COX-2 and mucosal PGE2 production at baseline. Increased severity of colitis in villin-TLR4 mice was characterized by enhanced expression of inflammatory mediators and increased neutrophilic infiltration. In human UC samples, TLR4 expression was upregulated in almost all CAC and progressively increases with grade of dysplasia. As a proof of principle, a TLR4/MD-2 antagonist antibody inhibited colitis-associated neoplasia in the mouse model. Our results show that regulation of TLR's can affect the outcome of both acute colitis and its consequences—cancer. Targeting TLR4 and other TLR's may ultimately play a role in prevention or treatment of colitis-associated cancer. PMID:21674704

Involvement of S100A8/A9-TLR4-NLRP3 Inflammasome Pathway in Contrast-Induced Acute Kidney Injury.

PubMed

Tan, Xuexian; Zheng, Xiaohe; Huang, Zena; Lin, Jiaqiong; Xie, Chuli; Lin, Yan

2017-01-01

Contrast-induced acute kidney injury (CIAKI) is a common cause of hospital-acquired acute kidney injury (AKI). S100A8/A9-TLR4-NLRP3 inflammasome pathway triggers inflammation, apoptosis and tissue injury in several AKI models. Nevertheless, the underlying mechanism of S100A8/A9-TLR4-NLRP3 inflammasome pathway in CIKAI is not clear. We aimed to investigate the possible role of S100A8/A9-TLR4-NLRP3 inflammasome in the pathophysiology of CIAKI. We treated male rats and NRK-52E cells by iopromide to establish in vivo and in vitro models of CIAKI. We collected serum and urine samples to detect renal function. We obtained kidney tissue for histological analysis and detection of protein concentration. We used inhibitor of TLR4 and NLRP3-siRNA to further testify their role in CIAKI in NRK-52E cells. Iopromide caused elevation of SCr, BUN and NGAL level, decrease of endogenous creatinine clearance, morphological injury and tubular apoptosis, enhanced IL-1β and IL-18 expression, and increased expression of S100A8/A9, TLR4 and NLRP3 inflammsome. In NRK-52E cells, iopromide caused enhanced apoptotic rates and ROS generation, which could be ameliorated by inhibitor of TLR4 and NLRP3-siRNA. Moreover, inhibition of TLR4 dampened NLRP3 expression. S100A8/A9-TLR4-NLRP3 inflammasome pathway represented a key mechanism of CI-AKI, which provided a potential therapeutic target. © 2017 The Author(s). Published by S. Karger AG, Basel.

TLR4 Methylation Moderates the Relationship Between Alcohol Use Severity and Gray Matter Loss.

PubMed

Karoly, Hollis C; Thayer, Rachel E; Hagerty, Sarah L; Hutchison, Kent E

2017-09-01

Alcohol use disorders (AUDs) are associated with decreased gray matter, and neuroinflammation is one mechanism through which alcohol may confer such damage, given that heavy alcohol use may promote neural damage via activation of toll-like receptor 4 (TLR4)-mediated inflammatory signaling cascades. We previously demonstrated that TLR4 is differentially methylated in AUD compared with control subjects, and the present study aims to extend this work by examining whether TLR4 methylation moderates the relationship between alcohol use and gray matter. We examined TLR4 methylation and gray matter thickness in a large sample (N = 707; 441 males) of adults (ages 18-56) reporting a range of AUD severity (mean Alcohol Use Disorders Identification Test score = 13.18; SD = 8.02). We used a series of ordinary least squares multiple regression equations to regress gray matter in four bilateral brain regions (precuneus, lateral orbitofrontal, inferior parietal, and superior temporal) on alcohol use, TLR4 methylation, and their interaction, controlling for demographic, psychological, and other substance use variables. After we corrected for multiple tests, a significant Alcohol × TLR4 Methylation interaction emerged in the equations modeling left precuneus and right inferior parietal gray matter. Follow-up analyses examining the nature of these interactions demonstrated a significant negative association between alcohol and precuneus and inferior parietal gray matter in individuals with low TLR4 methylation, but no relationship between alcohol and gray matter in the high methylation group. These findings suggest that TLR4 methylation may be protective against the damage conferred by alcohol on precuneus and inferior parietal gray matter, thereby implicating TLR4 for further investigation as a possible AUD treatment target.

Loss of TLR2 Worsens Spontaneous Colitis in MDR1A Deficiency through Commensally Induced Pyroptosis

PubMed Central

Ey, Birgit; Eyking, Annette; Klepak, Magdalena; Salzman, Nita H.; Göthert, Joachim R.; Rünzi, Michael; Schmid, Kurt W.; Gerken, Guido; Podolsky, Daniel K.

2013-01-01

Variants of the multidrug resistance gene (MDR1/ABCB1) have been associated with increased susceptibility to severe ulcerative colitis (UC). In this study, we investigated the role of TLR/IL-1R signaling pathways including the common adaptor MyD88 in the pathogenesis of chronic colonic inflammation in MDR1A deficiency. Double- or triple-null mice lacking TLR2, MD-2, MyD88, and MDR1A were generated in the FVB/N background. Deletion of TLR2 in MDR1A deficiency resulted in fulminant pancolitis with early expansion of CD11b+ myeloid cells and rapid shift toward TH1-dominant immune responses in the lamina propria. Colitis exacerbation in TLR2/MDR1A double-knockout mice required the unaltered commensal microbiota and the LPS coreceptor MD-2. Blockade of IL-1β activity by treatment with IL-1R antagonist (IL-1Ra; Anakinra) inhibited colitis acceleration in TLR2/MDR1A double deficiency; intestinal CD11b+Ly6C+-derived IL-1β production and inflammation entirely depended on MyD88. TLR2/MDR1A double-knockout CD11b+ myeloid cells expressed MD-2/TLR4 and hyperresponded to nonpathogenic Escherichia coli or LPS with reactive oxygen species production and caspase-1 activation, leading to excessive cell death and release of proinflammatory IL-1β, consistent with pyroptosis. Inhibition of reactive oxygen species–mediated lysosome degradation suppressed LPS hyperresponsiveness. Finally, active UC in patients carrying the TLR2-R753Q and MDR1-C3435T polymorphisms was associated with increased nuclear expression of caspase-1 protein and cell death in areas of acute inflammation, compared with active UC patients without these variants. In conclusion, we show that the combined defect of two UC susceptibility genes, MDR1A and TLR2, sets the stage for spontaneous and uncontrolled colitis progression through MD-2 and IL-1R signaling via MyD88, and we identify commensally induced pyroptosis as a potential innate immune effector in severe UC pathogenesis. PMID:23636052

Loss of TLR2 worsens spontaneous colitis in MDR1A deficiency through commensally induced pyroptosis.

PubMed

Ey, Birgit; Eyking, Annette; Klepak, Magdalena; Salzman, Nita H; Göthert, Joachim R; Rünzi, Michael; Schmid, Kurt W; Gerken, Guido; Podolsky, Daniel K; Cario, Elke

2013-06-01

Variants of the multidrug resistance gene (MDR1/ABCB1) have been associated with increased susceptibility to severe ulcerative colitis (UC). In this study, we investigated the role of TLR/IL-1R signaling pathways including the common adaptor MyD88 in the pathogenesis of chronic colonic inflammation in MDR1A deficiency. Double- or triple-null mice lacking TLR2, MD-2, MyD88, and MDR1A were generated in the FVB/N background. Deletion of TLR2 in MDR1A deficiency resulted in fulminant pancolitis with early expansion of CD11b(+) myeloid cells and rapid shift toward TH1-dominant immune responses in the lamina propria. Colitis exacerbation in TLR2/MDR1A double-knockout mice required the unaltered commensal microbiota and the LPS coreceptor MD-2. Blockade of IL-1β activity by treatment with IL-1R antagonist (IL-1Ra; Anakinra) inhibited colitis acceleration in TLR2/MDR1A double deficiency; intestinal CD11b(+)Ly6C(+)-derived IL-1β production and inflammation entirely depended on MyD88. TLR2/MDR1A double-knockout CD11b(+) myeloid cells expressed MD-2/TLR4 and hyperresponded to nonpathogenic Escherichia coli or LPS with reactive oxygen species production and caspase-1 activation, leading to excessive cell death and release of proinflammatory IL-1β, consistent with pyroptosis. Inhibition of reactive oxygen species-mediated lysosome degradation suppressed LPS hyperresponsiveness. Finally, active UC in patients carrying the TLR2-R753Q and MDR1-C3435T polymorphisms was associated with increased nuclear expression of caspase-1 protein and cell death in areas of acute inflammation, compared with active UC patients without these variants. In conclusion, we show that the combined defect of two UC susceptibility genes, MDR1A and TLR2, sets the stage for spontaneous and uncontrolled colitis progression through MD-2 and IL-1R signaling via MyD88, and we identify commensally induced pyroptosis as a potential innate immune effector in severe UC pathogenesis.

Differential Trafficking of TLR1 I602S Underlies Host Protection Against Pathogenic Mycobacteria§

PubMed Central

Hart, Bryan E.; Tapping, Richard I.

2012-01-01

We have recently identified I602S as a frequent single nucleotide polymorphism of human TLR1 which greatly inhibits cell surface trafficking, confers hyporesponsiveness to TLR1 agonists, and protects against the mycobacterial diseases leprosy and tuberculosis. Since mycobacteria are known to manipulate the TLR system to their advantage, we hypothesize that the hyporesponsive 602S variant may confer protection by enabling the host to overcome this immune subversion. We report that primary human monocytes and macrophages from homozygous TLR1 602S individuals are resistant to mycobacterial-induced downregulation of macrophage MHCII, CD64, and IFNγ responses compared to individuals who harbor the TLR1 602I variant. Additionally, when challenged with mycobacterial agonists, macrophages from TLR1 602S/S individuals resist induction of host arginase-1; an enzyme that depletes cellular arginine stores required for production of antimicrobial reactive nitrogen intermediates. The differences in cell activation mediated by TLR1 602S and TLR1 602I are observed upon stimulation with soluble mycobacterial-derived agonists but not with whole mycobacterial cells. Taken together, these results suggest that the TLR1 602S variant protects against mycobacterial disease by preventing soluble mycobacterial products, perhaps released from granulomas, from disarming myeloid cells prior to their encounter with whole mycobacteria. PMID:23105135

MyD88 and TLR4 Expression in Epithelial Ovarian Cancer

PubMed Central

Block, Matthew S.; Vierkant, Robert A.; Rambau, Peter F.; Winham, Stacey J.; Wagner, Philipp; Traficante, Nadia; Tołoczko, Aleksandra; Tiezzi, Daniel G.; Taran, Florin Andrei; Sinn, Peter; Sieh, Weiva; Sharma, Raghwa; Rothstein, Joseph H.; Cajal, Teresa Ramón y; Paz-Ares, Luis; Oszurek, Oleg; Orsulic, Sandra; Ness, Roberta B.; Nelson, Gregg; Modugno, Francesmary; Menkiszak, Janusz; McGuire, Valerie; McCauley, Bryan M.; Mack, Marie; Lubiński, Jan; Longacre, Teri A.; Li, Zheng; Lester, Jenny; Kennedy, Catherine J.; Kalli, Kimberly R.; Jung, Audrey Y.; Johnatty, Sharon E.; Jimenez-Linan, Mercedes; Jensen, Allan; Intermaggio, Maria P.; Hung, Jillian; Herpel, Esther; Hernandez, Brenda Y.; Hartkopf, Andreas D.; Harnett, Paul R.; Ghatage, Prafull; García-Bueno, José M.; Gao, Bo; Fereday, Sian; Eilber, Ursula; Edwards, Robert P.; de Sousa, Christiani B.; de Andrade, Jurandyr M.; Chudecka-Głaz, Anita; Chenevix-Trench, Georgia; Cazorla, Alicia; Brucker, Sara Y.; Alsop, Jennifer; Whittemore, Alice S.; Steed, Helen; Staebler, Annette; Moysich, Kirsten B.; Menon, Usha; Koziak, Jennifer M.; Kommoss, Stefan; Kjaer, Susanne K.; Kelemen, Linda E.; Karlan, Beth Y.; Huntsman, David G.; Høgdall, Estrid; Gronwald, Jacek; Goodman, Marc T.; Gilks, Blake; García, María José; Fasching, Peter A.; de Fazio, Anna; Deen, Suha; Chang-Claude, Jenny; Candido dos Reis, Francisco J.; Campbell, Ian G.; Brenton, James D.; Bowtell, David D.; Benítez, Javier; Pharoah, Paul D.P.; Köbel, Martin; Ramus, Susan J.; Goode, Ellen L.

2018-01-01

Objective To evaluate myeloid differentiation primary response gene 88 (MyD88) and Toll-like receptor 4 (TLR4) expression in relation to clinical features of epithelial ovarian cancer, histologic subtypes, and overall survival. Patients and Methods We conducted centralized immunohistochemical staining, semi-quantitative scoring, and survival analysis in 5263 patients participating in the Ovarian Tumor Tissue Analysis consortium. Patients were diagnosed between January 1, 1978, and December 31, 2014, including 2865 high-grade serous ovarian carcinomas (HGSOCs), with more than 12,000 person-years of follow-up time. Tissue microarrays were stained for MyD88 and TLR4, and staining intensity was classified using a 2-tiered system for each marker (weak vs strong). Results Expression of MyD88 and TLR4 was similar in all histotypes except clear cell ovarian cancer, which showed reduced expression compared with other histotypes (P<.001 for both). In HGSOC, strong MyD88 expression was modestly associated with shortened overall survival (hazard ratio [HR], 1.13; 95% CI, 1.01–1.26; P=.04) but was also associated with advanced stage (P<.001). The expression of TLR4 was not associated with survival. In low-grade serous ovarian cancer (LGSOC), strong expression of both MyD88 and TLR4 was associated with favorable survival (HR [95% CI], 0.49 [0.29–0.84] and 0.44 [0.21–0.89], respectively; P=.009 and P=.02, respectively). Conclusion Results are consistent with an association between strong MyD88 staining and advanced stage and poorer survival in HGSOC and demonstrate correlation between strong MyD88 and TLR4 staining and improved survival in LGSOC, highlighting the biological differences between the 2 serous histotypes. PMID:29502561

MyD88 and TLR4 Expression in Epithelial Ovarian Cancer.

PubMed

Block, Matthew S; Vierkant, Robert A; Rambau, Peter F; Winham, Stacey J; Wagner, Philipp; Traficante, Nadia; Tołoczko, Aleksandra; Tiezzi, Daniel G; Taran, Florin Andrei; Sinn, Peter; Sieh, Weiva; Sharma, Raghwa; Rothstein, Joseph H; Ramón Y Cajal, Teresa; Paz-Ares, Luis; Oszurek, Oleg; Orsulic, Sandra; Ness, Roberta B; Nelson, Gregg; Modugno, Francesmary; Menkiszak, Janusz; McGuire, Valerie; McCauley, Bryan M; Mack, Marie; Lubiński, Jan; Longacre, Teri A; Li, Zheng; Lester, Jenny; Kennedy, Catherine J; Kalli, Kimberly R; Jung, Audrey Y; Johnatty, Sharon E; Jimenez-Linan, Mercedes; Jensen, Allan; Intermaggio, Maria P; Hung, Jillian; Herpel, Esther; Hernandez, Brenda Y; Hartkopf, Andreas D; Harnett, Paul R; Ghatage, Prafull; García-Bueno, José M; Gao, Bo; Fereday, Sian; Eilber, Ursula; Edwards, Robert P; de Sousa, Christiani B; de Andrade, Jurandyr M; Chudecka-Głaz, Anita; Chenevix-Trench, Georgia; Cazorla, Alicia; Brucker, Sara Y; Alsop, Jennifer; Whittemore, Alice S; Steed, Helen; Staebler, Annette; Moysich, Kirsten B; Menon, Usha; Koziak, Jennifer M; Kommoss, Stefan; Kjaer, Susanne K; Kelemen, Linda E; Karlan, Beth Y; Huntsman, David G; Høgdall, Estrid; Gronwald, Jacek; Goodman, Marc T; Gilks, Blake; García, María José; Fasching, Peter A; de Fazio, Anna; Deen, Suha; Chang-Claude, Jenny; Candido Dos Reis, Francisco J; Campbell, Ian G; Brenton, James D; Bowtell, David D; Benítez, Javier; Pharoah, Paul D P; Köbel, Martin; Ramus, Susan J; Goode, Ellen L

2018-03-01

To evaluate myeloid differentiation primary response gene 88 (MyD88) and Toll-like receptor 4 (TLR4) expression in relation to clinical features of epithelial ovarian cancer, histologic subtypes, and overall survival. We conducted centralized immunohistochemical staining, semi-quantitative scoring, and survival analysis in 5263 patients participating in the Ovarian Tumor Tissue Analysis consortium. Patients were diagnosed between January 1, 1978, and December 31, 2014, including 2865 high-grade serous ovarian carcinomas (HGSOCs), with more than 12,000 person-years of follow-up time. Tissue microarrays were stained for MyD88 and TLR4, and staining intensity was classified using a 2-tiered system for each marker (weak vs strong). Expression of MyD88 and TLR4 was similar in all histotypes except clear cell ovarian cancer, which showed reduced expression compared with other histotypes (P<.001 for both). In HGSOC, strong MyD88 expression was modestly associated with shortened overall survival (hazard ratio [HR], 1.13; 95% CI, 1.01-1.26; P=.04) but was also associated with advanced stage (P<.001). The expression of TLR4 was not associated with survival. In low-grade serous ovarian cancer (LGSOC), strong expression of both MyD88 and TLR4 was associated with favorable survival (HR [95% CI], 0.49 [0.29-0.84] and 0.44 [0.21-0.89], respectively; P=.009 and P=.02, respectively). Results are consistent with an association between strong MyD88 staining and advanced stage and poorer survival in HGSOC and demonstrate correlation between strong MyD88 and TLR4 staining and improved survival in LGSOC, highlighting the biological differences between the 2 serous histotypes. Copyright © 2017 Mayo Foundation for Medical Education and Research. Published by Elsevier Inc. All rights reserved.

TLR4 has a TP53-dependent dual role in regulating breast cancer cell growth

PubMed Central

Haricharan, Svasti; Brown, Powel

2015-01-01

Breast cancer is a leading cause of cancer-related death, and it is important to understand pathways that drive the disease to devise effective therapeutic strategies. Our results show that Toll-like receptor 4 (TLR4) drives breast cancer cell growth differentially based on the presence of TP53, a tumor suppressor. TP53 is mutationally inactivated in most types of cancer and is mutated in 30–50% of diagnosed breast tumors. We demonstrate that TLR4 activation inhibits growth of TP53 wild-type cells, but promotes growth of TP53 mutant breast cancer cells by regulating proliferation. This differential effect is mediated by changes in tumor cell cytokine secretion. Whereas TLR4 activation in TP53 mutant breast cancer cells increases secretion of progrowth cytokines, TLR4 activation in TP53 wild-type breast cancer cells increases type I IFN (IFN-γ) secretion, which is both necessary and sufficient for mediating TLR4-induced growth inhibition. This study identifies a novel dichotomous role for TLR4 as a growth regulator and a modulator of tumor microenvironment in breast tumors. These results have translational relevance, demonstrating that TP53 mutant breast tumor growth can be suppressed by pharmacologic TLR4 inhibition, whereas TLR4 inhibitors may in fact promote growth of TP53 wild-type tumors. Furthermore, using data generated by The Cancer Genome Atlas consortium, we demonstrate that the effect of TP53 mutational status on TLR4 activity may extend to ovarian, colon, and lung cancers, among others, suggesting that the viability of TLR4 as a therapeutic target depends on TP53 status in many different tumor types. PMID:26063617

TLR4 has a TP53-dependent dual role in regulating breast cancer cell growth.

PubMed

Haricharan, Svasti; Brown, Powel

2015-06-23

Breast cancer is a leading cause of cancer-related death, and it is important to understand pathways that drive the disease to devise effective therapeutic strategies. Our results show that Toll-like receptor 4 (TLR4) drives breast cancer cell growth differentially based on the presence of TP53, a tumor suppressor. TP53 is mutationally inactivated in most types of cancer and is mutated in 30-50% of diagnosed breast tumors. We demonstrate that TLR4 activation inhibits growth of TP53 wild-type cells, but promotes growth of TP53 mutant breast cancer cells by regulating proliferation. This differential effect is mediated by changes in tumor cell cytokine secretion. Whereas TLR4 activation in TP53 mutant breast cancer cells increases secretion of progrowth cytokines, TLR4 activation in TP53 wild-type breast cancer cells increases type I IFN (IFN-γ) secretion, which is both necessary and sufficient for mediating TLR4-induced growth inhibition. This study identifies a novel dichotomous role for TLR4 as a growth regulator and a modulator of tumor microenvironment in breast tumors. These results have translational relevance, demonstrating that TP53 mutant breast tumor growth can be suppressed by pharmacologic TLR4 inhibition, whereas TLR4 inhibitors may in fact promote growth of TP53 wild-type tumors. Furthermore, using data generated by The Cancer Genome Atlas consortium, we demonstrate that the effect of TP53 mutational status on TLR4 activity may extend to ovarian, colon, and lung cancers, among others, suggesting that the viability of TLR4 as a therapeutic target depends on TP53 status in many different tumor types.

Substrate Stiffness Regulates Proinflammatory Mediator Production through TLR4 Activity in Macrophages

PubMed Central

Previtera, Michelle L.; Sengupta, Amitabha

2015-01-01

Clinical data show that disease adversely affects tissue elasticity or stiffness. While macrophage activity plays a critical role in driving disease pathology, there are limited data available on the effects of tissue stiffness on macrophage activity. In this study, the effects of substrate stiffness on inflammatory mediator production by macrophages were investigated. Bone marrow–derived macrophages were grown on polyacrylamide gels that mimicked the stiffness of a variety of soft biological tissues. Overall, macrophages grown on soft substrates produced less proinflammatory mediators than macrophages grown on stiff substrates when the endotoxin LPS was added to media. In addition, the pathways involved in stiffness–regulated proinflammation were investigated. The TLR4 signaling pathway was examined by evaluating TLR4, p–NF–κB p65, MyD88, and p–IκBα expression as well as p–NF–κB p65 translocation. Expression and translocation of the various signaling molecules were higher in macrophages grown on stiff substrates than on soft substrates. Furthermore, TLR4 knockout experiments showed that TLR4 activity enhanced proinflammation on stiff substrates. In conclusion, these results suggest that proinflammatory mediator production initiated by TLR4 is mechanically regulated in macrophages. PMID:26710072

Auranofin, as an anti-rheumatic gold compound suppresses LPS-induced homodimerization of TLR4

PubMed Central

Youn, Hyung S.; Lee, Joo Y.; Saitoh, Shin I.; Miyake, Kensuke; Hwang, Daniel H.

2009-01-01

Toll-like receptors (TLRs), which are activated by invading microorganisms or endogenous molecules, evoke immune and inflammatory responses. TLR activation is closely linked to the development of many chronic inflammatory diseases including rheumatoid arthritis. Auranofin, an Au(I) compound, is a well-known and long-used anti-rheumatic drug. However, the mechanism as to how auranofin relieves the symptom of rheumatoid arthritis has not been fully clarified. Our results demonstrated that auranofin suppressed TLR4-mediated activation of transcription factors, NF-κB and IRF3 and expression of COX-2, a pro-inflammatory enzyme. This suppression was well correlated with the inhibitory effect of auranofin on the homodimerization of TLR4 induced by an agonist. Furthermore, auranofin inhibited NF-κ activation induced by MyD88-dependent downstream signaling components of TLR4, MyD88, IKKβ, and p65. IRF3 activation induced by MyD88-independent signaling components, TRIF and TBK1, was also downregulated by auranofin. Our results first demonstrate that auranofin suppresses the multiple steps in TLR4 signaling, especially the homodimerization of TLR4. The results suggest that the suppression of TLR4 activity by auranofin may be the molecular mechanism through which auranofin exerts anti-rheumatic activity. PMID:17034761

1,25-Dihydroxyvitamin D3 up-regulates TLR10 while down-regulating TLR2, 4, and 5 in human monocyte THP-1.

PubMed

Verma, Rewa; Jung, Jong Hyeok; Kim, Jae Young

2014-05-01

In humans, there are ten Toll-like receptors (TLRs), among which TLR10 is the only orphan receptor whose function is unknown. In this study, we examined the effects of IFN-γ, LPS and 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on TLR10 expression of human monocyte THP-1 and compared them with those of other surface TLRs such as TLR2, 4 and 5 to differentiate TLR10 from other TLRs. Surface TLR10 expression on THP-1 was significantly enhanced by the addition of IFN-γ or LPS in a fashion similar to that of other TLRs. However, TLR10 expression was differentially regulated by 1,25(OH)2D3. Surface TLR10 expression on THP-1 was significantly enhanced at 24h, reaching approximately two times the control level at 48h after treatment with 100nM 1,25(OH)2D3, while that of TLR2, 4 and 5 decreased gradually in response to treatment over time. 1,25(OH)2D3 at concentrations above 1nM markedly enhanced surface TLR10 expression, but concentrations below 1nM did not. TLR10 mRNA expression was also increased by 1,25(OH)2D3. We next screened for putative binding sites of nuclear vitamin D receptor (VDR) and its counterpart RXR-α within promoter of TLR genes using a transcription factor binding site-prediction program. The results revealed that TLR10 is the only receptor among the tested TLRs that has both a VDR and RXR-α binding site within its proximal promoter. To identify possible involvement of VDR/RXR in the 1,25(OH)2D3-induced TLR10 up-regulation, we engaged the VDR synthesis inhibitor, dexamethasone, and the RXR antagonist, 1,8-dihydroxyanthraquinone. We found that TLR10 up-regulation was significantly blocked with pre-treatment of these inhibitors. These findings indicate that surface TLR10 expression is differentially regulated by 1,25(OH)2D3 and mainly regulated at the transcriptional level via VDR/RXR-α. Overall, results presented herein suggest that TLR10 functions differently from other known surface TLRs under certain circumstances. Further study using primary cells is

Role of innate immune receptors TLR2 and TLR4 as mediators of the inflammatory reaction in human visceral adipose tissue.

PubMed

Fusaru, Ana Marina; Stănciulescu, Camelia Elena; Surlin, V; Taisescu, C; Bold, Adriana; Pop, O T; Baniţă, Ileana Monica; Crăiţoiu, Stefania; Pisoschi, Cătălina Gabriela

2012-01-01

White adipose tissue from different locations is characterized by significant differences in the structure of adipocyte "secretoma". Fat accumulation in the central-visceral depots is usually associated with a chronic inflammatory state, which is complicated by the metabolic syndrome. Recently, the adipose tissue was emerged to have an essential role in the innate immunity, adipocytes being considered effector cells due to the presence of the Toll-like receptors (TLRs). In this study, we compared the expression of TNF-α, TLR2 and TLR4 in peripheral-subcutaneous and central-peritoneal adipose depots in three different conditions - lean, obese and obese diabetic - using immunohistochemistry. Our results suggest a correlation between the incidence of the stromal vascular cells and adipocytes TNF-α and TLR4 in the visceral depots in strong correlation with adipose tissue expansion. TLR2 positive cells were seen in the peripheral depots from all groups without any association with fat accumulation. These results focus on the existence of a new pathogenic pathway, the activation of TLR4, for the involvement of visceral adipose tissue in the activation and maintenance of the inflammatory cascade in obesity.

Increased A20-E3 ubiquitin ligase interactions in bid-deficient glia attenuate TLR3- and TLR4-induced inflammation.

PubMed

Kinsella, Sinéad; Fichtner, Michael; Watters, Orla; König, Hans-Georg; Prehn, Jochen H M

2018-05-02

Chronic pro-inflammatory signaling propagates damage to neural tissue and affects the rate of disease progression. Increased activation of Toll-like receptors (TLRs), master regulators of the innate immune response, is implicated in the etiology of several neuropathologies including amyotrophic lateral sclerosis, Alzheimer's disease, and Parkinson's disease. Previously, we identified that the Bcl-2 family protein BH3-interacting domain death agonist (Bid) potentiates the TLR4-NF-κB pro-inflammatory response in glia, and specifically characterized an interaction between Bid and TNF receptor associated factor 6 (TRAF6) in microglia in response to TLR4 activation. We assessed the activation of mitogen-activated protein kinase (MAPK) and interferon regulatory factor 3 (IRF3) inflammatory pathways in response to TLR3 and TLR4 agonists in wild-type (wt) and bid-deficient microglia and macrophages, using Western blot and qPCR, focusing on the response of the E3 ubiquitin ligases Pellino 1 (Peli1) and TRAF3 in the absence of microglial and astrocytic Bid. Additionally, by Western blot, we investigated the Bid-dependent turnover of Peli1 and TRAF3 in wt and bid -/- microglia using the proteasome inhibitor Bortezomib. Interactions between the de-ubiquitinating Smad6-A20 and the E3 ubiquitin ligases, TRAF3 and TRAF6, were determined by FLAG pull-down in TRAF6-FLAG or Smad6-FLAG overexpressing wt and bid-deficient mixed glia. We elucidated a positive role of Bid in both TIR-domain-containing adapter-inducing interferon-β (TRIF)- and myeloid differentiation primary response 88 (MyD88)-dependent pathways downstream of TLR4, concurrently implicating TLR3-induced inflammation. We identified that Peli1 mRNA levels were significantly reduced in PolyI:C- and lipopolysaccharide (LPS)-stimulated bid-deficient microglia, suggesting disturbed IRF3 activation. Differential regulation of TRAF3 and Peli1, both essential E3 ubiquitin ligases facilitating TRIF-dependent signaling, was

Recognition of LPS by TLR4: Potential for Anti-Inflammatory Therapies

PubMed Central

Nijland, Reindert; Hofland, Tom; van Strijp, Jos A. G.

2014-01-01

LPS molecules of marine bacteria show structures distinct from terrestrial bacteria, due to the different environment that marine bacteria live in. Because of these different structures, lipid A molecules from marine bacteria are most often poor stimulators of the Toll-like receptor 4 (TLR4) pathway. Due to their low stimulatory potential, these lipid A molecules are suggested to be applicable as antagonists of TLR4 signaling in sepsis patients, where this immune response is amplified and unregulated. Antagonizing lipid A molecules might be used for future therapies against sepsis, therapies that currently do not exist. In this review, we will discuss these differences in lipid A structures and their recognition by the immune system. The modifications present in marine lipid A structures are described, and their potential as LPS antagonists will be discussed. Finally, since clinical trials built on antagonizing lipid A molecules have proven unsuccessful, we propose to also focus on different aspects of the TLR4 signaling pathway when searching for new potential drugs. Furthermore, we put forward the notion that bacteria probably already produce inhibitors of TLR4 signaling, making these bacterial products interesting molecules to investigate for future sepsis therapies. PMID:25056632

Evidence for ProTα-TLR4/MD-2 binding: molecular dynamics and gravimetric assay studies.

PubMed

Omotuyi, Olaposi; Matsunaga, Hayato; Ueda, Hiroshi

2015-01-01

During preconditioning, lipopolysaccharide (LPS) selectively activates TLR4/MD-2/Toll/IL-1 receptor-domain-containing adaptor inducing IFN-β (TRIF) pathway instead of pro-inflammatory myeloid differentiation protein-88 (MyD88)/MyD88-adaptor-like protein (MAL) pathway. Extracellular prothymosin alpha (ProTα) is also known to selectively activate the TLR4/MD2/TRIF-IRF3 pathway in certain diseased conditions. In the current study, biophysical evidence for ProTα/TLR4/MD-2 complex formation and its interaction dynamics have been studied. Gravimetric assay was used to investigate ProTα/TLR4/MD-2 complex formation while molecular dynamics (MD) simulation was used to study its interaction dynamics. Through electrostatic interaction, full-length ProTα (F-ProTα) C-terminal peptide (aa 91 - 111) superficially interacts with similar TLR4/MD-2 (KD = 273.36 nm vs 16.07 μg/ml [LPS]) conformation with LPS at an overlapping three-dimensional space while F-ProTα is hinged to the TLR4 scaffold by one-amino acid shift-Mosoian domain (aa-51 - 90). Comparatively, F-ProTα better stabilizes MD-2 metastable states transition and mediates higher TLR4/MD-2 interaction than LPS. ProTα via its C-terminal peptide (aa 91 - 111) exhibits in vitro biophysical contact with TLR4/MD-2 complex conformation recognized by LPS at overlapping LPS-binding positions.

Vitamin K2 can suppress the expression of Toll-like receptor 2 (TLR2) and TLR4, and inhibit calcification of aortic intima in ApoE-/- mice as well as smooth muscle cells.

PubMed

Wang, Zhaojun; Wang, Zhongqun; Zhu, Jie; Long, Xinguang; Yan, Jinchuan

2018-02-01

Background and objectives Vascular calcification is a common complication in atherosclerosis. Accumulating evidence showed that Toll-like receptors (TLRs) mediate pro-inflammatory and atherosclerosis. Recent studies demonstrated that vascular calcification is one of the detrimental effects of vitamin K (Vit K) antagonists. However, the effects of Vit K on the expression of TLR2 and 4 and intimal calcification in artery remained unidentified. Methods and results Eighteen ApoE -/- mice were randomly divided into model group, Vit K-treated group, and control group. The mice of model and Vit K-treated group were fed with high-fat diet, while control group mice were fed with normal diet. Mice of Vit K-treated group were administered orally with vitamin K2 (40 mg.kg -1 .day -1 ) for 12 weeks. Twelve weeks later the aortic sections of mice were acquired and stained with hematoxylin and eosin and von Kossa, respectively. Calcium content and activity of alkaline phosphatase (ALP) at aortic tissues were measured. The expression levels of TLR2 and TLR4 in aorta sections were detected by immunohistochemisty and RT-PCR, respectively. The effects of Vit K on cellular calcification were further studied in A7r5 SMCs. Results demonstrated that high-fat diet induced typical atherosclerosis with intimal calcification in ApoE -/- mice, while in Vit K-treated group atherosclerosis and calcium deposits were not serious; Vit K2 also inhibited cellular calcification in A7r5 SMCs. Quantitative analysis showed that calcium and ALP activity at aortic tissues in the Vit K-treated mice were significantly lower than that of the model group ( P < 0.01); Compared to the control group, the expression levels of TLR2 and TLR4 in the model group were significantly higher ( P < 0.05), while in Vit K-treated group the levels of TLR2 and 4 were significantly lower than that in the model group. Furthermore, the content of calcium was positively related to the expression levels of TLR2 and TLR4

Parasite Specific Antibody Levels, Interferon-γ and TLR2 and TLR4 Transcripts in Blood from Dogs with Different Clinical Stages of Leishmaniosis

PubMed Central

Montserrat-Sangrà, Sara; Ordeix, Laura; Martínez-Orellana, Pamela; Solano-Gallego, Laia

2018-01-01

Canine leishmaniosis has a wide range of disease severity from mild (stage I), to severe (stages II–III), or very severe disease (stage IV). The objective of the study was to evaluate and compare serum antibody levels, Leishmania infantum specific IFN-γ production and TLR2 and TLR4 transcripts in non-stimulated blood from dogs with different clinical stages at the time of diagnosis as well as blood parasitemia. Enzyme-Linked ImmunoSorbent Assay (ELISAs) were performed to determine serum antibody levels and IFN-γ production and quantitative polymerase chain reaction (qPCRs) in order to determine blood parasite load and TLR2 and TLR4 transcripts. Older dogs were significantly affected by more severe disease with higher antibody levels and blood parasitemia than dogs with mild disease. IFN-γ production was significantly higher in dogs with stage I disease when compared to dogs with more severe disease. Relative quantification of TLR2 in dogs with mild disease was similar to that of control dogs. On the other hand, TLR2 transcripts were significantly higher in dogs with severe disease as compared with that from control healthy dogs. No differences were found in TLR4 relative quantification between groups. This study demonstrates that dogs with different clinical stages of leishmaniosis present different levels of biological markers indicative of different immune responses. PMID:29547503

Allelic Variation in TLR4 Is Linked to Susceptibility to Salmonella enterica Serovar Typhimurium Infection in Chickens

PubMed Central

Leveque, Gary; Forgetta, Vincenzo; Morroll, Shaun; Smith, Adrian L.; Bumstead, Nat; Barrow, Paul; Loredo-Osti, J. C.; Morgan, Kenneth; Malo, Danielle

2003-01-01

Toll-like receptor 4 (TLR4) is part of a group of evolutionarily conserved pattern recognition receptors involved in the activation of the immune system in response to various pathogens and in the innate defense against infection. We describe here the cloning and characterization of the avian orthologue of mammalian TLR4. Chicken TLR4 encodes a 843-amino-acid protein that contains a leucine-rich repeat extracellular domain, a short transmembrane domain typical of type I transmembrane proteins, and a Toll-interleukin-1R signaling domain characteristic of all TLR proteins. The chicken TLR4 protein shows 46% identity (64% similarity) to human TLR4 and 41% similarity to other TLR family members. Northern blot analysis reveals that TLR4 is expressed at approximately the same level in all tissues tested, including brain, thymus, kidney, intestine, muscle, liver, lung, bursa of Fabricius, heart, and spleen. The probe detected only one transcript of ca. 4.4 kb in length for all tissues except muscle where the size of TLR4 mRNA was ca. 9.6 kb. We have mapped TLR4 to microchromosome E41W17 in a region harboring the gene for tenascin C and known to be well conserved between the chicken and mammalian genomes. This region of the chicken genome was shown previously to harbor a Salmonella susceptibility locus. By using linkage analysis, TLR4 was shown to be linked to resistance to infection with Salmonella enterica serovar Typhimurium in chickens (likelihood ratio test of 10.2, P = 0.00138), suggesting a role of TLR4 in the host response of chickens to Salmonella infection. PMID:12595422

Polymorphisms in the Inflammatory Pathway Genes TLR2, TLR4, TLR9, LY96, NFKBIA, NFKB1, TNFA, TNFRSF1A, IL6R, IL10, IL23R, PTPN22, and PPARG Are Associated with Susceptibility of Inflammatory Bowel Disease in a Danish Cohort

PubMed Central

Bank, Steffen; Skytt Andersen, Paal; Burisch, Johan; Pedersen, Natalia; Roug, Stine; Galsgaard, Julie; Ydegaard Turino, Stine; Broder Brodersen, Jacob; Rashid, Shaista; Kaiser Rasmussen, Britt; Avlund, Sara; Bastholm Olesen, Thomas; Jürgen Hoffmann, Hans; Kragh Thomsen, Marianne; Østergaard Thomsen, Vibeke; Frydenberg, Morten; Andersen Nexø, Bjørn; Sode, Jacob; Vogel, Ulla; Andersen, Vibeke

2014-01-01

Background The inflammatory bowel diseases (IBD), Crohn's disease (CD) and ulcerative colitis (UC), result from the combined effects of susceptibility genes and environmental factors. Polymorphisms in genes regulating inflammation may explain part of the genetic heritage. Methods Using a candidate gene approach, 39 mainly functional single nucleotide polymorphisms (SNPs) in 26 genes regulating inflammation were assessed in a clinical homogeneous group of severely diseased patients consisting of 624 patients with CD, 411 patients with UC and 795 controls. The results were analysed using logistic regression. Results Sixteen polymorphisms in 13 genes involved in regulation of inflammation were associated with risk of CD and/or UC (p≤0.05). The polymorphisms TLR2 (rs1816702), NFKB1 (rs28362491), TNFRSF1A (rs4149570), IL6R (rs4537545), IL23R (rs11209026) and PTPN22 (rs2476601) were associated with risk of CD and the polymorphisms TLR2 (rs1816702), TLR4 (rs1554973 and rs12377632), TLR9 (rs352139), LY96 (rs11465996), NFKBIA (rs696), TNFA (rs1800629), TNFRSF1A (rs4149570), IL10 (rs3024505), IL23R (rs11209026), PTPN22 (rs2476601) and PPARG (rs1801282) were associated with risk of UC. When including all patients (IBD) the polymorphisms TLR2 (rs4696480 and rs1816702), TLR4 (rs1554973 and rs12377632), TLR9 (rs187084), TNFRSF1A (rs4149570), IL6R (rs4537545), IL10 (rs3024505), IL23R (rs11209026) and PTPN22 (rs2476601) were associated with risk. After Bonferroni correction for multiple testing, both the homozygous and the heterozygous variant genotypes of IL23R G>A(rs11209026) (ORCD,adj: 0.38, 95% CI: 0.21–0.67, p = 0.03; ORIBD,adj 0.43, 95% CI: 0.28–0.67, p = 0.007) and PTPN22 1858 G>A(rs2476601) (ORCD,unadj 0.54, 95% CI: 0.41–0.72, p = 7*10−4; ORIBD,unadj: 0.61, 95% CI: 0.48–0.77, p = 0.001) were associated with reduced risk of CD. Conclusion The biological effects of the studied polymorphisms suggest that genetically determined high inflammatory

Cathelicidins Inhibit Escherichia coli–Induced TLR2 and TLR4 Activation in a Viability-Dependent Manner

PubMed Central

Coorens, Maarten; Schneider, Viktoria A. F.; Meijerink, Marjolein; Wells, Jerry M.; Scheenstra, Maaike R.

2017-01-01

Activation of the immune system needs to be tightly regulated to provide protection against infections and, at the same time, to prevent excessive inflammation to limit collateral damage to the host. This tight regulation includes regulating the activation of TLRs, which are key players in the recognition of invading microbes. A group of short cationic antimicrobial peptides, called cathelicidins, have previously been shown to modulate TLR activation by synthetic or purified TLR ligands and may play an important role in the regulation of inflammation during infections. However, little is known about how these cathelicidins affect TLR activation in the context of complete and viable bacteria. In this article, we show that chicken cathelicidin-2 kills Escherichia coli in an immunogenically silent fashion. Our results show that chicken cathelicidin-2 kills E. coli by permeabilizing the bacterial inner membrane and subsequently binds the outer membrane–derived lipoproteins and LPS to inhibit TLR2 and TLR4 activation, respectively. In addition, other cathelicidins, including human, mouse, pig, and dog cathelicidins, which lack antimicrobial activity under cell culture conditions, only inhibit macrophage activation by nonviable E. coli. In total, this study shows that cathelicidins do not affect immune activation by viable bacteria and only inhibit inflammation when bacterial viability is lost. Therefore, cathelicidins provide a novel mechanism by which the immune system can discriminate between viable and nonviable Gram-negative bacteria to tune the immune response, thereby limiting collateral damage to the host and the risk for sepsis. PMID:28710255

Cotransfection of DC with TLR4 and MART-1 RNA induces MART-1-specific responses.

PubMed

Abdel-Wahab, Zeinab; Cisco, Robin; Dannull, Jens; Ueno, Tomio; Abdel-Wahab, Omar; Kalady, Matthew F; Onaitis, Mark W; Tyler, Douglas S; Pruitt, Scott K

2005-04-01

Cotransfection of dendritic cells (DC) with MART-1 and constitutively active TLR4 (caTLR4) RNA enhances the maturation of DC. Immature DC were cotransfected with RNA constructs encoding MART-1 and caTLR4, and CTL responses were analyzed. Cotransfection of DC with MART-1 + caTLR4 enhanced the expression of CD80 and CD83 surface markers and increased the secretion of cytokines IL-6, IL-12, and TNFalpha. Neither the native nor the A27L-modified MART-1 RNA could induce significant DC maturation or cytokine secretion. More importantly, DC cotransfected with caTLR4 + MART-1 RNA induced MART-1-specific CTL responses of a higher magnitude than DC transfected with either the native or A27L MART-1 RNA. When the MART-1 RNA-transfected DC were treated with DC-maturing cytokines, the induced CTL were less frequent and less lytic than those induced with MART-1 + caTLR4. A 2- to 100-fold increase in MART-1 tetramer+ cells and 2- to 10-fold increases in IFNgamma secretion and cytotoxicity were seen in CTL induced with MART-1 + caTLR4 compared to CTL induced with either MART-1 or A27L RNA. CTL induced with the mixed RNA displayed high percentages of CD8+ cells coexpressing CD45RA, CD56, and 2B4 antigens. Transfection with caTLR4 alone induced DC maturation, but did not induce lytic CTL, suggesting that CTL responses were induced solely by MART-1 epitopes. caTLR4 increases the CTL-inducing capacity of DC generating a lytic response specific for the accompanying antigen. These results demonstrate the possibility of enhancing the immunogenicity of the native MART-1 and other RNA derived from weakly immunogenic tumors in DC-based immunotherapy.

Pathogen induction of CXCR4/TLR2 cross-talk impairs host defense function

PubMed Central

Hajishengallis, George; Wang, Min; Liang, Shuang; Triantafilou, Martha; Triantafilou, Kathy

2008-01-01

We report a mechanism of microbial evasion of Toll-like receptor (TLR)-mediated immunity that depends on CXCR4 exploitation. Specifically, the oral/systemic pathogen Porphyromonas gingivalis induces cross-talk between CXCR4 and TLR2 in human monocytes or mouse macrophages and undermines host defense. This is accomplished through its surface fimbriae, which induce CXCR4/TLR2 co-association in lipid rafts and interact with both receptors: Binding to CXCR4 induces cAMP-dependent protein kinase A (PKA) signaling, which in turn inhibits TLR2-mediated proinflammatory and antimicrobial responses to the pathogen. This outcome enables P. gingivalis to resist clearance in vitro and in vivo and thus to promote its adaptive fitness. However, a specific CXCR4 antagonist abrogates this immune evasion mechanism and offers a promising counterstrategy for the control of P. gingivalis periodontal or systemic infections. PMID:18765807

Increased Thymic Cell Turnover under Boron Stress May Bypass TLR3/4 Pathway in African Ostrich

PubMed Central

Huang, Hai-bo; Xiao, Ke; Lu, Shun; Yang, Ke-li; Ansari, Abdur Rahman; Khaliq, Haseeb; Song, Hui; Zhong, Juming; Liu, Hua-zhen; Peng, Ke-mei

2015-01-01

Previous studies revealed that thymus is a targeted immune organ in malnutrition, and high-boron stress is harmful for immune organs. African ostrich is the living fossil of ancient birds and the food animals in modern life. There is no report about the effect of boron intake on thymus of ostrich. The purpose of present study was to evaluate the effect of excessive boron stress on ostrich thymus and the potential role of TLR3/4 signals in this process. Histological analysis demonstrated that long-term boron stress (640 mg/L for 90 days) did not disrupt ostrich thymic structure during postnatal development. However, the numbers of apoptotic cells showed an increased tendency, and the expression of autophagy and proliferation markers increased significantly in ostrich thymus after boron treatment. Next, we examined the expression of TLR3 and TLR4 with their downstream molecular in thymus under boron stress. Since ostrich genome was not available when we started the research, we first cloned ostrich TLR3 TLR4 cDNA from thymus. Ostrich TLR4 was close to white-throated Tinamou. Whole avian TLR4 codons were under purify selection during evolution, whereas 80 codons were under positive selection. TLR3 and TLR4 were expressed in ostrich thymus and bursa of fabricius as was revealed by quantitative real-time PCR (qRT-PCR). TLR4 expression increased with age but significantly decreased after boron treatment, whereas TLR3 expression showed the similar tendency. Their downstream molecular factors (IRF1, JNK, ERK, p38, IL-6 and IFN) did not change significantly in thymus, except that p100 was significantly increased under boron stress when analyzed by qRT-PCR or western blot. Taken together, these results suggest that ostrich thymus developed resistance against long-term excessive boron stress, possibly by accelerating intrathymic cell death and proliferation, which may bypass the TLR3/4 pathway. In addition, attenuated TLRs activity may explain the reduced inflammatory

Increased Thymic Cell Turnover under Boron Stress May Bypass TLR3/4 Pathway in African Ostrich.

PubMed

Huang, Hai-bo; Xiao, Ke; Lu, Shun; Yang, Ke-li; Ansari, Abdur Rahman; Khaliq, Haseeb; Song, Hui; Zhong, Juming; Liu, Hua-zhen; Peng, Ke-mei

2015-01-01

Previous studies revealed that thymus is a targeted immune organ in malnutrition, and high-boron stress is harmful for immune organs. African ostrich is the living fossil of ancient birds and the food animals in modern life. There is no report about the effect of boron intake on thymus of ostrich. The purpose of present study was to evaluate the effect of excessive boron stress on ostrich thymus and the potential role of TLR3/4 signals in this process. Histological analysis demonstrated that long-term boron stress (640 mg/L for 90 days) did not disrupt ostrich thymic structure during postnatal development. However, the numbers of apoptotic cells showed an increased tendency, and the expression of autophagy and proliferation markers increased significantly in ostrich thymus after boron treatment. Next, we examined the expression of TLR3 and TLR4 with their downstream molecular in thymus under boron stress. Since ostrich genome was not available when we started the research, we first cloned ostrich TLR3 TLR4 cDNA from thymus. Ostrich TLR4 was close to white-throated Tinamou. Whole avian TLR4 codons were under purify selection during evolution, whereas 80 codons were under positive selection. TLR3 and TLR4 were expressed in ostrich thymus and bursa of fabricius as was revealed by quantitative real-time PCR (qRT-PCR). TLR4 expression increased with age but significantly decreased after boron treatment, whereas TLR3 expression showed the similar tendency. Their downstream molecular factors (IRF1, JNK, ERK, p38, IL-6 and IFN) did not change significantly in thymus, except that p100 was significantly increased under boron stress when analyzed by qRT-PCR or western blot. Taken together, these results suggest that ostrich thymus developed resistance against long-term excessive boron stress, possibly by accelerating intrathymic cell death and proliferation, which may bypass the TLR3/4 pathway. In addition, attenuated TLRs activity may explain the reduced inflammatory

Innately activated TLR4 signal in the nucleus accumbens is sustained by CRF amplification loop and regulates impulsivity.

PubMed

Balan, Irina; Warnock, Kaitlin T; Puche, Adam; Gondre-Lewis, Marjorie C; Aurelian, Laure

2018-03-01

Cognitive impulsivity is a heritable trait believed to represent the behavior that defines the volition to initiate alcohol drinking. We have previously shown that a neuronal Toll-like receptor 4 (TLR4) signal located in the central amygdala (CeA) and ventral tegmental area (VTA) controls the initiation of binge drinking in alcohol-preferring P rats, and TLR4 expression is upregulated by alcohol-induced corticotropin-releasing factor (CRF) at these sites. However, the function of the TLR4 signal in the nucleus accumbens shell (NAc-shell), a site implicated in the control of reward, drug-seeking behavior and impulsivity and the contribution of other signal-associated genes, are still poorly understood. Here we report that P rats have an innately activated TLR4 signal in NAc-shell neurons that co-express the α2 GABA A receptor subunit and CRF prior to alcohol exposure. This signal is not present in non-alcohol drinking NP rats. The TLR4 signal is sustained by a CRF amplification loop, which includes TLR4-mediated CRF upregulation through PKA/CREB activation and CRF-mediated TLR4 upregulation through the CRF type 1 receptor (CRFR1) and the MAPK/ERK pathway. NAc-shell Infusion of a neurotropic, non-replicating herpes simplex virus vector for TLR4-specific small interfering RNA (pHSVsiTLR4) inhibits TLR4 expression and cognitive impulsivity, implicating the CRF-amplified TLR4 signal in impulsivity regulation. Copyright © 2017 Elsevier Inc. All rights reserved.

Cross-talk between toll-like receptor 4 (TLR4) and proteinase-activated receptor 2 (PAR(2) ) is involved in vascular function.

PubMed

Bucci, M; Vellecco, V; Harrington, L; Brancaleone, V; Roviezzo, F; Mattace Raso, G; Ianaro, A; Lungarella, G; De Palma, R; Meli, R; Cirino, G

2013-01-01

Proteinase-activated receptors (PARs) and toll-like receptors (TLRs) are involved in innate immune responses. The aim of this study was to evaluate the possible cross-talk between PAR(2) and TLR4 in vessels in physiological condition and how it varies following stimulation of TLR4 by using in vivo and ex vivo models. Thoracic aortas were harvested from both naïve and endotoxaemic rats for in vitro studies. Arterial blood pressure was monitored in anaesthetized rats in vivo. LPS was used as a TLR4 agonist while PAR(2) activating peptide (AP) was used as a PAR(2) agonist. Aortas harvested from TLR4(-/-) mice were also used to characterize the PAR(2) response. PAR(2) , but not TLR4, expression was enhanced in aortas of endotoxaemic rats. PAR(2) AP-induced vasorelaxation was increased in aortic rings of LPS-treated rats. TLR4 inhibitors, curcumine and resveratrol, reduced PAR(2) AP-induced vasorelaxation and PAR(2) AP-induced hypotension in both naïve and endotoxaemic rats. Finally, in aortic rings from TLR4(-/-) mice, the expression of PAR(2) was reduced and the PAR(2) AP-induced vasodilatation impaired compared with those from wild-type mice and both resveratrol and curcumine were ineffective. Cross-talk between PAR(2) and TLR4 contributes to vascular homeostasis. © 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.

Cross-talk between toll-like receptor 4 (TLR4) and proteinase-activated receptor 2 (PAR2) is involved in vascular function

PubMed Central

Bucci, M; Vellecco, V; Harrington, L; Brancaleone, V; Roviezzo, F; Mattace Raso, G; Ianaro, A; Lungarella, G; De Palma, R; Meli, R; Cirino, G

2013-01-01

Background and Purpose Proteinase-activated receptors (PARs) and toll-like receptors (TLRs) are involved in innate immune responses. The aim of this study was to evaluate the possible cross-talk between PAR2 and TLR4 in vessels in physiological condition and how it varies following stimulation of TLR4 by using in vivo and ex vivo models. Experimental Approach Thoracic aortas were harvested from both naïve and endotoxaemic rats for in vitro studies. Arterial blood pressure was monitored in anaesthetized rats in vivo. LPS was used as a TLR4 agonist while PAR2 activating peptide (AP) was used as a PAR2 agonist. Aortas harvested from TLR4–/– mice were also used to characterize the PAR2 response. Key Results PAR2, but not TLR4, expression was enhanced in aortas of endotoxaemic rats. PAR2AP-induced vasorelaxation was increased in aortic rings of LPS-treated rats. TLR4 inhibitors, curcumine and resveratrol, reduced PAR2AP-induced vasorelaxation and PAR2AP-induced hypotension in both naïve and endotoxaemic rats. Finally, in aortic rings from TLR4–/– mice, the expression of PAR2 was reduced and the PAR2AP-induced vasodilatation impaired compared with those from wild-type mice and both resveratrol and curcumine were ineffective. Conclusions and Implications Cross-talk between PAR2 and TLR4 contributes to vascular homeostasis. PMID:22957757

Palmitate induces VSMC apoptosis via toll like receptor (TLR)4/ROS/p53 pathway.

PubMed

Zhang, Yuanjun; Xia, Guanghao; Zhang, Yaqiong; Liu, Juxiang; Liu, Xiaowei; Li, Weihua; Lv, Yaya; Wei, Suhong; Liu, Jing; Quan, Jinxing

2017-08-01

Toll-like receptor 4 (TLR4) has been implicated in vascular inflammation, as well as in the pathogenesis of atherosclerosis and diabetes. Vascular smooth muscle cell (VSMC) apoptosis has been shown to induce plaque vulnerability in atherosclerosis. Previous studies reported that palmitate induced apoptosis in VSMCs; however, the role of TLR4 in palmitate-induced apoptosis in VSMCs has not yet been defined. In this study, we investigated whether or not palmitate-induced apoptosis depended on the activation of the TLR4 pathway. VSMCs were treated with or without palmitate, CRISPR/Cas9z-mediated genome editing methods were used to deplete TLR4 expression, while NADPH oxidase inhibitors were used to inhibit reactive oxygen species (ROS) generation. Cell apoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, ROS was measured using the 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) method, the mRNA and protein expression levels of caspase 3, caspase 9, BCL-2 and p53 were studied by real-time polymerase chain reaction (RT-PCR) and ELISA. Palmitate significantly promotes VSMC apoptosis, ROS generation, and expression of caspase 3, caspase 9 and p53; while NADPH oxidase inhibitor pretreatment markedly attenuated these effects. Moreover, knockdown of TLR4 significantly blocked palmitate-induced ROS generation and VSMC apoptosis accompanied by inhibition of caspase 3, caspase 9, p53 expression and restoration of BCL-2 expression. Our results suggest that palmitate-induced apoptosis depends on the activation of the TLR4/ROS/p53 signaling pathway, and that TLR4 may be a potential therapeutic target for the prevention and treatment of atherosclerosis. Copyright © 2017 Elsevier B.V. All rights reserved.

Diversity of the TLR4 Immunity Receptor in Czech Native Cattle Breeds Revealed Using the Pacific Biosciences Sequencing Platform.

PubMed

Novák, Karel; Pikousová, Jitka; Czerneková, Vladimíra; Mátlová, Věra

2017-07-03

The allelic variants of immunity genes in historical breeds likely reflect local infection pressure and therefore represent a reservoir for breeding. Screening to determine the diversity of the Toll-like receptor gene TLR4 was conducted in two conserved cattle breeds: Czech Red and Czech Red Pied. High-throughput sequencing of pooled PCR amplicons using the PacBio platform revealed polymorphisms, which were subsequently confirmed via genotyping techniques. Eight SNPs found in coding and adjacent regions were grouped into 18 haplotypes, representing a significant portion of the known diversity in the global breed panel and presumably exceeding diversity in production populations. Notably, the ancient Czech Red breed appeared to possess greater haplotype diversity than the Czech Red Pied breed, a Simmental variant, although the haplotype frequencies might have been distorted by significant crossbreeding and bottlenecks in the history of Czech Red cattle. The differences in haplotype frequencies validated the phenotypic distinctness of the local breeds. Due to the availability of Czech Red Pied production herds, the effect of intensive breeding on TLR diversity can be evaluated in this model. The advantages of the Pacific Biosciences technology for the resequencing of long PCR fragments with subsequent direct phasing were independently validated.

TLR4 signaling shapes B cell dynamics via MyD88-dependent pathways and Rac GTPases.

PubMed

Barrio, Laura; Saez de Guinoa, Julia; Carrasco, Yolanda R

2013-10-01

B cells use a plethora of TLR to recognize pathogen-derived ligands. These innate signals have an important function in the B cell adaptive immune response and modify their trafficking and tissue location. The direct role of TLR signaling on B cell dynamics nonetheless remains almost entirely unknown. In this study, we used a state-of-the-art two-dimensional model combined with real-time microscopy to study the effect of TLR4 stimulation on mouse B cell motility in response to chemokines. We show that a minimum stimulation period is necessary for TLR4 modification of B cell behavior. TLR4 stimulation increased B cell polarization, migration, and directionality; these increases were dependent on the MyD88 signaling pathway and did not require ERK or p38 MAPK activity downstream of TLR4. In addition, TLR4 stimulation enhanced Rac GTPase activity and promoted sustained Rac activation in response to chemokines. These results increase our understanding of the regulation of B cell dynamics by innate signals and the underlying molecular mechanisms.

TLR4 ligands LPS and MPLA differentially regulate effector and memory CD8+ T cell differentiation

PubMed Central

Cui, Weiguo; Joshi, Nikhil S.; Liu, Ying; Meng, Hailong; Kleinstein, Steven H; Kaech, Susan M.

2014-01-01

Vaccines formulated with non-replicating pathogens require adjuvants to help bolster immunogenicity. The role of adjuvants in antibody production has been well studied, but how they influence memory CD8+ T cell differentiation remains poorly defined. Here we implemented dendritic cell (DC)-mediated immunization to study the effects of commonly used adjuvants, TLR ligands, on effector and memory CD8+ T cell differentiation in mice. Intriguingly, we found that the TLR4 ligand LPS was far more superior to other TLR ligands in generating memory CD8+ T cells upon immunization. LPS boosted clonal expansion similar to the other adjuvants, but fewer of the activated CD8+ T cells died during contraction, generating a larger pool of memory cells. Surprisingly, monophosphoryl lipid A (MPLA), another TLR4 ligand, enhanced clonal expansion of effector CD8+ T cells, but also promoted their terminal differentiation and contraction; thus, fewer memory CD8+ T cells formed and MPLA-primed animals were less protected against secondary infection compared to those primed with LPS. Furthermore, gene expression profiling revealed that LPS-primed effector cells displayed a stronger pro-memory gene expression signature, whereas the gene expression profile of MPLA-primed effector cells aligned closer with terminal effector CD8+ T cells. Lastly, we demonstrated that the LPS-TLR4-derived “pro-memory” signals were MyD88, but not Trif, dependent. This study reveals the influential power of adjuvants on the quantity and quality of CD8+ T cell memory, and that attention to adjuvant selection is crucial because boosting effector cell expansion may not always equate with more memory T cells or greater protection. PMID:24659688

Endotoxin Exposure during Sensitization to Blomia tropicalis Allergens Shifts TH2 Immunity Towards a TH17-Mediated Airway Neutrophilic Inflammation: Role of TLR4 and TLR2

PubMed Central

Barboza, Renato; Câmara, Niels Olsen Saraiva; Gomes, Eliane; Sá-Nunes, Anderson; Florsheim, Esther; Mirotti, Luciana; Labrada, Alexis; Alcântara-Neves, Neuza Maria; Russo, Momtchilo

2013-01-01

Experimental evidence and epidemiological studies indicate that exposure to endotoxin lipopolysaccharide (eLPS) or other TLR agonists prevent asthma. We have previously shown in the OVA-model of asthma that eLPS administration during alum-based allergen sensitization blocked the development of lung TH2 immune responses via MyD88 pathway and IL-12/IFN-γ axis. In the present work we determined the effect of eLPS exposure during sensitization to a natural airborne allergen extract derived from the house dust mite Blomia tropicalis (Bt). Mice were subcutaneously sensitized with Bt allergens co-adsorbed onto alum with or without eLPS and challenged twice intranasally with Bt. Cellular and molecular parameters of allergic lung inflammation were evaluated 24 h after the last Bt challenge. Exposure to eLPS but not to ultrapure LPS (upLPS) preparation during sensitization to Bt allergens decreased the influx of eosinophils and increased the influx of neutrophils to the airways. Inhibition of airway eosinophilia was not observed in IFN-γdeficient mice while airway neutrophilia was not observed in IL-17RA-deficient mice as well in mice lacking MyD88, CD14, TLR4 and, surprisingly, TLR2 molecules. Notably, exposure to a synthetic TLR2 agonist (PamCSK4) also induced airway neutrophilia that was dependent on TLR2 and TLR4 molecules. In the OVA model, exposure to eLPS or PamCSK4 suppressed OVA-induced airway inflammation. Our results suggest that B. tropicalis allergens engage TLR4 that potentiates TLR2 signaling. This dual TLR activation during sensitization results in airway neutrophilic inflammation associated with increased frequency of lung TH17 cells. Our work highlight the complex interplay between bacterial products, house dust mite allergens and TLR signaling in the induction of different phenotypes of airway inflammation. PMID:23805294

[TLR-4 involvement in pyroptosis of mice with pulmonary inflammation infected by Actinobacillus pleuropneumoniae].

PubMed

Hu, Peipei; Huang, Fushen; Niu, Junchao; Tang, Zhaoshan

2015-05-04

Pyroptosis is a caspase-1 dependent programmed cell death and involves pathogenesis of infectious diseases by releasing many pro-inflammatory cytokines to induced inflammation. TLR-4 plays an important role in mediating pathogenesis of some infectious diseases. In this study, we detected the expression of TLR-4 and some molecules (e. g caspase-1, TNF-α, IL-1β, IL-6, IL-18 ) related with pyroptosis to determine its involvement and mechanisms of pulmonary inflammation in mice infected by A. pleuropneumoniae. Mice were intranasally infected by A. pleuropneumoniae and killed 48 hours post infection. Pulmonary gross lesion and histological pathology by H-E were observed. Expression levels of caspase-1 , caspase-3, TNF-α, IL-1β, IL-6, IL-18, and TLR-4 in lung of mice were detected by RT-PCR and qPCR. Serious pulmonary hemorrhage and inflammation in infected mice were observed. Expression levels of caspase-1, caspase-3, TNF-α, IL-1β, IL-6, IL-18 and TLR-4 increased, and expression levels of caspase-3 were not changed in lung of infected mice. TLR-4 might be involved in pulmonary inflammation of mice infected by A. pleuropneumoniae. After induced by activated TLR-4 some cells in this lesion expressed pro-inflammatory cytokines. These cytokines would induce pulmonary inflammation. This lesion might involve pyroptosis with caspase-1 expression.

Robust TLR4-induced gene expression patterns are not an accurate indicator of human immunity

PubMed Central

2010-01-01

Background Activation of Toll-like receptors (TLRs) is widely accepted as an essential event for defence against infection. Many TLRs utilize a common signalling pathway that relies on activation of the kinase IRAK4 and the transcription factor NFκB for the rapid expression of immunity genes. Methods 21 K DNA microarray technology was used to evaluate LPS-induced (TLR4) gene responses in blood monocytes from a child with an IRAK4-deficiency. In vitro responsiveness to LPS was confirmed by real-time PCR and ELISA and compared to the clinical predisposition of the child and IRAK4-deficient mice to Gram negative infection. Results We demonstrated that the vast majority of LPS-responsive genes in IRAK4-deficient monocytes were greatly suppressed, an observation that is consistent with the described role for IRAK4 as an essential component of TLR4 signalling. The severely impaired response to LPS, however, is inconsistent with a remarkably low incidence of Gram negative infections observed in this child and other children with IRAK4-deficiency. This unpredicted clinical phenotype was validated by demonstrating that IRAK4-deficient mice had a similar resistance to infection with Gram negative S. typhimurium as wildtype mice. A number of immunity genes, such as chemokines, were expressed at normal levels in human IRAK4-deficient monocytes, indicating that particular IRAK4-independent elements within the repertoire of TLR4-induced responses are expressed. Conclusions Sufficient defence to Gram negative immunity does not require IRAK4 or a robust, 'classic' inflammatory and immune response. PMID:20105294

A role for intestinal TLR4-driven inflammatory response during activity-based anorexia

PubMed Central

Belmonte, Liliana; Achamrah, Najate; Nobis, Séverine; Guérin, Charlène; Riou, Gaëtan; Bôle-Feysot, Christine; Boyer, Olivier; Richard, Vincent; Rego, Jean Claude Do; Déchelotte, Pierre; Goichon, Alexis; Coëffier, Moïse

2016-01-01

Anorexia nervosa (AN) is associated with low-grade systemic inflammation and altered gut microbiota. However, the molecular origin of the inflammation remains unknown. Toll-like receptors are key regulators of innate immune response and their activation seems also to be involved in the control of food intake. We used activity-based anorexia (ABA) model to investigate the role of TLR4 and its contribution in anorexia-associated low-grade inflammation. Here, we found that ABA affected early the intestinal inflammatory status and the hypothalamic response. Indeed, TLR4 was upregulated both on colonic epithelial cells and intestinal macrophages, leading to elevated downstream mucosal cytokine production. These mucosal changes occurred earlier than hypothalamic changes driving to increased levels of IL-1β and IL-1R1 as well as increased levels of plasma corticosterone. Paradoxically, TLR4-deficient mice exhibited greater vulnerability to ABA with increased mortality rate, suggesting a major contribution of TLR4-mediated responses during ABA-induced weight loss. PMID:27779218

A role for intestinal TLR4-driven inflammatory response during activity-based anorexia.

PubMed

Belmonte, Liliana; Achamrah, Najate; Nobis, Séverine; Guérin, Charlène; Riou, Gaëtan; Bôle-Feysot, Christine; Boyer, Olivier; Richard, Vincent; Rego, Jean Claude Do; Déchelotte, Pierre; Goichon, Alexis; Coëffier, Moïse

2016-10-25

Anorexia nervosa (AN) is associated with low-grade systemic inflammation and altered gut microbiota. However, the molecular origin of the inflammation remains unknown. Toll-like receptors are key regulators of innate immune response and their activation seems also to be involved in the control of food intake. We used activity-based anorexia (ABA) model to investigate the role of TLR4 and its contribution in anorexia-associated low-grade inflammation. Here, we found that ABA affected early the intestinal inflammatory status and the hypothalamic response. Indeed, TLR4 was upregulated both on colonic epithelial cells and intestinal macrophages, leading to elevated downstream mucosal cytokine production. These mucosal changes occurred earlier than hypothalamic changes driving to increased levels of IL-1β and IL-1R1 as well as increased levels of plasma corticosterone. Paradoxically, TLR4-deficient mice exhibited greater vulnerability to ABA with increased mortality rate, suggesting a major contribution of TLR4-mediated responses during ABA-induced weight loss.

Gene polymorphisms and febrile neutropenia in acute leukemia--no association with IL-4, CCR-5, IL-1RA, but the MBL-2, ACE, and TLR-4 are associated with the disease in Turkish patients: a preliminary study.

PubMed

Pehlivan, Mustafa; Sahin, Handan Haydaroğlu; Ozdilli, Kurşat; Onay, Hüseyin; Ozcan, Ali; Ozkinay, Ferda; Pehlivan, Sacide

2014-07-01

The aim of this study was to investigate the mannose-binding lectin 2 (MBL-2), interleukin (IL)-4, Toll-like receptor 4 (TLR-4), angiotensin converting enzyme (ACE), chemokine receptor 5 (CCR-5), and IL-1 receptor antagonist (RA) gene polymorphisms (GPs) in acute leukemias (ALs) and to evaluate their roles in febrile neutropenia (FN) resulting from chemotherapy. The study included 60 AL patients hospitalized between the period of July 2001 and August 2006. Polymorphisms for the genes ACE(I/D), CCR-5, IL-1RA, MBL-2, TLR-4, and IL-4 were typed by polymerase chain reaction (PCR) and/or PCR-restriction fragment length polymerase. Genotype frequencies for these genes were compared in the patient and control groups. The relationships between the genotypes and the body distribution of infections, pathogens, the duration of neutropenia, and febrile episodes in AL patients were evaluated. No significant differences in either the genotype distribution or the allelic frequencies of TLR-4, IL-4, CCR-5, IL-1RN GPs were observed between patients and healthy controls. The AB/BB genotype (53.3%) in the MBL-2 gene was found to be significantly higher in the AL patients compared with control groups. There were correlations between the presence of MBL-2, TLR-4, and ACE polymorphisms and clinical parameters due to FN. Overall, bacteremia was more common in MBL BB and ACE DD. Gram-positive bacteremia was more common in ACE for ID versus DD genotype. Gram-negative bacteremia was more common for both the MBL-2 AB/BB genotype and TLR-4 AG genotype. Median durations of febrile episodes were significantly shorter in ACE DD and MBL AB/BB. Although TLR-4, ACE, and MBL-2 GPs have been extensively investigated in different clinical pictures, this is the first study to evaluate the role of these polymorphisms in the genetic etiopathogenesis of FN in patients with ALs. As a conclusion, TLR-4, ACE, and MBL-2 genes might play roles in the genetic etiopathogenesis of FN in patients with ALs.

Genetic Polymorphisms of TLR4 and MICA are Associated with Severity of Trachoma Disease in Tanzania

PubMed Central

Abbas, Muneer; Berka, Noureddine; Khraiwesh, Mozna; Ramadan, Ali; Apprey, Victor; Furbert-Harris, Paulette; Quinn, Thomas; Brim, Hassan; Dunston, Georgia

2016-01-01

Aim To examine the association of TLR4 Asp299Gly and MICA exon 5 microsatellites polymorphisms with severity of trachoma in a sub-Saharan East Africa population of Tanzanian villagers. Methods The samples were genotyped for MICA exon 5 microsatellites and the TLR4 299 A/G polymorphism by Restriction Fragment Length Polymorphism (RFLP), and GeneScan®, respectively. The association of TLR4 Asp299Gly and MICA exon 5 microsatellites with inflammatory trachoma (TI) and trichiasis (TI) were examined. Results The results showed an association between TLR4 and MICA polymorphisms and trachoma disease severity, as well as with protection. TLR4 an allele was significantly associated with inflammatory trachoma (p=0.0410), while the G allele (p=0.0410) was associated with protection. Conclusion TLR4 and MICA may modulate the risk of severity to trachoma disease by modulating the immune response to Ct. In addition; the increased frequency of MICA-A9 heterozygote in controls may suggest a positive selection of these alleles in adaptation to environments where Ct is endemic. PMID:27559544

How important are rare variants in common disease?

PubMed

Saint Pierre, Aude; Génin, Emmanuelle

2014-09-01

Genome-wide association studies have uncovered hundreds of common genetic variants involved in complex diseases. However, for most complex diseases, these common genetic variants only marginally contribute to disease susceptibility. It is now argued that rare variants located in different genes could in fact play a more important role in disease susceptibility than common variants. These rare genetic variants were not captured by genome-wide association studies using single nucleotide polymorphism-chips but with the advent of next-generation sequencing technologies, they have become detectable. It is now possible to study their contribution to common disease by resequencing samples of cases and controls or by using new genotyping exome arrays that cover rare alleles. In this review, we address the question of the contribution of rare variants in common disease by taking the examples of different diseases for which some resequencing studies have already been performed, and by summarizing the results of simulation studies conducted so far to investigate the genetic architecture of complex traits in human. So far, empirical data have not allowed the exclusion of many models except the most extreme ones involving only a small number of rare variants with large effects contributing to complex disease. To unravel the genetic architecture of complex disease, case-control data will not be sufficient, and alternative study designs need to be proposed together with methodological developments. © The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Identification of Francisella tularensis lipoproteins that stimulate the toll-like receptor (TLR) 2/TLR1 heterodimer.

PubMed

Thakran, Shalini; Li, Hanfen; Lavine, Christy L; Miller, Mark A; Bina, James E; Bina, Xiaowen R; Re, Fabio

2008-02-15

The innate immune response to Francisella tularensis is primarily mediated by TLR2, though the bacterial products that stimulate this receptor remain unknown. Here we report the identification of two Francisella lipoproteins, TUL4 and FTT1103, which activate TLR2. We demonstrate that TUL4 and FTT1103 stimulate chemokine production in human and mouse cells in a TLR2-dependent way. Using an assay that relies on chimeric TLR proteins, we show that TUL4 and FTT1103 stimulate exclusively the TLR2/TLR1 heterodimer. Our results also show that yet unidentified Francisella proteins, possibly unlipi-dated, have the ability to stimulate the TLR2/TLR6 heterodimer. Through domain-exchange analysis, we determined that an extended region that comprises LRR 9-17 in the extra-cellular portion of TLR1 mediates response to Francisella lipoproteins and triacylated lipopeptide. Substitution of the corresponding LRR of TLR6 with the LRR derived from TLR1 enables TLR6 to recognize TUL4, FTT1103, and triacylated lipopeptide. This study identifies for the first time specific Fran-cisella products capable of stimulating a proinflammatory response and the cellular receptors they trigger.

MD-2 is required for disulfide HMGB1–dependent TLR4 signaling

PubMed Central

Wang, Haichao; Ju, Zhongliang; Ragab, Ahmed A.; Lundbäck, Peter; Long, Wei; Valdes-Ferrer, Sergio I.; He, Mingzhu; Pribis, John P.; Li, Jianhua; Lu, Ben; Gero, Domokos; Szabo, Csaba; Antoine, Daniel J.; Harris, Helena E.; Golenbock, Doug T.; Meng, Jianmin; Roth, Jesse; Chavan, Sangeeta S.; Andersson, Ulf; Billiar, Timothy R.; Al-Abed, Yousef

2015-01-01

Innate immune receptors for pathogen- and damage-associated molecular patterns (PAMPs and DAMPs) orchestrate inflammatory responses to infection and injury. Secreted by activated immune cells or passively released by damaged cells, HMGB1 is subjected to redox modification that distinctly influences its extracellular functions. Previously, it was unknown how the TLR4 signalosome distinguished between HMGB1 isoforms. Here we demonstrate that the extracellular TLR4 adaptor, myeloid differentiation factor 2 (MD-2), binds specifically to the cytokine-inducing disulfide isoform of HMGB1, to the exclusion of other isoforms. Using MD-2–deficient mice, as well as MD-2 silencing in macrophages, we show a requirement for HMGB1-dependent TLR4 signaling. By screening HMGB1 peptide libraries, we identified a tetramer (FSSE, designated P5779) as a specific MD-2 antagonist preventing MD-2–HMGB1 interaction and TLR4 signaling. P5779 does not interfere with lipopolysaccharide-induced cytokine/chemokine production, thus preserving PAMP-mediated TLR4–MD-2 responses. Furthermore, P5779 can protect mice against hepatic ischemia/reperfusion injury, chemical toxicity, and sepsis. These findings reveal a novel mechanism by which innate systems selectively recognize specific HMGB1 isoforms. The results may direct toward strategies aimed at attenuating DAMP-mediated inflammation while preserving antimicrobial immune responsiveness. PMID:25559892

Polymorphisms of the TLR4 gene and risk of gastric cancer.

PubMed

Huang, Lina; Yuan, Kexin; Liu, Jingjing; Ren, Xiyun; Dong, Xiaoqun; Tian, Wenjing; Jia, Yunhe

2014-03-01

Toll-like receptor 4 (TLR4) is an important lipo-polysaccharide (LPS) receptor in gastric epithelial cell signaling transduction and plays critical roles in the development and progression of gastric cancer (GC). We investigated the effects of TLR4 gene polymorphisms and gene-environmental interactions on the risk of GC in Northeastern China. We genotyped two single-nucleotide polymorphisms (SNPs) in TLR4 (rs10116253 and rs1927911) in 217 GC patients and 294 cancer-free controls using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Odds ratio (OR) and 95% confidence intervals (CIs) were estimated by unconditional logistic-regression models. Individuals carrying CC genotype of rs10116253 and TT genotype of rs1927911 had a significantly decreased risk of GC (adjusted OR=0.33, 95% CI 0.18-0.60, P<0.001 and adjusted OR=0.37, 95% CI 0.21-0.67, P=0.001 respectively), compared with TT genotype of rs10116253 and CC genotype of rs1927911. In addition, the SNP effects were additive to the effects of some known environmental factors without any interaction between them in the susceptibility to GC. Our data suggested that TLR4 gene polymorphisms may be associated with a decreased risk of GC in Chinese population. And these SNPs and their combined effects with environmental factors may be associated with the risk of GC. Copyright © 2013 Elsevier B.V. All rights reserved.

Lipopolysaccharide induces proliferation and osteogenic differentiation of adipose-derived mesenchymal stromal cells in vitro via TLR4 activation

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

Herzmann, Nicole; Salamon, Achim; Fiedler, Tomas

Multipotent mesenchymal stromal cells (MSC) are capable of multi-lineage differentiation and support regenerative processes. In bacterial infections, resident MSC can come intocontact with and need to react to bacterial components. Lipopolysaccharide (LPS), a typical structure of Gram-negative bacteria, increases the proliferation and osteogenic differentiation of MSC. LPS is usually recognized by the toll-like receptor (TLR) 4 and induces pro-inflammatory reactions in numerous cell types. In this study, we quantified the protein expression of TLR4 and CD14 on adipose-derived MSC (adMSC) in osteogenic differentiation and investigated the effect of TLR4 activation by LPS on NF-κB activation, proliferation and osteogenic differentiation ofmore » adMSC. We found that TLR4 is expressed on adMSC whereas CD14 is not, and that osteogenic differentiation induced an increase of the amount of TLR4 protein whereas LPS stimulation did not. Moreover, we could show that NF-κB activation via TLR4 occurs upon LPS treatment. Furthermore, we were able to show that competitive inhibition of TLR4 completely abolished the stimulatory effect of LPS on the proliferation and osteogenic differentiation of adMSC. In addition, the inhibition of TLR4 leads to the complete absence of osteogenic differentiation of adMSC, even when osteogenically stimulated. Thus, we conclude that LPS induces proliferation and osteogenic differentiation of adMSC in vitro through the activation of TLR4 and that the TLR4 receptor seems to play a role during osteogenic differentiation of adMSC.« less

MD-2-mediated Ionic Interactions between Lipid A and TLR4 Are Essential for Receptor Activation*

PubMed Central

Meng, Jianmin; Lien, Egil; Golenbock, Douglas T.

2010-01-01

Lipopolysaccharide (LPS) activates innate immune responses through TLR4·MD-2. LPS binds to the MD-2 hydrophobic pocket and bridges the dimerization of two TLR4·MD-2 complexes to activate intracellular signaling. However, exactly how lipid A, the endotoxic moiety of LPS, activates myeloid lineage cells remains unknown. Lipid IVA, a tetra-acylated lipid A precursor, has been used widely as a model for lipid A activation. For unknown reasons, lipid IVA activates proinflammatory responses in rodent cells but inhibits the activity of LPS in human cells. Using stable TLR4-expressing cell lines and purified monomeric MD-2, as well as MD-2-deficient bone marrow-derived macrophages, we found that both mouse TLR4 and mouse MD-2 are required for lipid IVA activation. Computational studies suggested that unique ionic interactions exist between lipid IVA and TLR4 at the dimerization interface in the mouse complex only. The negatively charged 4′-phosphate on lipid IVA interacts with two positively charged residues on the opposing mouse, but not human, TLR4 (Lys367 and Arg434) at the dimerization interface. When replaced with their negatively charged human counterparts Glu369 and Gln436, mouse TLR4 was no longer responsive to lipid IVA. In contrast, human TLR4 gained lipid IVA responsiveness when ionic interactions were enabled by charge reversal at the dimerization interface, defining the basis of lipid IVA species specificity. Thus, using lipid IVA as a selective lipid A agonist, we successfully decoupled and coupled two sequential events required for intracellular signaling: receptor engagement and dimerization, underscoring the functional role of ionic interactions in receptor activation. PMID:20018893

Common FABP4 genetic variants and plasma levels of fatty acid binding protein 4 in older adults.

PubMed

Mukamal, Kenneth J; Wilk, Jemma B; Biggs, Mary L; Jensen, Majken K; Ix, Joachim H; Kizer, Jorge R; Tracy, Russell P; Zieman, Susan J; Mozaffarian, Dariush; Psaty, Bruce M; Siscovick, David S; Djoussé, Luc

2013-11-01

We examined common variants in the fatty acid binding protein 4 gene (FABP4) and plasma levels of FABP4 in adults aged 65 and older from the Cardiovascular Health Study. We genotyped rs16909187, rs1054135, rs16909192, rs10808846, rs7018409, rs2290201, and rs6992708 and measured circulating FABP4 levels among 3190 European Americans and 660 African Americans. Among European Americans, the minor alleles of six single nucleotide polymorphisms (SNP) were associated with lower FABP4 levels (all p ≤ 0.01). Among African Americans, the SNP with the lowest minor allele frequency was associated with lower FABP4 levels (p = 0.015). The C-A haplotype of rs16909192 and rs2290201 was associated with lower FABP4 levels in both European Americans (frequency = 16 %; p = 0.001) and African Americans (frequency = 8 %; p = 0.04). The haplotype combined a SNP in the first intron with one in the 3'untranslated region. However, the alleles associated with lower FABP4 levels were associated with higher fasting glucose in meta-analyses from the MAGIC consortium. These results demonstrate associations of common SNP and haplotypes in the FABP4 gene with lower plasma FABP4 but higher fasting glucose levels.

Measuring missing heritability: Inferring the contribution of common variants

PubMed Central

Golan, David; Lander, Eric S.; Rosset, Saharon

2014-01-01

Genome-wide association studies (GWASs), also called common variant association studies (CVASs), have uncovered thousands of genetic variants associated with hundreds of diseases. However, the variants that reach statistical significance typically explain only a small fraction of the heritability. One explanation for the “missing heritability” is that there are many additional disease-associated common variants whose effects are too small to detect with current sample sizes. It therefore is useful to have methods to quantify the heritability due to common variation, without having to identify all causal variants. Recent studies applied restricted maximum likelihood (REML) estimation to case–control studies for diseases. Here, we show that REML considerably underestimates the fraction of heritability due to common variation in this setting. The degree of underestimation increases with the rarity of disease, the heritability of the disease, and the size of the sample. Instead, we develop a general framework for heritability estimation, called phenotype correlation–genotype correlation (PCGC) regression, which generalizes the well-known Haseman–Elston regression method. We show that PCGC regression yields unbiased estimates. Applying PCGC regression to six diseases, we estimate the proportion of the phenotypic variance due to common variants to range from 25% to 56% and the proportion of heritability due to common variants from 41% to 68% (mean 60%). These results suggest that common variants may explain at least half the heritability for many diseases. PCGC regression also is readily applicable to other settings, including analyzing extreme-phenotype studies and adjusting for covariates such as sex, age, and population structure. PMID:25422463

CXCR4-CXCL12-CXCR7, TLR2-TLR4, and PD-1/PD-L1 in colorectal cancer liver metastases from neoadjuvant-treated patients.

PubMed

D'Alterio, Crescenzo; Nasti, Guglielmo; Polimeno, Marianeve; Ottaiano, Alessandro; Conson, Manuel; Circelli, Luisa; Botti, Giovanni; Scognamiglio, Giosuè; Santagata, Sara; De Divitiis, Chiara; Nappi, Anna; Napolitano, Maria; Tatangelo, Fabiana; Pacelli, Roberto; Izzo, Francesco; Vuttariello, Emilia; Botti, Gerardo; Scala, Stefania

2016-01-01

A neoadjuvant clinical trial was previously conducted in patients with resectable colorectal cancer liver metastases (CRLM). At a median follow up of 28 months, 20/33 patients were dead of disease, 8 were alive with disease and 5 were alive with no evidence of disease. To shed further insight into biological features accounting for different outcomes, the expression of CXCR4-CXCL12-CXCR7, TLR2-TLR4, and the programmed death receptor-1 (PD-1)/programmed death-1 ligand (PD-L1) was evaluated in excised liver metastases. Expression profiles were assessed through qPCR in metastatic and unaffected liver tissue of 33 CRLM neoadjuvant-treated patients. CXCR4 and CXCR7, TLR2/TLR4, and PD-1/PD-L1 mRNA were significantly overexpressed in metastatic compared to unaffected liver tissues. CXCR4 protein was negative/low in 10/31, and high in 21/31, CXCR7 was negative/low in 16/31 and high in 15/31, CXCL12 was negative/low in 14/31 and high in 17/31 CRLM. PD-1 was negative in 19/30 and positive in 11/30, PD-L1 was negative/low in 24/30 and high in 6/30 CRLM. Stromal PD-L1 expression, affected the progression-free survival (PFS) in the CRLM population. Patients overexpressing CXCR4 experienced a worse PFS and cancer specific survival (CSS) ( p = 0.001 and p = 0.0008); in these patients, KRAS mutation identified a subgroup with a significantly worse CSS ( p < 0.01). Thus, CXCR4 and PD-L1 expression discriminate patients with the worse PFS within the CRLM evaluated patients. Within the CXCR4 high expressing patients carrying Mut-KRAS in CRLM identifies the worst prognostic group. Thus, CXCR4 targeting plus anti-PD-1 therapy should be explored to improve the prognosis of Mut-KRAS-high CXCR4-CRLMs.

Apigenin protects blood-brain barrier and ameliorates early brain injury by inhibiting TLR4-mediated inflammatory pathway in subarachnoid hemorrhage rats.

PubMed

Zhang, Tingting; Su, Jingyuan; Guo, Bingyu; Wang, Kaiwen; Li, Xiaoming; Liang, Guobiao

2015-09-01

Early brain injury (EBI) following subarachnoid hemorrhage (SAH) is associated with high morbidity and mortality. Inflammation has been considered as the major contributor to brain damage after SAH. SAH induces a systemic increase in pro-inflammatory cytokines and chemokines. Disruption of blood-brain barrier (BBB) facilitates the influx of inflammatory cells. It has been reported that the activation of toll-like receptor 4 (TLR4)/NF-κB signaling pathway plays a vital role in the central nervous system diseases. Apigenin, a common plant flavonoid, possesses anti-inflammation effect. In this study, we focused on the effects of apigenin on EBI following SAH and its anti-inflammation mechanism. Our results showed that apigenin (20mg/kg) administration significantly attenuated EBI (including brain edema, BBB disruption, neurological deficient, severity of SAH, and cell apoptosis) after SAH in rats by suppressing the expression of TLR4, NF-κB and their downstream pro-inflammatory cytokines in the cortex and by up-regulating the expression of tight junction proteins of BBB. Double immunofluorescence staining demonstrated that TLR4 was activated following SAH in neurons, microglia cells, and endothelial cells but not in astrocytes. Apigenin could suppress the activation of TLR4 induced by SAH and inhibit apoptosis of cells in the cortex. These results suggested that apigenin could attenuate EBI after SAH in rats by suppressing TLR4-mediated inflammation and protecting against BBB disruption. Copyright © 2015 Elsevier B.V. All rights reserved.

Humanized TLR4/MD-2 mice reveal LPS recognition differentially impacts susceptibility to Yersinia pestis and Salmonella enterica.

PubMed

Hajjar, Adeline M; Ernst, Robert K; Fortuno, Edgardo S; Brasfield, Alicia S; Yam, Cathy S; Newlon, Lindsay A; Kollmann, Tobias R; Miller, Samuel I; Wilson, Christopher B

2012-01-01

Although lipopolysaccharide (LPS) stimulation through the Toll-like receptor (TLR)-4/MD-2 receptor complex activates host defense against Gram-negative bacterial pathogens, how species-specific differences in LPS recognition impact host defense remains undefined. Herein, we establish how temperature dependent shifts in the lipid A of Yersinia pestis LPS that differentially impact recognition by mouse versus human TLR4/MD-2 dictate infection susceptibility. When grown at 37°C, Y. pestis LPS is hypo-acylated and less stimulatory to human compared with murine TLR4/MD-2. By contrast, when grown at reduced temperatures, Y. pestis LPS is more acylated, and stimulates cells equally via human and mouse TLR4/MD-2. To investigate how these temperature dependent shifts in LPS impact infection susceptibility, transgenic mice expressing human rather than mouse TLR4/MD-2 were generated. We found the increased susceptibility to Y. pestis for "humanized" TLR4/MD-2 mice directly paralleled blunted inflammatory cytokine production in response to stimulation with purified LPS. By contrast, for other Gram-negative pathogens with highly acylated lipid A including Salmonella enterica or Escherichia coli, infection susceptibility and the response after stimulation with LPS were indistinguishable between mice expressing human or mouse TLR4/MD-2. Thus, Y. pestis exploits temperature-dependent shifts in LPS acylation to selectively evade recognition by human TLR4/MD-2 uncovered with "humanized" TLR4/MD-2 transgenic mice.

The effect of lipopolysaccharides on the expression of CD14 and TLR4 in rat Kupffer cells.

PubMed

Feng, Jun-Ming; Shi, Jing-Quan; Liu, You-Sheng

2003-05-01

To assess the effect of lipopolysaccharides (LPS) on the expression of CD14 and TLR4 in rat Kupffer cells (KCs). In rat KCs induced by LPS, the changes of CD14 and TLR4 expression were measured by RT-PCR and immunohistochemistry, and the expressions of TNF-alphamRNA, IL-6mRNA or the concentrations of TNF-alpha, IL-6 were estimated by in situ hybridization, radioimmunoassay, and others. The expressions of CD14 and TLR4 in KCs induced by LPS were markedly increased in a dose-dependent manner (10 mg/L-1 microg/L) or in a time-dependent manner (0.5 h-24 h), with the peaked expression of CD14 at 3-6 hours. The expressions of CD14 and TLR4 in KCs stimulated by the active mediators from KCs which had been exposed to LPS for 1 hour were obviously increased. There is a close relationship between LPS or the active mediators from KCs induced by LPS and the expressions of CD14, TLR4. It is implied that the increase of TLR4, CD14 expression may be induced by LPS within 1-3 hours, and further increase of TLR4, CD14 expression may be correlated with the cytokines produced by KCs.

Morphine amplifies mechanical allodynia via TLR4 in a rat model of spinal cord injury

PubMed Central

Ellis, Amanda; Grace, Peter M.; Wieseler, Julie; Favret, Jacob; Springer, Kendra; Skarda, Bryce; Hutchinson, Mark R.; Falci, Scott; Rice, Kenner C.; Maier, Steven F.; Watkins, Linda R.

2016-01-01

Central neuropathic pain (CNP) is a pervasive, debilitating problem that impacts thousands of people living with central nervous system disorders, including spinal cord injury (SCI). Current therapies for treating this type of pain are ineffective and often have dose-limiting side effects. Although opioids are one of the most commonly used CNP treatments, recent animal literature has indicated that administering opioids shortly after a traumatic injury can actually have deleterious effects on long-term health and recovery. In order to study the deleterious effects of administering morphine shortly after trauma, we employed our low thoracic (T13) dorsal root avulsion model (Spinal Neuropathic Avulsion Pain, SNAP). Administering a weeklong course of 10 mg/kg/day morphine beginning 24 hr after SNAP resulted in amplified mechanical allodynia. Co-administering the non-opioid toll-like receptor 4 (TLR4) antagonist (+)-naltrexone throughout the morphine regimen prevented morphine-induced amplification of SNAP. Exploration of changes induced by early post-trauma morphine revealed that this elevated gene expression of TLR4, TNF, IL-1β, and NLRP3, as well as IL-1β protein at the site of spinal cord injury. These data suggest that a short course of morphine administered early after spinal trauma can exacerbate CNP in the long term. TLR4 initiates this phenomenon and, as such, may be potential therapeutic targets for preventing the deleterious effects of administering opioids after traumatic injury. PMID:27519154

Accumulation mode particles and LPS exposure induce TLR-4 dependent and independent inflammatory responses in the lung.

PubMed

Fonceca, Angela M; Zosky, Graeme R; Bozanich, Elizabeth M; Sutanto, Erika N; Kicic, Anthony; McNamara, Paul S; Knight, Darryl A; Sly, Peter D; Turner, Debra J; Stick, Stephen M

2018-01-22

Accumulation mode particles (AMP) are formed from engine combustion and make up the inhalable vapour cloud of ambient particulate matter pollution. Their small size facilitates dispersal and subsequent exposure far from their original source, as well as the ability to penetrate alveolar spaces and capillary walls of the lung when inhaled. A significant immuno-stimulatory component of AMP is lipopolysaccharide (LPS), a product of Gram negative bacteria breakdown. As LPS is implicated in the onset and exacerbation of asthma, the presence or absence of LPS in ambient particulate matter (PM) may explain the onset of asthmatic exacerbations to PM exposure. This study aimed to delineate the effects of LPS and AMP on airway inflammation, and potential contribution to airways disease by measuring airway inflammatory responses induced via activation of the LPS cellular receptor, Toll-like receptor 4 (TLR-4). The effects of nebulized AMP, LPS and AMP administered with LPS on lung function, cellular inflammatory infiltrate and cytokine responses were compared between wildtype mice and mice not expressing TLR-4. The presence of LPS administered with AMP appeared to drive elevated airway resistance and sensitivity via TLR-4. Augmented TLR4 driven eosinophilia and greater TNF-α responses observed in AMP-LPS treated mice independent of TLR-4 expression, suggests activation of allergic responses by TLR4 and non-TLR4 pathways larger than those induced by LPS administered alone. Treatment with AMP induced macrophage recruitment independent of TLR-4 expression. These findings suggest AMP-LPS as a stronger stimulus for allergic inflammation in the airways then LPS alone.

Role of TLR4 in the Modulation of Central Amygdala GABA Transmission by CRF Following Restraint Stress.

PubMed

Varodayan, F P; Khom, S; Patel, R R; Steinman, M Q; Hedges, D M; Oleata, C S; Homanics, G E; Roberto, M; Bajo, M

2018-01-04

Stress induces neuroimmune responses via Toll-like receptor 4 (TLR4) activation. Here, we investigated the role of TLR4 in the effects of the stress peptide corticotropin-releasing factor (CRF) on GABAergic transmission in the central nucleus of the amygdala (CeA) following restraint stress. Tlr4 knock out (KO) and wild-type rats were exposed to no stress (naïve), a single restraint stress (1 h) or repeated restraint stress (1 h per day for 3 consecutive days). After 1 h recovery from the final stress session, whole-cell patch-clamp electrophysiology was used to investigate the effects of CRF (200 nM) on CeA GABAA-mediated spontaneous inhibitory postsynaptic currents (sIPSCs). TLR4 does not regulate baseline GABAergic transmission in the CeA of naive and stress-treated animals. However, CRF significantly increased the mean sIPSC frequencies (indicating enhanced GABA release) across all genotypes and stress treatments, except for the Tlr4 KO rats that experienced repeated restraint stress. Overall, our results suggest a limited role for TLR4 in CRF's modulation of CeA GABAergic synapses in naïve and single stress rats, though TLR4-deficient rats that experienced repeated psychological stress exhibit a blunted CRF cellular response. TLR4 has a limited role in CRF's activation of the CeA under basal conditions, but interacts with the CRF system to regulate GABAergic synapse function in animals that experience repeated psychological stress. © The Author(s) 2018. Medical Council on Alcohol and Oxford University Press. All rights reserved.

Maternal western diet causes inflammatory milk and TLR2/4-dependent neonatal toxicity

PubMed Central

Du, Yang; Yang, Marie; Lee, Syann; Behrendt, Cassie L.; Hooper, Lora V.; Saghatelian, Alan; Wan, Yihong

2012-01-01

For all newborn mammals, mother's milk is the perfect nourishment, crucial for their postnatal development. Here we report that, unexpectedly, maternal western diet consumption in mice causes the production of toxic milk that contains excessive long chain and saturated fatty acids, which triggers ceramide accumulation and inflammation in the nursing neonates, manifested as alopecia. This neonatal toxicity requires Toll-like-receptors (TLR), but not gut microbiota, because TLR2/4 deletion or TLR4 inhibition confers resistance, whereas germ-free mice remain sensitive. These findings unravel maternal western diet-induced inflammatory milk secretion as a novel aspect of the metabolic syndrome at the maternal offspring interface. PMID:22713870

Maternal western diet causes inflammatory milk and TLR2/4-dependent neonatal toxicity.

PubMed

Du, Yang; Yang, Marie; Lee, Syann; Behrendt, Cassie L; Hooper, Lora V; Saghatelian, Alan; Wan, Yihong

2012-06-15

For all newborn mammals, mother's milk is the perfect nourishment, crucial for their postnatal development. Here we report that, unexpectedly, maternal western diet consumption in mice causes the production of toxic milk that contains excessive long chain and saturated fatty acids, which triggers ceramide accumulation and inflammation in the nursing neonates, manifested as alopecia. This neonatal toxicity requires Toll-like-receptors (TLR), but not gut microbiota, because TLR2/4 deletion or TLR4 inhibition confers resistance, whereas germ-free mice remain sensitive. These findings unravel maternal western diet-induced inflammatory milk secretion as a novel aspect of the metabolic syndrome at the maternal offspring interface.

A Role for TLR4 in Clostridium difficile Infection and the Recognition of Surface Layer Proteins

PubMed Central

Ryan, Anthony; Lynch, Mark; Smith, Sinead M.; Amu, Sylvie; Nel, Hendrik J.; McCoy, Claire E.; Dowling, Jennifer K.; Draper, Eve; O'Reilly, Vincent; McCarthy, Ciara; O'Brien, Julie; Ní Eidhin, Déirdre; O'Connell, Mary J.; Keogh, Brian; Morton, Charles O.; Rogers, Thomas R.; Fallon, Padraic G.; O'Neill, Luke A.

2011-01-01

Clostridium difficile is the etiological agent of antibiotic-associated diarrhoea (AAD) and pseudomembranous colitis in humans. The role of the surface layer proteins (SLPs) in this disease has not yet been fully explored. The aim of this study was to investigate a role for SLPs in the recognition of C. difficile and the subsequent activation of the immune system. Bone marrow derived dendritic cells (DCs) exposed to SLPs were assessed for production of inflammatory cytokines, expression of cell surface markers and their ability to generate T helper (Th) cell responses. DCs isolated from C3H/HeN and C3H/HeJ mice were used in order to examine whether SLPs are recognised by TLR4. The role of TLR4 in infection was examined in TLR4-deficient mice. SLPs induced maturation of DCs characterised by production of IL-12, TNFα and IL-10 and expression of MHC class II, CD40, CD80 and CD86. Furthermore, SLP-activated DCs generated Th cells producing IFNγ and IL-17. SLPs were unable to activate DCs isolated from TLR4-mutant C3H/HeJ mice and failed to induce a subsequent Th cell response. TLR4−/− and Myd88−/−, but not TRIF−/− mice were more susceptible than wild-type mice to C. difficile infection. Furthermore, SLPs activated NFκB, but not IRF3, downstream of TLR4. Our results indicate that SLPs isolated from C. difficile can activate innate and adaptive immunity and that these effects are mediated by TLR4, with TLR4 having a functional role in experimental C. difficile infection. This suggests an important role for SLPs in the recognition of C. difficile by the immune system. PMID:21738466

TLR2 and TLR4 signaling pathways are required for recombinant Brucella abortus BCSP31-induced cytokine production, functional upregulation of mouse macrophages, and the Th1 immune response in vivo and in vitro.

PubMed

Li, Jia-Yun; Liu, Yuan; Gao, Xiao-Xue; Gao, Xiang; Cai, Hong

2014-09-01

Brucella abortus is a zoonotic Gram-negative pathogen that causes brucelosis in ruminants and humans. Toll-like receptors (TLRs) recognize Brucella abortus and initiate antigen-presenting cell activities that affect both innate and adaptive immunity. In this study, we focused on recombinant Brucella cell-surface protein 31 (rBCSP31) to determine its effects on mouse macrophages. Our results demonstrated that rBCSP31 induced TNF-α, IL-6 and IL-12p40 production, which depended on the activation of mitogen-activated protein kinases (MAPKs) by stimulating the rapid phosphorylation of p38 and JNK and the activation of transcription factor NF-κB in macrophages. In addition, continuous exposure (>24 h) of RAW264.7 cells to rBCSP31 significantly enhanced IFN-γ-induced expression of MHC-II and the ability to present rBCSP31 peptide to CD4(+) T cells. Furthermore, we found that rBCSP31 could interact with both TLR2 and TLR4. The rBCSP31-induced cytokine production by macrophages from TLR2(-/-) and TLR4(-/-) mice was lower than that from C57BL/6 macrophages, and the activation of NF-κB and MAPKs was attenuated in macrophages from TLR2(-/-) and TLR4(-/-) mice. In addition, CD4(+) T cells from C57BL/6 mice immunized with rBCSP31 produced higher levels of IFN-γ and IL-2 compared with CD4(+) T cells from TLR2(-/-) and TLR4(-/-) mice. Macrophages from immunized C57BL/6 mice produced higher levels of IL-12p40 than those from TLR2(-/-) and TLR4(-/-) mice. Furthermore, immunization with rBCSP31 provided better protection in C57BL/6 mice than in TLR2(-/-) and TLR4(-/-) mice after B. abortus 2308 challenge. These results indicate that rBCSP31 is a TLR2 and TLR4 agonist that induces cytokine production, upregulates macrophage function and induces the Th1 immune response.

Ursolic acid isolated from Uncaria rhynchophylla activates human dendritic cells via TLR2 and/or TLR4 and induces the production of IFN-gamma by CD4+ naïve T cells.

PubMed

Jung, Tae-Young; Pham, Thanh Nhan Nguyen; Umeyama, Akemi; Shoji, Noboru; Hashimoto, Toshihiro; Lee, Je-Jung; Takei, Masao

2010-09-25

Ursolic acid is triterpene isolated from Uncaria rhynchophylla and is a pharmacologically active substance. The induction of dendritic cell maturation is critical for the induction of Ag-specific T-lymphocyte response and may be essential for the development of human vaccine relying on T cell immunity. In this study, we investigated that the effect of Ursolic acid on the phenotypic and functional maturation of human monocyte-derived dendritic cells in vitro. Dendritic cells harvested on day 8 were examined using functional assay. The expression levels of CD1a, CD80, CD83, CD86, HLA-DR and CCR7 on Ursolic acid-primed dendritic cells was slightly enhanced. Ursolic acid dose-dependently enhanced the T cell stimulatory capacity in an allogeneic mixed lymphocyte reaction, as measured by T cell proliferation. The production of IL-12p70 induced by Ursolic acid-primed dendritic cells was inhibited by the anti-Toll-like receptor-2 (TLR2) mAb and anti-TLR4 mAb. Moreover, Ursolic acid-primed dendritic cells expressed levels of mRNA coding for both TLR2 and TLR4. The majority of cells produced considerable interferon-gamma (IFN-gamma), but also small amounts of interleukin (IL-4)-4. Ursolic acid-primed dendritic cells have an intermediate migratory capacity towards CCL19 and CCL21. These results suggest that Ursolic acid modulates human dendritic cells function in a fashion that favors Th1 polarization via the activation of IL-12p70 dependent on TLR2 and/or TLR4, and may be used on dendritic cells-based vaccines for cancer immunotherapy. 2010 Elsevier B.V. All rights reserved.

Saturated and unsaturated fat induce hepatic insulin resistance independently of TLR-4 signaling and ceramide synthesis in vivo.

PubMed

Galbo, Thomas; Perry, Rachel J; Jurczak, Michael J; Camporez, João-Paulo G; Alves, Tiago C; Kahn, Mario; Guigni, Blas A; Serr, Julie; Zhang, Dongyan; Bhanot, Sanjay; Samuel, Varman T; Shulman, Gerald I

2013-07-30

Hepatic insulin resistance is a principal component of type 2 diabetes, but the cellular and molecular mechanisms responsible for its pathogenesis remain unknown. Recent studies have suggested that saturated fatty acids induce hepatic insulin resistance through activation of the toll-like receptor 4 (TLR-4) receptor in the liver, which in turn transcriptionally activates hepatic ceramide synthesis leading to inhibition of insulin signaling. In this study, we demonstrate that TLR-4 receptor signaling is not directly required for saturated or unsaturated fat-induced hepatic insulin resistance in both TLR-4 antisense oligonucleotide treated and TLR-4 knockout mice, and that ceramide accumulation is not dependent on TLR-4 signaling or a primary event in hepatic steatosis and impairment of insulin signaling. Further, we show that both saturated and unsaturated fats lead to hepatic accumulation of diacylglycerols, activation of PKCε, and impairment of insulin-stimulated IRS-2 signaling. These data demonstrate that saturated fat-induced insulin resistance is independent of TLR-4 activation and ceramides.

Saturated and unsaturated fat induce hepatic insulin resistance independently of TLR-4 signaling and ceramide synthesis in vivo

PubMed Central

Galbo, Thomas; Perry, Rachel J.; Jurczak, Michael J.; Camporez‎, João-Paulo G.; Alves, Tiago C.; Kahn, Mario; Guigni, Blas A.; Serr, Julie; Zhang, Dongyan; Bhanot, Sanjay; Samuel, Varman T.; Shulman, Gerald I.

2013-01-01

Hepatic insulin resistance is a principal component of type 2 diabetes, but the cellular and molecular mechanisms responsible for its pathogenesis remain unknown. Recent studies have suggested that saturated fatty acids induce hepatic insulin resistance through activation of the toll-like receptor 4 (TLR-4) receptor in the liver, which in turn transcriptionally activates hepatic ceramide synthesis leading to inhibition of insulin signaling. In this study, we demonstrate that TLR-4 receptor signaling is not directly required for saturated or unsaturated fat-induced hepatic insulin resistance in both TLR-4 antisense oligonucleotide treated and TLR-4 knockout mice, and that ceramide accumulation is not dependent on TLR-4 signaling or a primary event in hepatic steatosis and impairment of insulin signaling. Further, we show that both saturated and unsaturated fats lead to hepatic accumulation of diacylglycerols, activation of PKCε, and impairment of insulin-stimulated IRS-2 signaling. These data demonstrate that saturated fat-induced insulin resistance is independent of TLR-4 activation and ceramides. PMID:23840067

Heterodimerization of TLR2 with TLR1 or TLR6 expands the ligand spectrum but does not lead to differential signaling.

PubMed

Farhat, Katja; Riekenberg, Sabine; Heine, Holger; Debarry, Jennifer; Lang, Roland; Mages, Jörg; Buwitt-Beckmann, Ute; Röschmann, Kristina; Jung, Günther; Wiesmüller, Karl-Heinz; Ulmer, Artur J

2008-03-01

TLR are primary triggers of the innate immune system by recognizing various microorganisms through conserved pathogen-associated molecular patterns. TLR2 is the receptor for a functional recognition of bacterial lipopeptides (LP) and is up-regulated during various disorders such as chronic obstructive pulmonary disease and sepsis. This receptor is unique in its ability to form heteromers with TLR1 or TLR6 to mediate intracellular signaling. According to the fatty acid pattern as well as the assembling of the polypeptide tail, LP can signal through TLR2 in a TLR1- or TLR6-dependent manner. There are also di- and triacylated LP, which stimulate TLR1-deficient cells and TLR6-deficient cells. In this study, we investigated whether heterodimerization evolutionarily developed to broaden the ligand spectrum or to induce different immune responses. We analyzed the signal transduction pathways activated through the different TLR2 dimers using the three LP, palmitic acid (Pam)octanoic acid (Oct)(2)C-(VPGVG)(4)VPGKG, fibroblast-stimulating LP-1, and Pam(2)C-SK(4). Dominant-negative forms of signaling molecules, immunoblotting of MAPK, as well as microarray analysis indicate that all dimers use the same signaling cascade, leading to an identical pattern of gene activation. We conclude that heterodimerization of TLR2 with TLR1 or TLR6 evolutionarily developed to expand the ligand spectrum to enable the innate immune system to recognize the numerous, different structures of LP present in various pathogens. Thus, although mycoplasma and Gram-positive and Gram-negative bacteria may activate different TLR2 dimers, the development of different signal pathways in response to different LP does not seem to be of vital significance for the innate defense system.

Studies of the TLR4-associated protein MD-2 using yeast-display and mutational analyses

PubMed Central

Mattis, Daiva M.; Chervin, Adam; Ranoa, Diana; Kelley, Stacy; Tapping, Richard; Kranz, David M.

2015-01-01

Bacterial lipopolysaccharide (LPS) activates the innate immune system by forming a complex with myeloid differentiation factor 2 (MD-2) and Toll-like receptor 4 (TLR4), which is present on antigen presenting cells. MD-2 plays an essential role in this activation of the innate immune system as a member of the ternary complex, TLR4:MD-2:LPS. With the goal of further understanding the molecular details of the interaction of MD-2 with LPS and TLR4, and possibly toward engineering dominant negative regulators of the MD-2 protein, here we subjected MD-2 to a mutational analysis using yeast display. The approach included generation of site-directed alanine mutants, and ligand-driven selections of MD-2 mutant libraries. Our findings showed that: 1) proline mutations in the F119-K132 loop that binds LPS were strongly selected for enhanced yeast surface stability, 2) there was a preference for positive-charged side chains (R/K) at residue 120 for LPS binding, and negative-charged side chains (D/E) for TLR4 binding, 3) aromatic residues were strongly preferred at F119 and F121 for LPS binding, and 4) an MD-2 mutant (T84N/D101A/S118A/S120D/K122P) exhibited increased binding to TLR4 but decreased binding to LPS. These studies revealed the impact of specific residues and regions of MD-2 on the binding of LPS and TLR4, and they provide a framework for further directed evolution of the MD-2 protein. PMID:26320630

SOCS2 overexpression alleviates diabetic nephropathy in rats by inhibiting the TLR4/NF-κB pathway

PubMed Central

Yang, Suxia; Zhang, Junwei; Wang, Shiying; Zhao, Xinxin; Shi, Jun

2017-01-01

Suppressor of cytokine signaling 2 (SOCS2) was reported to be involved in the development of Diabetic Nephropathy (DN). However, its underlying mechanism remains undefined. Western blot was carried out to determine the expressions of SOCS2, Toll-like receptors 4 (TLR4) and nuclear factor kappa B (NF-κB) pathway-related proteins in DN patients, streptozotocin (STZ)-induced DN rats and high glucose (HG)-stimulated podocytes. The effects of SOCS2 overexpression on renal injury, the inflammatory cytokines production, renal pathological changes, apoptosis and the TLR4/NF-κB pathway in DN rats or HG-stimulated podocytes were investigated. TLR4 antagonist TAK-242 and NF-κB inhibitor PDTC were used to confirm the functional mechanism of SOCS2 overexpression in HG-stimulated podocytes. SOCS2 was down-regulated, while TLR4 and NF-κB were up-regulated in renal tissues of DN patients and DN rats. Ad-SOCS2 infection alleviated STZ-induced renal injury and pathological changes and inhibited STZ-induced IL-6, IL-1β and MCP-1 generation and activation of the TLR4/NF-κB pathway in DN rats. SOCS2 overexpression attenuated apoptosis, suppressed the inflammatory cytokines expression, and inactivated the TLR4/NF-κB pathway in HG-stimulated podocytes. Suppression of the TLR4/NF-κB pathway enhanced the inhibitory effect of SOCS2 overexpression on apoptosis and inflammatory cytokines expressions in HG-stimulated podocytes. SOCS2 overexpression alleviated the development of DN by inhibiting the TLR4/NF-κB pathway, contributing to developing new therapeutic strategies against DN. PMID:29207635

CD14 and TLR4 are expressed early in tammar (Macropus eugenii) neonate development.

PubMed

Daly, Kerry A; Lefévre, Christophe; Nicholas, Kevin; Deane, Elizabeth; Williamson, Peter

2008-04-01

Marsupials are born in a relatively underdeveloped state and develop during a period of intensive maturation in the postnatal period. During this period, the young marsupial lacks a competent immune system, but manages to survive despite the potential of exposure to environmental pathogens. Passive immune transfer via the milk is one well-recognised strategy to compensate the neonate, but there also may be innate immune mechanisms in place. In this study, CD14 and Toll-like receptor 4 (TLR4), integral molecular components of pathogen recognition, were identified and characterised for the first time in a marsupial, the tammar wallaby (Macropus eugenii). Functional motifs of tammar CD14 and the toll/interleukin receptor (TIR) domain of TLR4 were highly conserved. The lipopolysaccharide (LPS) binding residues and the TLR4 interaction site of CD14 were conserved in all marsupials. The TIR signalling domain had 84% identity within marsupials and 77% with eutherians. Stimulation of adult tammar leukocytes resulted in the induction of a biphasic pattern of CD14 and TLR4 expression, and coincided with increased production of the pro-inflammatory cytokine TNF-alpha. Differential patterns of expression of CD14 and TLR4 were observed in tammar pouch young early in development, suggesting that early maturation of the innate immune system in these animals may have developed as an immune survival strategy to protect the marsupial neonate from exposure to microbial pathogens.

The HMGB1-TLR4 axis contributes to myocardial ischemia/reperfusion injury via regulation of cardiomyocyte apoptosis.

PubMed

Ding, Hua-Sheng; Yang, Jun; Chen, Ping; Yang, Jian; Bo, Sun-Qing; Ding, Jia-Wang; Yu, Qin-Qin

2013-09-15

Toll-like receptor 4 (TLR4) and its ligand high mobility group box 1 (HMGB1), are known for playing central roles in ischemia-reperfusion injury in myocardium. However, the detailed mechanisms of TLR4 and HMGB1 are not fully understood. The aim of this study was to investigate the effects and possible mechanisms of the HMGB1-TLR4 axis and cardiomyocyte apoptosis on myocardial ischemic damage. Artificial oxygen ventilated anesthetized C3H/HeN mice and C3H/HeJ mice were subjected to 30 min of left anterior descending coronary artery occlusion followed by 6h of reperfusion. The myocardial infarct size, HMGB1 levels, apoptosis index, Bax, Bcl-2 and TNF-α mRNA levels were assessed. The results showed that a lowered amount of cardiomyocyte apoptosis and infarct size in the myocardium of TLR4-mutant mice after myocardial I/R and that TLR4 deficiency notably inhibited the expression of HMGB1 and TNF-a, both of which were up-regulated by ischemia/reperfusion. These findings suggest that the HMGB1-TLR4 axis plays a pathogenic role in triggering cardiomyocyte apoptosis during myocardial I/R injury and that the possible mechanism for this process is the result of released cytokines and inflammatory response involved in the HMGB1/TLR4-related pathway. Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.

TLR2 and TLR4 signaling pathways are required for recombinant Brucella abortus BCSP31-induced cytokine production, functional upregulation of mouse macrophages, and the Th1 immune response in vivo and in vitro

PubMed Central

Li, Jia-Yun; Liu, Yuan; Gao, Xiao-Xue; Gao, Xiang; Cai, Hong

2014-01-01

Brucella abortus is a zoonotic Gram-negative pathogen that causes brucelosis in ruminants and humans. Toll-like receptors (TLRs) recognize Brucella abortus and initiate antigen-presenting cell activities that affect both innate and adaptive immunity. In this study, we focused on recombinant Brucella cell-surface protein 31 (rBCSP31) to determine its effects on mouse macrophages. Our results demonstrated that rBCSP31 induced TNF-α, IL-6 and IL-12p40 production, which depended on the activation of mitogen-activated protein kinases (MAPKs) by stimulating the rapid phosphorylation of p38 and JNK and the activation of transcription factor NF-κB in macrophages. In addition, continuous exposure (>24 h) of RAW264.7 cells to rBCSP31 significantly enhanced IFN-γ-induced expression of MHC-II and the ability to present rBCSP31 peptide to CD4+ T cells. Furthermore, we found that rBCSP31 could interact with both TLR2 and TLR4. The rBCSP31-induced cytokine production by macrophages from TLR2−/− and TLR4−/− mice was lower than that from C57BL/6 macrophages, and the activation of NF-κB and MAPKs was attenuated in macrophages from TLR2−/− and TLR4−/− mice. In addition, CD4+ T cells from C57BL/6 mice immunized with rBCSP31 produced higher levels of IFN-γ and IL-2 compared with CD4+ T cells from TLR2−/− and TLR4−/− mice. Macrophages from immunized C57BL/6 mice produced higher levels of IL-12p40 than those from TLR2−/− and TLR4−/− mice. Furthermore, immunization with rBCSP31 provided better protection in C57BL/6 mice than in TLR2−/− and TLR4−/− mice after B. abortus 2308 challenge. These results indicate that rBCSP31 is a TLR2 and TLR4 agonist that induces cytokine production, upregulates macrophage function and induces the Th1 immune response. PMID:24769793

Correlation of TLR4 and KLF7 in Inflammation Induced by Obesity.

PubMed

Wang, Cuizhe; Ha, Xiaodan; Li, Wei; Xu, Peng; Gu, Yajuan; Wang, Tingting; Wang, Yan; Xie, Jianxin; Zhang, Jun

2017-02-01

Objective Recent studies have revealed a link between toll-like receptors (TLRs), Kruppel-like factors (KLFs), and the adipose tissue inflammation associated with obesity. TLR4 is associated with chronic inflammation in obesity. KLF7 is known to play an important role in the differentiation of adipocytes, but its role in visceral adipose tissue inflammation has not yet been investigated. Thus, the objective of this study was to determine the correlation of TLR4 and KLF7 in inflammation induced by obesity. Methods A total of 32 Wistar male rat subjects were fed in the center for experimental animals of Shihezi University. The rats were divided into normal control (NC) and high-fat diet (HFD) group. Surgical instruments were used to collect rats' visceral adipose tissue samples in the 10th week after HFD feeding. Ninety-five Uygur subjects between 20 and 90 years old were enrolled in the present study. The subjects were divided into two groups: the normal control group (NC, 18.0 kg/m 2  ≤ BMI ≤ 23.9 kg/m 2 , n = 50) and the obesity group (OB, BMI ≥ 28 kg/m 2 , n = 45), and visceral adipose tissue was collected from the subjects. Anthropometric and clinical parameters were measured using standard procedures; biochemical indices were detected using the glucose oxidase-peroxidase method and a standardized automatic biochemistry analyzer; the plasma levels of inflammatory factors and adipocytokines were measured by enzyme-linked immunosorbent assay (ELISA); the mRNA and protein expression levels of key genes involved in the inflammatory signaling pathway were measured by real-time PCR and Western blot. Results In rats, compared with the NC group, the weight, Lee's index, waist circumference, visceral fat mass, and the plasma level of Glu, TG, FFA, and TNF-α were higher in the HFD group, while the plasma levels of LPT and APN were significantly lower in the HFD group in the 10th week. Furthermore, compared with the NC group, visceral adipose

Identification of the HSPB4/TLR2/NF-κB axis in macrophage as a therapeutic target for sterile inflammation of the cornea

PubMed Central

Oh, Joo Youn; Choi, Hosoon; Lee, Ryang Hwa; Roddy, Gavin W; Ylöstalo, Joni H; Wawrousek, Eric; Prockop, Darwin J

2012-01-01

Sterile inflammation underlies many diseases of the cornea including serious chemical burns and the common dry eye syndrome. In search for therapeutic targets for corneal inflammation, we defined the kinetics of neutrophil infiltration in a model of sterile injury to the cornea and identified molecular and cellular mechanisms triggering inflammatory responses. Neutrophil infiltration occurred in two phases: a small initial phase (Phase I) that began within 15 min after injury, and a larger second phase (Phase II) that peaked at 24–48 h. Temporal analysis suggested that the neuropeptide secretoneurin initiated Phase I without involvement of resident macrophages. Phase II was initiated by the small heat shock protein HSPB4 that was released from injured keratocytes and that activated resident macrophages via the TLR2/NF-κB pathway. The Phase II inflammation was responsible for vision-threatening opacity and was markedly suppressed by different means of inhibition of the HSPB4/TLR2/NF-κB axis: in mice lacking HSPB4 or TLR2, by antibodies to HSPB4 or by TNF-α stimulated gene/protein 6 that CD44-dependently inhibits the TLR2/NF-κB pathway. Therefore, our data identified the HSPB4/TLR2/NF-κB axis in macrophages as an effective target for therapy of corneal inflammation. PMID:22359280

Fine-Mapping of Common Genetic Variants Associated with Colorectal Tumor Risk Identified Potential Functional Variants

PubMed Central

Gala, Manish; Abecasis, Goncalo; Bezieau, Stephane; Brenner, Hermann; Butterbach, Katja; Caan, Bette J.; Carlson, Christopher S.; Casey, Graham; Chang-Claude, Jenny; Conti, David V.; Curtis, Keith R.; Duggan, David; Gallinger, Steven; Haile, Robert W.; Harrison, Tabitha A.; Hayes, Richard B.; Hoffmeister, Michael; Hopper, John L.; Hudson, Thomas J.; Jenkins, Mark A.; Küry, Sébastien; Le Marchand, Loic; Leal, Suzanne M.; Newcomb, Polly A.; Nickerson, Deborah A.; Potter, John D.; Schoen, Robert E.; Schumacher, Fredrick R.; Seminara, Daniela; Slattery, Martha L.; Hsu, Li; Chan, Andrew T.; White, Emily; Berndt, Sonja I.; Peters, Ulrike

2016-01-01

Genome-wide association studies (GWAS) have identified many common single nucleotide polymorphisms (SNPs) associated with colorectal cancer risk. These SNPs may tag correlated variants with biological importance. Fine-mapping around GWAS loci can facilitate detection of functional candidates and additional independent risk variants. We analyzed 11,900 cases and 14,311 controls in the Genetics and Epidemiology of Colorectal Cancer Consortium and the Colon Cancer Family Registry. To fine-map genomic regions containing all known common risk variants, we imputed high-density genetic data from the 1000 Genomes Project. We tested single-variant associations with colorectal tumor risk for all variants spanning genomic regions 250-kb upstream or downstream of 31 GWAS-identified SNPs (index SNPs). We queried the University of California, Santa Cruz Genome Browser to examine evidence for biological function. Index SNPs did not show the strongest association signals with colorectal tumor risk in their respective genomic regions. Bioinformatics analysis of SNPs showing smaller P-values in each region revealed 21 functional candidates in 12 loci (5q31.1, 8q24, 11q13.4, 11q23, 12p13.32, 12q24.21, 14q22.2, 15q13, 18q21, 19q13.1, 20p12.3, and 20q13.33). We did not observe evidence of additional independent association signals in GWAS-identified regions. Our results support the utility of integrating data from comprehensive fine-mapping with expanding publicly available genomic databases to help clarify GWAS associations and identify functional candidates that warrant more onerous laboratory follow-up. Such efforts may aid the eventual discovery of disease-causing variant(s). PMID:27379672

TLR4-HMGB1 signaling pathway affects the inflammatory reaction of autoimmune myositis by regulating MHC-I.

PubMed

Wan, Zemin; Zhang, Xiujuan; Peng, Anping; He, Min; Lei, Zhenhua; Wang, Yunxiu

2016-12-01

To analyze the effects of TLR4 on the expression of the HMGB1, MHC-I and downstream cytokines IL-6 and TNF-α, and to investigate the biological role of the TLR4-HMGB1 signaling pathway in the development of the autoimmune myositis. We built mice models with experimental autoimmune myositis (EAM) and used the inverted screen experiment to measure their muscle endurance; we also examined inflammatory infiltration of muscle tissues after HE staining; and we assessed the expression of MHC-I using immunohistochemistry. In addition, peripheral blood mononuclear cells (PBMC) were extracted and flow cytometry was utilized to detect the effect of IFN-γ on the expression of MHC-I. Furthermore, PBMCs were treated with IFN-γ, anti-TLR4, anti-HMGB1 and anti-MHC-I. Real-time PCR and western blotting were employed to examine the expressions of TLR4, HMGB1 and MHC-I in different groups. The ELISA method was also utilized to detect the expression of the downstream cytokines TNF-α and IL-6. The expressions of TLR4, HMGB1 and MHC-I in muscle tissues from mice with EAM were significantly higher than those in the control group (all P<0.05). After IFN-γ treatment, the expressions of TLR4, HMGB1, MHC-I, TNF-α and IL-6 in PBMCs significantly increased (all P<0.05). The treatment of anti-TLR4, anti-HMGB1 and anti-MHC-I could significantly downregulate the expression of MHC-I (all P<0.05). In addition, anti-TLR4 and anti-HMGB1 significantly reduced the expression of TNF-α and IL-6 (all P<0.05). The TLR4-HMGB1 signaling pathway affects the process of autoimmune myositis inflammation by regulating the expression of MHC-I and other pro-inflammatory cytokines. Copyright © 2016 Elsevier B.V. All rights reserved.

Down-regulation of Toll-like Receptor TLR4 Is Associated with HPV DNA Integration in Penile Carcinoma.

PubMed

Damasdi, Miklos; Kovacs, Krisztina; Farkas, Nelli; Jakab, Ferenc; Kovacs, Gyula

2017-10-01

Development of penile cancers is attributed to HPV-related carcinogenesis. Our aim was to analyze HPV positivity and TLR4, p16 ink4a and p53 expression. HPV presence was assessed with virus-specific TaqMan PCR and HPV Genotyping Test in 31 penile cancers. Immunohistochemistry was carried out on tissue microarray. TLR4 expression was detected in 4 of the 16 HPV positive and 13 of the 15 HPV negative tumors. We found a significant inverse correlation between HPV positivity and TLR4 expression (p=0.0006). Ten of the 16 HPV-positive but none of the 15 HPV-negative tumors expressed p16INK4a. A significant correlation was seen between p53 expression and lack of HPV DNA (p=0.0191) as well as between TLR4 and p53 expression (p=0.0198) in penile cancers. Our findings suggest a protective role of TLR4 expression against HPV DNA integration and the viral and non-viral carcinogenesis of penile cancer. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Systemic Inflammatory Response Syndrome After Major Abdominal Surgery Predicted by Early Upregulation of TLR4 and TLR5.

PubMed

Lahiri, Rajiv; Derwa, Yannick; Bashir, Zora; Giles, Edward; Torrance, Hew D T; Owen, Helen C; O'Dwyer, Michael J; O'Brien, Alastair; Stagg, Andrew J; Bhattacharya, Satyajit; Foster, Graham R; Alazawi, William

2016-05-01

To study innate immune pathways in patients undergoing hepatopancreaticobiliary surgery to understand mechanisms leading to enhanced inflammatory responses and identifying biomarkers of adverse clinical consequences. Patients undergoing major abdominal surgery are at risk of life-threatening systemic inflammatory response syndrome (SIRS) and sepsis. Early identification of at-risk patients would allow tailored postoperative care and improve survival. Two separate cohorts of patients undergoing major hepatopancreaticobiliary surgery were studied (combined n = 69). Bloods were taken preoperatively, on day 1 and day 2 postoperatively. Peripheral blood mononuclear cells and serum were separated and immune phenotype and function assessed ex vivo. Early innate immune dysfunction was evident in 12 patients who subsequently developed SIRS (postoperative day 6) compared with 27 who did not, when no clinical evidence of SIRS was apparent (preoperatively or days 1 and 2). Serum interleukin (IL)-6 concentration and monocyte Toll-like receptor (TLR)/NF-κB/IL-6 functional pathways were significantly upregulated and overactive in patients who developed SIRS (P < 0.0001). Interferon α-mediated STAT1 phosphorylation was higher preoperatively in patients who developed SIRS. Increased TLR4 and TLR5 gene expression in whole blood was demonstrated in a separate validation cohort of 30 patients undergoing similar surgery. Expression of TLR4/5 on monocytes, particularly intermediate CD14CD16 monocytes, on day 1 or 2 predicted SIRS with accuracy 0.89 to 1.0 (areas under receiver operator curves). These data demonstrate the mechanism for IL-6 overproduction in patients who develop postoperative SIRS and identify markers that predict patients at risk of SIRS 5 days before the onset of clinical signs.

Efficient production of pronuclear embryos in breeding and nonbreeding season for generating transgenic sheep overexpressing TLR4.

PubMed

Li, Yan; Lian, Di; Deng, Shoulong; Zhang, Xiaosheng; Zhang, Jinlong; Li, Wenting; Bai, Hai; Wang, Zhixian; Wu, Hongping; Fu, Juncai; Han, Hongbing; Feng, Jianzhong; Liu, Guoshi; Lian, Ling; Lian, Zhengxing

2016-01-01

Brucella is a zoonotic Gram-negative pathogen that causes abortion and infertility in ruminants and humans. TLR4 is the receptor for LPS which can recognize Brucella and initiate antigen-presenting cell activities that affect both innate and adaptive immunity. Consequently, transgenic sheep over-expressing TLR4 are an suitable model to investigate the effects of TLR4 on preventing Brucellosis. In this study, we generated transgenic sheep overexpressing TLR4 and aimed to evaluate the effects of different seasons (breeding and non-breeding season) on superovulation and the imported exogenous gene on growth. In total of 43 donor ewes and 166 recipient ewes in breeding season, 37 donor ewes and 144 recipient ewes in non-breeding season were selected for super-ovulation and injected embryo transfer to generate transgenic sheep. Our results indicated the no. of embryos recovered of donors and the rate of pronuclear embryos did not show any significant difference between breeding and non-breeding seasons (P > 0.05). The positive rate of exogenous TLR4 tested were 21.21 % and 22.58 % in breeding and non-breeding season by Southern blot. The expression level of TLR4 in the transgenic sheep was 1.5 times higher than in the non-transgenic group (P < 0.05). The lambs overexpressing TLR4 had similar growth performance with non-transgenic lambs, and the blood physiological parameters of transgenic and non-transgenic were both in the normal range and did not show any difference. Here we establish an efficient platform for the production of transgenic sheep by the microinjection of pronuclear embryos during the whole year. The over-expression of TLR4 had no adverse effect on the growth of the sheep.

TLR4 ligands lipopolysaccharide and monophosphoryl lipid a differentially regulate effector and memory CD8+ T Cell differentiation.

PubMed

Cui, Weiguo; Joshi, Nikhil S; Liu, Ying; Meng, Hailong; Kleinstein, Steven H; Kaech, Susan M

2014-05-01

Vaccines formulated with nonreplicating pathogens require adjuvants to help bolster immunogenicity. The role of adjuvants in Ab production has been well studied, but how they influence memory CD8(+) T cell differentiation remains poorly defined. In this study we implemented dendritic cell-mediated immunization to study the effects of commonly used adjuvants, TLR ligands, on effector and memory CD8(+) T cell differentiation in mice. Intriguingly, we found that the TLR4 ligand LPS was far more superior to other TLR ligands in generating memory CD8(+) T cells upon immunization. LPS boosted clonal expansion similar to the other adjuvants, but fewer of the activated CD8(+) T cells died during contraction, generating a larger pool of memory cells. Surprisingly, monophosphoryl lipid A (MPLA), another TLR4 ligand, enhanced clonal expansion of effector CD8(+) T cells, but it also promoted their terminal differentiation and contraction; thus, fewer memory CD8(+) T cells formed, and MPLA-primed animals were less protected against secondary infection compared with those primed with LPS. Furthermore, gene expression profiling revealed that LPS-primed effector cells displayed a stronger pro-memory gene expression signature, whereas the gene expression profile of MPLA-primed effector cells aligned closer with terminal effector CD8(+) T cells. Lastly, we demonstrated that the LPS-TLR4-derived "pro-memory" signals were MyD88, but not Toll/IL-1R domain-containing adapter inducing IFN-β, dependent. This study reveals the influential power of adjuvants on the quantity and quality of CD8(+) T cell memory, and that attention to adjuvant selection is crucial because boosting effector cell expansion may not always equate with more memory T cells or greater protection.

Adaptive evolution and functional constraint at TLR4 during the secondary aquatic adaptation and diversification of cetaceans.

PubMed

Shen, Tong; Xu, Shixia; Wang, Xiaohong; Yu, Wenhua; Zhou, Kaiya; Yang, Guang

2012-03-24

Cetaceans (whales, dolphins and porpoises) are a group of adapted marine mammals with an enigmatic history of transition from terrestrial to full aquatic habitat and rapid radiation in waters around the world. Throughout this evolution, the pathogen stress-response proteins must have faced challenges from the dramatic change of environmental pathogens in the completely different ecological niches cetaceans occupied. For this reason, cetaceans could be one of the most ideal candidate taxa for studying evolutionary process and associated driving mechanism of vertebrate innate immune systems such as Toll-like receptors (TLRs), which are located at the direct interface between the host and the microbial environment, act at the first line in recognizing specific conserved components of microorganisms, and translate them rapidly into a defense reaction. We used TLR4 as an example to test whether this traditionally regarded pattern recognition receptor molecule was driven by positive selection across cetacean evolutionary history. Overall, the lineage-specific selection test showed that the dN/dS (ω) values along most (30 out of 33) examined cetartiodactylan lineages were less than 1, suggesting a common effect of functional constraint. However, some specific codons made radical changes, fell adjacent to the residues interacting with lipopolysaccharides (LPS), and showed parallel evolution between independent lineages, suggesting that TLR4 was under positive selection. Especially, strong signatures of adaptive evolution on TLR4 were identified in two periods, one corresponding to the early evolutionary transition of the terrestrial ancestors of cetaceans from land to semi-aquatic (represented by the branch leading to whale + hippo) and from semi-aquatic to full aquatic (represented by the ancestral branch leading to cetaceans) habitat, and the other to the rapid diversification and radiation of oceanic dolphins. This is the first study thus far to characterize the TLR

Adaptive evolution and functional constraint at TLR4 during the secondary aquatic adaptation and diversification of cetaceans

PubMed Central

2012-01-01

Background Cetaceans (whales, dolphins and porpoises) are a group of adapted marine mammals with an enigmatic history of transition from terrestrial to full aquatic habitat and rapid radiation in waters around the world. Throughout this evolution, the pathogen stress-response proteins must have faced challenges from the dramatic change of environmental pathogens in the completely different ecological niches cetaceans occupied. For this reason, cetaceans could be one of the most ideal candidate taxa for studying evolutionary process and associated driving mechanism of vertebrate innate immune systems such as Toll-like receptors (TLRs), which are located at the direct interface between the host and the microbial environment, act at the first line in recognizing specific conserved components of microorganisms, and translate them rapidly into a defense reaction. Results We used TLR4 as an example to test whether this traditionally regarded pattern recognition receptor molecule was driven by positive selection across cetacean evolutionary history. Overall, the lineage-specific selection test showed that the dN/dS (ω) values along most (30 out of 33) examined cetartiodactylan lineages were less than 1, suggesting a common effect of functional constraint. However, some specific codons made radical changes, fell adjacent to the residues interacting with lipopolysaccharides (LPS), and showed parallel evolution between independent lineages, suggesting that TLR4 was under positive selection. Especially, strong signatures of adaptive evolution on TLR4 were identified in two periods, one corresponding to the early evolutionary transition of the terrestrial ancestors of cetaceans from land to semi-aquatic (represented by the branch leading to whale + hippo) and from semi-aquatic to full aquatic (represented by the ancestral branch leading to cetaceans) habitat, and the other to the rapid diversification and radiation of oceanic dolphins. Conclusions This is the first study thus

Common NOTCH3 Variants and Cerebral Small-Vessel Disease.

PubMed

Rutten-Jacobs, Loes C A; Traylor, Matthew; Adib-Samii, Poneh; Thijs, Vincent; Sudlow, Cathie; Rothwell, Peter M; Boncoraglio, Giorgio; Dichgans, Martin; Bevan, Steve; Meschia, James; Levi, Christopher; Rost, Natalia S; Rosand, Jonathan; Hassan, Ahamad; Markus, Hugh S

2015-06-01

The most common monogenic cause of cerebral small-vessel disease is cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy, caused by NOTCH3 gene mutations. It has been hypothesized that more common variants in NOTCH3 may also contribute to the risk of sporadic small-vessel disease. Previously, 4 common variants (rs10404382, rs1043994, rs10423702, and rs1043997) were found to be associated with the presence of white matter hyperintensity in hypertensive community-dwelling elderly. We investigated the association of common single nucleotide polymorphisms (SNPs) in NOTCH3 in 1350 patients with MRI-confirmed lacunar stroke and 7397 controls, by meta-analysis of genome-wide association study data sets. In addition, we investigated the association of common SNPs in NOTCH3 with MRI white matter hyperintensity volumes in 3670 white patients with ischemic stroke. In each analysis, we considered all SNPs within the NOTCH3 gene, and within 50-kb upstream and downstream of the coding region. A total of 381 SNPs from the 1000 genome population with a mean allele frequency>0.01 were included in the analysis. A significance level of P<0.0015 was used, adjusted for the effective number of independent SNPs in the region using the Galwey method. We found no association of any common variants in NOTCH3 (including rs10404382, rs1043994, rs10423702, and rs1043997) with lacunar stroke or white matter hyperintensity volume. We repeated our analysis stratified for hypertension but again found no association. Our study does not support a role for common NOTCH3 variation in the risk of sporadic small-vessel disease. © 2015 The Authors.

The Roles of Bacteria and TLR4 in Rat and Murine Models of Necrotizing Enterocolitis1

PubMed Central

Jilling, Tamas; Simon, Dyan; Lu, Jing; Meng, Fan Jing; Li, Dan; Schy, Robert; Thomson, Richard B.; Soliman, Antoine; Arditi, Moshe; Caplan, Michael S.

2009-01-01

Bacteria are thought to contribute to the pathogenesis of necrotizing enterocolitis (NEC), but it is unknown whether their interaction with the epithelium can participate in the initiation of mucosal injury or they can act only following translocation across a damaged intestinal barrier. Our aims were to determine whether bacteria and intestinal epithelial TLR4 play roles in a well-established neonatal rat model and a novel neonatal murine model of NEC. Neonatal rats, C57BL/6J, C3HeB/FeJ (TLR4 wild type), and C3H/HeJ (TLR4 mutant) mice were delivered by Cesarean section and were subjected to formula feeding and cold asphyxia stress or were delivered naturally and were mother-fed. NEC incidence was evaluated by histological scoring, and gene expression was quantified using quantitative real-time PCR from cDNA generated from intestinal total RNA or from RNA obtained by laser capture microdissection. Spontaneous feeding catheter colonization or supplementation of cultured bacterial isolates to formula increased the incidence of experimental NEC. During the first 72 h of life, i.e., the time frame of NEC development in this model, intestinal TLR4 mRNA gradually decreases in mother-fed but increases in formula feeding and cold asphyxia stress, correlating with induced inducible NO synthase. TLR4, inducible NO synthase, and inflammatory cytokine induction occurred in the intestinal epithelium but not in the submucosa. NEC incidence was diminished in C3H/HeJ mice, compared with C3HeB/FeJ mice. In summary, bacteria and TLR4 play significant roles in experimental NEC, likely via an interaction of intraluminal bacteria and aberrantly overexpressed TLR4 in enterocytes. PMID:16920968

Intraspinal TLR4 activation promotes iron storage but does not protect neurons or oligodendrocytes from progressive iron-mediated damage.

PubMed

Goldstein, Evan Z; Church, Jamie S; Pukos, Nicole; Gottipati, Manoj K; Popovich, Phillip G; McTigue, Dana M

2017-12-01

Iron is essential for basic cellular functions but in excess is highly toxic. For this reason, free iron and iron storage are controlled in the periphery by elaborate regulatory mechanisms. In contrast, iron regulation in the central nervous system (CNS) is not well defined. Given that excess iron is present after trauma, hemorrhagic stroke and neurodegeneration, understanding normal iron regulation and promoting iron uptake in CNS pathology is crucial. Peripherally, toll-like receptor 4 (TLR4) activation promotes iron sequestration by macrophages. Notably, iron-rich sites of CNS pathology typically contain TLR4 agonists, which may promote iron uptake. Indeed, our recent work showed impaired iron storage after acute spinal cord injury in mice with TLR4 deficiency. Here we used a reductionist model to ask if TLR4 activation in the CNS stimulates iron uptake and promotes neuroprotection from iron-induced toxicity. For this, we measured the ability of microglia/macrophages to sequester exogenous iron and prevent pathology with and without concomitant intraspinal TLR4 activation. Results show that, similar to the periphery, activating intraspinal TLR4 via focal LPS injection increased mRNA encoding iron uptake and storage proteins and promoted iron sequestration into ferritin-expressing macrophages. However, this did not prevent oligodendrocyte and neuron loss. Moreover, replacement of oligodendrocytes by progenitor cells - a normally robust response to in vivo macrophage TLR4 activation - was significantly reduced if iron was present concomitant with TLR4 activation. Thus, while TLR4 signaling promotes CNS iron uptake, future work needs to determine ways to enhance iron removal without blocking the reparative effects of innate immune receptor signaling. Copyright © 2017 Elsevier Inc. All rights reserved.

Albumin stimulates renal tubular inflammation through an HSP70-TLR4 axis in mice with early diabetic nephropathy

PubMed Central

Jheng, Huei-Fen; Tsai, Pei-Jane; Chuang, Yi-Lun; Shen, Yi-Ting; Tai, Ting-An; Chen, Wen-Chung; Chou, Chuan-Kai; Ho, Li-Chun; Tang, Ming-Jer; Lai, Kuei-Tai A.; Sung, Junne-Ming; Tsai, Yau-Sheng

2015-01-01

ABSTRACT Increased urinary albumin excretion is not simply an aftermath of glomerular injury, but is also involved in the progression of diabetic nephropathy (DN). Whereas Toll-like receptors (TLRs) are incriminated in the renal inflammation of DN, whether and how albumin is involved in the TLR-related renal inflammatory response remains to be clarified. Here, we showed that both TLR2 and TLR4, one of their putative endogenous ligands [heat shock protein 70 (HSP70)] and nuclear factor-κB promoter activity were markedly elevated in the kidneys of diabetic mice. A deficiency of TLR4 but not of TLR2 alleviated albuminuria, tubulointerstitial fibrosis and inflammation induced by diabetes. The protection against renal injury in diabetic Tlr4−/− mice was associated with reduced tubular injuries and preserved cubilin levels, rather than amelioration of glomerular lesions. In vitro studies revealed that albumin, a stronger inducer than high glucose (HG), induced the release of HSP70 from proximal tubular cells. HSP70 blockade ameliorated albumin-induced inflammatory mediators. HSP70 triggered the production of inflammatory mediators in a TLR4-dependent manner. Moreover, HSP70 inhibition in vivo ameliorated diabetes-induced albuminuria, inflammatory response and tubular injury. Finally, we found that individuals with DN had higher levels of TLR4 and HSP70 in the dilated tubules than non-diabetic controls. Thus, activation of the HSP70-TLR4 axis, stimulated at least in part by albumin, in the tubular cell is a newly identified mechanism associated with induction of tubulointerstitial inflammation and aggravation of pre-existing microalbuminuria in the progression of DN. PMID:26398934

Mapping of a microbial protein domain involved in binding and activation of the TLR2/TLR1 heterodimer.

PubMed

Liang, Shuang; Hosur, Kavita B; Lu, Shanyun; Nawar, Hesham F; Weber, Benjamin R; Tapping, Richard I; Connell, Terry D; Hajishengallis, George

2009-03-01

The pentameric B subunit of type IIb Escherichia coli enterotoxin (LT-IIb-B(5)), a doughnut-shaped oligomeric protein from enterotoxigenic E. coli, activates the TLR2/TLR1 heterodimer (TLR2/1). We investigated the molecular basis of the LT-IIb-B(5) interaction with TLR2/1 to define the structure-function relationship of LT-IIb-B(5) and, moreover, to gain an insight into how TLR2/1 recognizes large, nonacylated protein ligands that cannot fit within its lipid-binding pockets, as previously shown for the Pam(3)CysSerLys(4) (Pam(3)CSK(4)) lipopeptide. We first identified four critical residues in the upper region of the LT-IIb-B(5) pore. Corresponding point mutants (M69E, A70D, L73E, S74D) were defective in binding TLR2 or TLR1 and could not activate APCs, despite retaining full ganglioside-binding capacity. Point mutations in the TLR2/1 dimer interface, as determined in the crystallographic structure of the TLR2/1-Pam(3)CSK(4) complex, resulted in diminished activation by both Pam(3)CSK(4) and LT-IIb-B(5). Docking analysis of the LT-IIb-B(5) interaction with this apparently predominant activation conformation of TLR2/1 revealed that LT-IIb-B(5) might primarily contact the convex surface of the TLR2 central domain. Although the TLR1/LT-IIb-B(5) interface is relatively smaller, the leucine-rich repeat motifs 9-12 in the central domain of TLR1 were found to be critical for cooperative TLR2-induced cell activation by LT-IIb-B(5). Moreover, the putative LT-IIb-B(5) binding site overlaps partially with that of Pam(3)CSK(4); consistent with this, Pam(3)CSK(4) suppressed TLR2 binding of LT-IIb-B(5), albeit not as potently as self-competitive inhibition. We identified the upper pore region of LT-IIb-B(5) as a TLR2/1 interactive domain, which contacts the heterodimeric receptor at a site that is distinct from, although it overlaps with, that of Pam(3)CSK(4).

Co-factors Required for TLR7- and TLR9- dependent Innate Immune Responses

PubMed Central

Chiang, Chih-yuan; Engel, Alex; Opaluch, Amanda M.; Ramos, Irene; Maestre, Ana M.; Secundino, Ismael; De Jesus, Paul D.; Nguyen, Quy T.; Welch, Genevieve; Bonamy, Ghislain M.C.; Miraglia, Loren J.; Orth, Anthony P.; Nizet, Victor; Fernandez-Sesma, Ana; Zhou, Yingyao; Barton, Gregory M.; Chanda, Sumit K.

2012-01-01

SUMMARY Pathogens commonly utilize endocytic pathways to gain cellular access. The endosomal pattern recognition receptors TLR7 and TLR9 detect pathogen-encoded nucleic acids to initiate MyD88-dependent pro-inflammatory responses to microbial infection. Using genome-wide RNAi screening and integrative systems-based analysis we identify 190 co-factors required for TLR7- and TLR9-directed signaling responses. A set of co-factors were cross-profiled for their activities downstream of several immunoreceptors, and then functionally mapped based on the known architecture of NF-κB signaling pathways. Protein complexes and pathways involved in ubiquitin-protein ligase activities, sphingolipid metabolism, chromatin modifications, and ancient stress responses were found to modulate innate recognition of endosomal nucleic acids. Additionally, hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) was characterized as necessary for ubiquitin-dependent TLR9 targeting to the endolysosome. Proteins and pathways identified here should prove useful in delineating strategies to manipulate innate responses for treatment of autoimmune disorders and microbial infection. PMID:22423970

Impact of TLR4 on behavioral and cognitive dysfunctions associated with alcohol-induced neuroinflammatory damage.

PubMed

Pascual, María; Baliño, Pablo; Alfonso-Loeches, Silvia; Aragón, Carlos M G; Guerri, Consuelo

2011-06-01

Toll-like receptors (TLRs) play an important role in the innate immune response, and emerging evidence indicates their role in brain injury and neurodegeneration. Our recent results have demonstrated that ethanol is capable of activating glial TLR4 receptors and that the elimination of these receptors in mice protects against ethanol-induced glial activation, induction of inflammatory mediators and apoptosis. This study was designed to assess whether ethanol-induced inflammatory damage causes behavioral and cognitive consequences, and if behavioral alterations are dependent of TLR4 functions. Here we show in mice drinking alcohol for 5months, followed by a 15-day withdrawal period, that activation of the astroglial and microglial cells in frontal cortex and striatum is maintained and that these events are associated with cognitive and anxiety-related behavioral impairments in wild-type (WT) mice, as demonstrated by testing the animals with object memory recognition, conditioned taste aversion and dark and light box anxiety tasks. Mice lacking TLR4 receptors are protected against ethanol-induced inflammatory damage, and behavioral associated effects. We further assess the possibility of the epigenetic modifications participating in short- or long-term behavioral effects associated with neuroinflammatory damage. We show that chronic alcohol treatment decreases H4 histone acetylation and histone acetyltransferases activity in frontal cortex, striatum and hippocampus of WT mice. Alterations in chromatin structure were not observed in TLR4(-/-) mice. These results provide the first evidence of the role that TLR4 functions play in the behavioral consequences of alcohol-induced inflammatory damage and suggest that the epigenetic modifications mediated by TLR4 could contribute to short- or long-term alcohol-induced behavioral or cognitive dysfunctions. Copyright © 2011 Elsevier Inc. All rights reserved.

Changes in endometrial transcription of TLR2, TLR4, and CD14 during the first-week postpartum in dairy cows with retained placenta.

PubMed

Martins, Telma M; Muniz, Clarice S; Andrade, Virgílio B; Paixão, Tatiane A; Santos, Renato L; Borges, Álan M

2016-04-15

Changes in the endometrial transcription of pattern recognition receptors may increase the susceptibility to postpartum uterine infections in Holstein cows with retained placenta. To test this hypothesis, nine cows with retained placenta and ten cows without retained placenta were submitted to endometrial biopsies at the first and seventh days postpartum. Cows were monitored weekly with clinical and gynecological examinations until 42 days postpartum. Samples of the uterine contents were collected weekly for aerobic bacteria isolation. All cows had endometrial transcription of Toll-like receptors (TLRs) 1/6, 2, 4, 5, and 9; nucleotide-binding oligomerization domain (NOD)-like receptors 1 and 2; and the coreceptors cluster of differentiation 14 (CD14) and myeloid differentiation protein-2 (MD-2), as measured on the first and seventh days postpartum. Escherichia coli was the most common bacterium isolated from the uterine contents of cows with or without retained placenta until 21 days postpartum. Transcription levels of TLR2, TLR4, and CD14 in Holstein cows with retained placenta significantly decreased (P < 0.05) between the first and the seventh day postpartum. Conversely, cows without retained placenta did not have any significant changes in transcription levels between these time points. Copyright © 2016 Elsevier Inc. All rights reserved.

Single Nucleotide Polymorphisms in IL8 and TLR4 Genes as Candidates for Digital Dermatitis Resistance/Susceptibility in Holstein Cattle.

PubMed

El-Shafaey, El-Sayed; Ateya, Ahmed; Ramadan, Hazem; Saleh, Rasha; Elseady, Yousef; Abo El Fadl, Eman; El-Khodery, Sabry

2017-04-03

Relatedness between single nucleotide polymorphisms in IL8 and TLR4 genes and digital dermatitis resistance/susceptibility was investigated in seventy Holstein dairy cows. Animals were assigned into two groups, affected group (n = 35) and resistant group (n = 35) based on clinical signs and previous history of farm clinical records. Blood samples were collected for DNA extraction to ampliy fragments of 267-bp and 382-bp for IL8 and TLR4 genes, respectively. PCR-DNA sequencing revealed three SNPs in each of IL8 and TLR4 genes. The identified SNPs associated with digital dermatitis resistance were C94T, A220G, and T262A for IL8 and C118T for TLR4. However, the G349C and C355A SNPs in TLR4 gene were associated with digital dermatitis susceptibility. Chi-square analysis for comparison the distribution of all identified SNPs in both IL8 and TLR4 genes between resistant and affected animals showed no significant variation among the identified SNPs in IL8 gene. Meanwhile, there was a significant variation in case of TLR4 gene. As a pilot study, the present results revealed that identified SNPs in IL8 and TLR4 genes can be used as a genetic marker and predisposing factor for resistance/susceptibility to digital dermatitis in dairy cows. However, TLR4 gene may be a potential candidate for such disease.

Mycobacterium indicus pranii (MIP) mediated host protective intracellular mechanisms against tuberculosis infection: Involvement of TLR-4 mediated signaling.

PubMed

Das, Shibali; Chowdhury, Bidisha Paul; Goswami, Avranil; Parveen, Shabina; Jawed, Junaid; Pal, Nishith; Majumdar, Subrata

2016-12-01

Mycobacterium tuberculosis infection inflicts the disease Tuberculosis (TB), which is fatal if left untreated. During M. tuberculosis infection, the pathogen modulates TLR-4 receptor down-stream signaling, indicating the possible involvement of TLR-4 in the regulation of the host immune response. Mycobacterium indicus pranii (MIP) possesses immuno-modulatory properties which induces the pro-inflammatory responses via induction of TLR-4-mediated signaling. Here, we observed the immunomodulatory properties of MIP against tuberculosis infection. We have studied the detailed signaling mechanisms employed by MIP in order to restore the host immune response against the in vitro tuberculosis infection. We observed that in infected macrophages MIP treatment significantly increased the TLR-4 expression as well as activation of its downstream signaling, facilitating the activation of P38 MAP kinase. MIP treatment was able to activate NF-κB via involvement of TLR-4 signaling leading to the enhanced pro-inflammatory cytokine and NO generation in the infected macrophages and generation of protective immune response. Therefore, we may suggest that, TLR4 may represent a novel therapeutic target for the activation of the innate immune response during Tuberculosis infection. Copyright © 2016. Published by Elsevier Ltd.

CXCR4–CXCL12–CXCR7, TLR2–TLR4, and PD-1/PD-L1 in colorectal cancer liver metastases from neoadjuvant-treated patients

PubMed Central

D'Alterio, Crescenzo; Nasti, Guglielmo; Polimeno, Marianeve; Ottaiano, Alessandro; Conson, Manuel; Circelli, Luisa; Botti, Giovanni; Scognamiglio, Giosuè; Santagata, Sara; De Divitiis, Chiara; Nappi, Anna; Napolitano, Maria; Tatangelo, Fabiana; Pacelli, Roberto; Izzo, Francesco; Vuttariello, Emilia; Botti, Gerardo; Scala, Stefania

2016-01-01

ABSTRACT A neoadjuvant clinical trial was previously conducted in patients with resectable colorectal cancer liver metastases (CRLM). At a median follow up of 28 months, 20/33 patients were dead of disease, 8 were alive with disease and 5 were alive with no evidence of disease. To shed further insight into biological features accounting for different outcomes, the expression of CXCR4–CXCL12–CXCR7, TLR2–TLR4, and the programmed death receptor-1 (PD-1)/programmed death-1 ligand (PD-L1) was evaluated in excised liver metastases. Expression profiles were assessed through qPCR in metastatic and unaffected liver tissue of 33 CRLM neoadjuvant-treated patients. CXCR4 and CXCR7, TLR2/TLR4, and PD-1/PD-L1 mRNA were significantly overexpressed in metastatic compared to unaffected liver tissues. CXCR4 protein was negative/low in 10/31, and high in 21/31, CXCR7 was negative/low in 16/31 and high in 15/31, CXCL12 was negative/low in 14/31 and high in 17/31 CRLM. PD-1 was negative in 19/30 and positive in 11/30, PD-L1 was negative/low in 24/30 and high in 6/30 CRLM. Stromal PD-L1 expression, affected the progression-free survival (PFS) in the CRLM population. Patients overexpressing CXCR4 experienced a worse PFS and cancer specific survival (CSS) (p = 0.001 and p = 0.0008); in these patients, KRAS mutation identified a subgroup with a significantly worse CSS (p < 0.01). Thus, CXCR4 and PD-L1 expression discriminate patients with the worse PFS within the CRLM evaluated patients. Within the CXCR4 high expressing patients carrying Mut-KRAS in CRLM identifies the worst prognostic group. Thus, CXCR4 targeting plus anti-PD-1 therapy should be explored to improve the prognosis of Mut-KRAS-high CXCR4-CRLMs. PMID:28123896

Polymorphisms at the innate immune receptor TLR2 are associated with Borrelia infection in a wild rodent population

PubMed Central

Tschirren, Barbara; Andersson, Martin; Scherman, Kristin; Westerdahl, Helena; Mittl, Peer R. E.; Råberg, Lars

2013-01-01

The discovery of the key role of Toll-like receptors (TLRs) in initiating innate immune responses and modulating adaptive immunity has revolutionized our understanding of vertebrate defence against pathogens. Yet, despite their central role in pathogen recognition and defence initiation, there is little information on how variation in TLRs influences disease susceptibility in natural populations. Here, we assessed the extent of naturally occurring polymorphisms at TLR2 in wild bank voles (Myodes glareolus) and tested for associations between TLR2 variants and infection with Borrelia afzelii, a common tick-transmitted pathogen in rodents and one of the causative agents of human Lyme disease. Bank voles in our population had 15 different TLR2 haplotypes (10 different haplotypes at the amino acid level), which grouped in three well-separated clusters. In a large-scale capture–mark–recapture study, we show that voles carrying TLR2 haplotypes of one particular cluster (TLR2c2) were almost three times less likely to be Borrelia infected than animals carrying other haplotypes. Moreover, neutrality tests suggested that TLR2 has been under positive selection. This is, to our knowledge, the first demonstration of an association between TLR polymorphism and parasitism in wildlife, and a striking example that genetic variation at innate immune receptors can have a large impact on host resistance. PMID:23554395

Polymorphisms at the innate immune receptor TLR2 are associated with Borrelia infection in a wild rodent population.

PubMed

Tschirren, Barbara; Andersson, Martin; Scherman, Kristin; Westerdahl, Helena; Mittl, Peer R E; Råberg, Lars

2013-05-22

The discovery of the key role of Toll-like receptors (TLRs) in initiating innate immune responses and modulating adaptive immunity has revolutionized our understanding of vertebrate defence against pathogens. Yet, despite their central role in pathogen recognition and defence initiation, there is little information on how variation in TLRs influences disease susceptibility in natural populations. Here, we assessed the extent of naturally occurring polymorphisms at TLR2 in wild bank voles (Myodes glareolus) and tested for associations between TLR2 variants and infection with Borrelia afzelii, a common tick-transmitted pathogen in rodents and one of the causative agents of human Lyme disease. Bank voles in our population had 15 different TLR2 haplotypes (10 different haplotypes at the amino acid level), which grouped in three well-separated clusters. In a large-scale capture-mark-recapture study, we show that voles carrying TLR2 haplotypes of one particular cluster (TLR2c2) were almost three times less likely to be Borrelia infected than animals carrying other haplotypes. Moreover, neutrality tests suggested that TLR2 has been under positive selection. This is, to our knowledge, the first demonstration of an association between TLR polymorphism and parasitism in wildlife, and a striking example that genetic variation at innate immune receptors can have a large impact on host resistance.

The Structural Basis for Endotoxin-induced Allosteric Regulation of the Toll-like Receptor 4 (TLR4) Innate Immune Receptor*

PubMed Central

Paramo, Teresa; Piggot, Thomas J.; Bryant, Clare E.; Bond, Peter J.

2013-01-01

As part of the innate immune system, Toll-like receptor 4 (TLR4) recognizes bacterial cell surface lipopolysaccharide (LPS) by forming a complex with a lipid-binding co-receptor, MD-2. In the presence of agonist, TLR4·MD-2 dimerizes to form an active receptor complex, leading to initiation of intracellular inflammatory signals. TLR4 is of great biomedical interest, but its pharmacological manipulation is complicated because even subtle variations in the structure of LPS can profoundly impact the resultant immunological response. Here, we use atomically detailed molecular simulations to gain insights into the nature of the molecular signaling mechanism. We first demonstrate that MD-2 is extraordinarily flexible. The “clamshell-like” motions of its β-cup fold enable it to sensitively match the volume of its hydrophobic cavity to the size and shape of the bound lipid moiety. We show that MD-2 allosterically transmits this conformational plasticity, in a ligand-dependent manner, to a phenylalanine residue (Phe-126) at the cavity mouth previously implicated in TLR4 activation. Remarkably, within the receptor complex, we observe spontaneous transitions between active and inactive signaling states of Phe-126, and we confirm that Phe-126 is indeed the “molecular switch” in endotoxic signaling. PMID:24178299

The structural basis for endotoxin-induced allosteric regulation of the Toll-like receptor 4 (TLR4) innate immune receptor.

PubMed

Paramo, Teresa; Piggot, Thomas J; Bryant, Clare E; Bond, Peter J

2013-12-20

As part of the innate immune system, Toll-like receptor 4 (TLR4) recognizes bacterial cell surface lipopolysaccharide (LPS) by forming a complex with a lipid-binding co-receptor, MD-2. In the presence of agonist, TLR4·MD-2 dimerizes to form an active receptor complex, leading to initiation of intracellular inflammatory signals. TLR4 is of great biomedical interest, but its pharmacological manipulation is complicated because even subtle variations in the structure of LPS can profoundly impact the resultant immunological response. Here, we use atomically detailed molecular simulations to gain insights into the nature of the molecular signaling mechanism. We first demonstrate that MD-2 is extraordinarily flexible. The "clamshell-like" motions of its β-cup fold enable it to sensitively match the volume of its hydrophobic cavity to the size and shape of the bound lipid moiety. We show that MD-2 allosterically transmits this conformational plasticity, in a ligand-dependent manner, to a phenylalanine residue (Phe-126) at the cavity mouth previously implicated in TLR4 activation. Remarkably, within the receptor complex, we observe spontaneous transitions between active and inactive signaling states of Phe-126, and we confirm that Phe-126 is indeed the "molecular switch" in endotoxic signaling.

Kukoamine B promotes TLR4-independent lipopolysaccharide uptake in murine hepatocytes.

PubMed

Yang, Dong; Zheng, Xinchuan; Wang, Ning; Fan, Shijun; Yang, Yongjun; Lu, Yongling; Chen, Qian; Liu, Xin; Zheng, Jiang

2016-09-06

Free bacterial lipopolysaccharide (LPS) is generally removed from the bloodstream through hepatic uptake via TLR4, the LPS pattern recognition receptor, but mechanisms for internalization and clearance of conjugated LPS are less clear. Kukoamine B (KB) is a novel cationic alkaloid that interferes with LPS binding to TLR4. In this study, KB accelerated blood clearance of LPS. KB also enhanced LPS distribution in the hepatic tissues of C57 BL/6 mice, along with LPS uptake in primary hepatocytes and HepG2 cells. By contrast, KB inhibited LPS internalization in Kupffer and RAW 264.7 cells. Loss of TLR4 did not affect LPS uptake into KB-treated hepatocytes. We also detected selective upregulation of the asialoglycoprotein receptor (ASGPR) upon KB treatment, and ASGPR colocalized with KB in cultured hepatocytes. Molecular docking showed that KB bound to ASGPR in a manner similar to GalNAc, a known ASGPR agonist. GalNAc dose-dependently reduced KB internalization, suggesting it competes with KB for ASGPR binding, and ASGPR knockdown also impaired LPS uptake into hepatocytes. Finally, while KB enhanced LPS uptake, it was protective against LPS-induced inflammation and hepatocyte injury. Our study provides a new mechanism for conjugated LPS hepatic uptake induced by the LPS neutralizer KB and mediated by membrane ASGPR binding.

Increased TLR4 and TREM-1 expression on monocytes and neutrophils in preterm birth: further evidence of a proinflammatory state.

PubMed

Yan, Huan; Li, Hong; Zhu, Linlin; Gao, Junjun; Li, Pengyun; Zhang, Zhan

2018-03-25

Increased inflammation is considered as a risk factor and a promoter of preterm birth (PTB). Monocytes and neutrophils are the main sources of cytokines in the early inflammatory phase. So far, very few studies have indicated CD14/TLR4 and TREM-1 on the monocytes and neutrophils as important targets in PTB. TLR4 and TREM-1 on CD14 + maternal and cord blood monocytes and neutrophils were detected using flow cytometry in 48 normal term women, 48 PTB with chorioamnionitis (CCA) women, and 40 PTB without CCA women. In the fetal membranes, mRNA and protein levels of the CD14/TLR4-TREM-1 signaling pathway, CD14, TLR4, NF-κBp65, and TREM-1 were analyzed by qRT-PCR and western blot. ELISA was further used to detect TLR4 and TREM-1 levels in maternal and cord serums. Compared with the normal term and PTB without CCA women, we found that (1) TLR4 and TREM-1 levels on CD14 + maternal and cord blood monocytes and neutrophils in the PTB with CCA group were elevated (p < .001); (2) the protein and mRNA expressions of CA14, TLR4, NF-κBp65, and TREM-1 of the PTB with CCA group were upregulated (p < .001); (3) Maternal and cord serum concentrations of TLR4 and TREM-1 in the PTB with CCA group were greater (p < .001). The high levels of TLR4 and TREM-1 surface expression were observed on CD14 + maternal and cord blood monocyte and neutrophils, confirming their proinflammatory profiles in PTB with CCA. TLR4 and TREM-1 on monocyte and neutrophils might have a role in infection-related PTB.

P-MAPA immunotherapy potentiates the effect of cisplatin on serous ovarian carcinoma through targeting TLR4 signaling.

PubMed

de Almeida Chuffa, Luiz Gustavo; de Moura Ferreira, Grazielle; Lupi, Luiz Antonio; da Silva Nunes, Iseu; Fávaro, Wagner José

2018-01-17

Toll-like receptors (TLRs) are transmembrane proteins expressed on the surface of ovarian cancer (OC) and immune cells. Identifying the specific roles of the TLR-mediated signaling pathways in OC cells is important to guide new treatments. Because immunotherapies have emerged as the adjuvant treatment for patients with OC, we investigated the effect of a promising immunotherapeutic strategy based on protein aggregate magnesium-ammonium phospholinoleate-palmitoleate anhydride (P-MAPA) combined with cisplatin (CIS) on the TLR2 and TLR4 signaling pathways via myeloid differentiation factor 88 (MyD88) and TLR-associated activator of interferon (TRIF) in an in vivo model of OC. Tumors were chemically induced by a single injection of 100 μg of 7,12-dimethylbenz(a)anthracene (DMBA) directly under the left ovarian bursa in Fischer 344 rats. After the rats developed serous papillary OC, they were given P-MAPA, CIS or the combination P-MAPA+CIS as therapies. To understand the effects of the treatments, we assessed the tumor size, histopathology, and the TLR2- and TLR4-mediated inflammatory responses. Although CIS therapy was more effective than P-MAPA in reducing the tumor size, P-MAPA immunotherapy significantly increased the expressions of TLR2 and TLR4. More importantly, the combination of P-MAPA with CIS showed a greater survival rate compared to CIS alone, and exhibited a significant reduction in tumor volume compared to P-MAPA alone. The combination therapy also promoted the increase in the levels of the following OC-related proteins: TLR4, MyD88, TRIF, inhibitor of phosphorylated NF-kB alpha (p-IkBα), and nuclear factor kappa B (NF-kB p65) in both cytoplasmic and nuclear sites. While P-MAPA had no apparent effect on tumor necrosis factor alpha (TNF-α) and interleukin (IL)-6, it seems to increase interferon-γ (IFN-γ), which may induce the Thelper (Th1)-mediated immune response. Collectively, our results suggest that P-MAPA immunotherapy combined with cisplatin

β2-glycoprotein I, lipopolysaccharide and endothelial TLR4: three players in the two hit theory for anti-phospholipid-mediated thrombosis.

PubMed

Raschi, Elena; Chighizola, Cecilia B; Grossi, Claudia; Ronda, Nicoletta; Gatti, Rita; Meroni, Pier Luigi; Borghi, M Orietta

2014-12-01

The thrombogenic effect of β2-glycoprotein I (β2GPI)-dependent anti-phospholipid antibodies (aPL) in animal models was found to be LPS dependent. Since β2GPI behaves as LPS scavenger, LPS/β2GPI complex was suggested to account for in vitro cell activation through LPS/TLR4 involvement being LPS the actual bridge ligand between β2GPI and TLR4 at least in monocytes/macrophages. However, no definite information is available on the interaction among β2GPI, LPS and endothelial TLR4 in spite of the main role of endothelial cells (EC) in clotting. To analyse at the endothelial level the need of LPS, we investigated the in vitro interaction of β2GPI with endothelial TLR4 and we assessed the role of LPS in such an interaction. To do this, we evaluated the direct binding and internalization of β2GPI by confocal microscopy in living TLR4-MD2 transfected CHO cells (CHO/TLR4-MD2) and β2GPI binding to CHO/TLR4-MD2 cells and human umbilical cord vein EC (HUVEC) by flow cytometry and cell-ELISA using anti-β2GPI monoclonal antibodies in the absence or presence of various concentrations of exogenous LPS. To further investigate the role of TLR4, we performed anti-β2GPI antibody binding and adhesion molecule up-regulation in TLR4-silenced HUVEC. Confocal microscopy studies show that β2GPI does interact with TLR4 at the cell membrane and is internalized in cytoplasmic granules in CHO/TLR4-MD2 cells. β2GPI binding to CHO/TLR4-MD2 cells and HUVEC is also confirmed by flow cytometry and cell-ELISA, respectively. The interaction between β2GPI and TLR4 is confirmed by the reduction of anti-β2GPI antibody binding and by the up-regulation of E-selectin or ICAM-1 by TLR4 silencing in HUVEC. β2GPI binding is not affected by LPS at concentrations comparable to those found in both β2GPI and antibody preparations. Only higher amount of LPS that can activate EC and up-regulate TLR4 expression are found to increase the binding. Our findings demonstrate that β2GPI interacts

Common genetic variants influence human subcortical brain structures

PubMed Central

Hibar, Derrek P.; Stein, Jason L.; Renteria, Miguel E.; Arias-Vasquez, Alejandro; Desrivières, Sylvane; Jahanshad, Neda; Toro, Roberto; Wittfeld, Katharina; Abramovic, Lucija; Andersson, Micael; Aribisala, Benjamin S.; Armstrong, Nicola J.; Bernard, Manon; Bohlken, Marc M.; Boks, Marco P.; Bralten, Janita; Brown, Andrew A.; Chakravarty, M. Mallar; Chen, Qiang; Ching, Christopher R. K.; Cuellar-Partida, Gabriel; den Braber, Anouk; Giddaluru, Sudheer; Goldman, Aaron L.; Grimm, Oliver; Guadalupe, Tulio; Hass, Johanna; Woldehawariat, Girma; Holmes, Avram J.; Hoogman, Martine; Janowitz, Deborah; Jia, Tianye; Kim, Sungeun; Klein, Marieke; Kraemer, Bernd; Lee, Phil H.; Olde Loohuis, Loes M.; Luciano, Michelle; Macare, Christine; Mather, Karen A.; Mattheisen, Manuel; Milaneschi, Yuri; Nho, Kwangsik; Papmeyer, Martina; Ramasamy, Adaikalavan; Risacher, Shannon L.; Roiz-Santiañez, Roberto; Rose, Emma J.; Salami, Alireza; Sämann, Philipp G.; Schmaal, Lianne; Schork, Andrew J.; Shin, Jean; Strike, Lachlan T.; Teumer, Alexander; van Donkelaar, Marjolein M. J.; van Eijk, Kristel R.; Walters, Raymond K.; Westlye, Lars T.; Whelan, Christopher D.; Winkler, Anderson M.; Zwiers, Marcel P.; Alhusaini, Saud; Athanasiu, Lavinia; Ehrlich, Stefan; Hakobjan, Marina M. H.; Hartberg, Cecilie B.; Haukvik, Unn K.; Heister, Angelien J. G. A. M.; Hoehn, David; Kasperaviciute, Dalia; Liewald, David C. M.; Lopez, Lorna M.; Makkinje, Remco R. R.; Matarin, Mar; Naber, Marlies A. M.; McKay, D. Reese; Needham, Margaret; Nugent, Allison C.; Pütz, Benno; Royle, Natalie A.; Shen, Li; Sprooten, Emma; Trabzuni, Daniah; van der Marel, Saskia S. L.; van Hulzen, Kimm J. E.; Walton, Esther; Wolf, Christiane; Almasy, Laura; Ames, David; Arepalli, Sampath; Assareh, Amelia A.; Bastin, Mark E.; Brodaty, Henry; Bulayeva, Kazima B.; Carless, Melanie A.; Cichon, Sven; Corvin, Aiden; Curran, Joanne E.; Czisch, Michael; de Zubicaray, Greig I.; Dillman, Allissa; Duggirala, Ravi; Dyer, Thomas D.; Erk, Susanne; Fedko, Iryna O.; Ferrucci, Luigi; Foroud, Tatiana M.; Fox, Peter T.; Fukunaga, Masaki; Gibbs, J. Raphael; Göring, Harald H. H.; Green, Robert C.; Guelfi, Sebastian; Hansell, Narelle K.; Hartman, Catharina A.; Hegenscheid, Katrin; Heinz, Andreas; Hernandez, Dena G.; Heslenfeld, Dirk J.; Hoekstra, Pieter J.; Holsboer, Florian; Homuth, Georg; Hottenga, Jouke-Jan; Ikeda, Masashi; Jack, Clifford R.; Jenkinson, Mark; Johnson, Robert; Kanai, Ryota; Keil, Maria; Kent, Jack W.; Kochunov, Peter; Kwok, John B.; Lawrie, Stephen M.; Liu, Xinmin; Longo, Dan L.; McMahon, Katie L.; Meisenzahl, Eva; Melle, Ingrid; Mohnke, Sebastian; Montgomery, Grant W.; Mostert, Jeanette C.; Mühleisen, Thomas W.; Nalls, Michael A.; Nichols, Thomas E.; Nilsson, Lars G.; Nöthen, Markus M.; Ohi, Kazutaka; Olvera, Rene L.; Perez-Iglesias, Rocio; Pike, G. Bruce; Potkin, Steven G.; Reinvang, Ivar; Reppermund, Simone; Rietschel, Marcella; Romanczuk-Seiferth, Nina; Rosen, Glenn D.; Rujescu, Dan; Schnell, Knut; Schofield, Peter R.; Smith, Colin; Steen, Vidar M.; Sussmann, Jessika E.; Thalamuthu, Anbupalam; Toga, Arthur W.; Traynor, Bryan J.; Troncoso, Juan; Turner, Jessica A.; Valdés Hernández, Maria C.; van ’t Ent, Dennis; van der Brug, Marcel; van der Wee, Nic J. A.; van Tol, Marie-Jose; Veltman, Dick J.; Wassink, Thomas H.; Westman, Eric; Zielke, Ronald H.; Zonderman, Alan B.; Ashbrook, David G.; Hager, Reinmar; Lu, Lu; McMahon, Francis J.; Morris, Derek W.; Williams, Robert W.; Brunner, Han G.; Buckner, Randy L.; Buitelaar, Jan K.; Cahn, Wiepke; Calhoun, Vince D.; Cavalleri, Gianpiero L.; Crespo-Facorro, Benedicto; Dale, Anders M.; Davies, Gareth E.; Delanty, Norman; Depondt, Chantal; Djurovic, Srdjan; Drevets, Wayne C.; Espeseth, Thomas; Gollub, Randy L.; Ho, Beng-Choon; Hoffmann, Wolfgang; Hosten, Norbert; Kahn, René S.; Le Hellard, Stephanie; Meyer-Lindenberg, Andreas; Müller-Myhsok, Bertram; Nauck, Matthias; Nyberg, Lars; Pandolfo, Massimo; Penninx, Brenda W. J. H.; Roffman, Joshua L.; Sisodiya, Sanjay M.; Smoller, Jordan W.; van Bokhoven, Hans; van Haren, Neeltje E. M.; Völzke, Henry; Walter, Henrik; Weiner, Michael W.; Wen, Wei; White, Tonya; Agartz, Ingrid; Andreassen, Ole A.; Blangero, John; Boomsma, Dorret I.; Brouwer, Rachel M.; Cannon, Dara M.; Cookson, Mark R.; de Geus, Eco J. C.; Deary, Ian J.; Donohoe, Gary; Fernández, Guillén; Fisher, Simon E.; Francks, Clyde; Glahn, David C.; Grabe, Hans J.; Gruber, Oliver; Hardy, John; Hashimoto, Ryota; Hulshoff Pol, Hilleke E.; Jönsson, Erik G.; Kloszewska, Iwona; Lovestone, Simon; Mattay, Venkata S.; Mecocci, Patrizia; McDonald, Colm; McIntosh, Andrew M.; Ophoff, Roel A.; Paus, Tomas; Pausova, Zdenka; Ryten, Mina; Sachdev, Perminder S.; Saykin, Andrew J.; Simmons, Andy; Singleton, Andrew; Soininen, Hilkka; Wardlaw, Joanna M.; Weale, Michael E.; Weinberger, Daniel R.; Adams, Hieab H. H.; Launer, Lenore J.; Seiler, Stephan; Schmidt, Reinhold; Chauhan, Ganesh; Satizabal, Claudia L.; Becker, James T.; Yanek, Lisa; van der Lee, Sven J.; Ebling, Maritza; Fischl, Bruce; Longstreth, W. T.; Greve, Douglas; Schmidt, Helena; Nyquist, Paul; Vinke, Louis N.; van Duijn, Cornelia M.; Xue, Luting; Mazoyer, Bernard; Bis, Joshua C.; Gudnason, Vilmundur; Seshadri, Sudha; Ikram, M. Arfan; Martin, Nicholas G.; Wright, Margaret J.; Schumann, Gunter; Franke, Barbara; Thompson, Paul M.; Medland, Sarah E.

2015-01-01

The highly complex structure of the human brain is strongly shaped by genetic influences1. Subcortical brain regions form circuits with cortical areas to coordinate movement2, learning, memory3 and motivation4, and altered circuits can lead to abnormal behaviour and disease2. To investigate how common genetic variants affect the structure of these brain regions, here we conduct genome-wide association studies of the volumes of seven subcortical regions and the intracranial volume derived from magnetic resonance images of 30,717 individuals from 50 cohorts. We identify five novel genetic variants influencing the volumes of the putamen and caudate nucleus. We also find stronger evidence for three loci with previously established influences on hippocampal volume5 and intracranial volume6. These variants show specific volumetric effects on brain structures rather than global effects across structures. The strongest effects were found for the putamen, where a novel intergenic locus with replicable influence on volume (rs945270; P = 1.08 × 10−33; 0.52% variance explained) showed evidence of altering the expression of the KTN1 gene in both brain and blood tissue. Variants influencing putamen volume clustered near developmental genes that regulate apoptosis, axon guidance and vesicle transport. Identification of these genetic variants provides insight into the causes of variability inhuman brain development, and may help to determine mechanisms of neuropsychiatric dysfunction. PMID:25607358

Mapping of a Microbial Protein Domain Involved in Binding and Activation of the TLR2/TLR1 Heterodimer 1

PubMed Central

Liang, Shuang; Hosur, Kavita B.; Lu, Shanyun; Nawar, Hesham F.; Weber, Benjamin R.; Tapping, Richard I.; Connell, Terry D.; Hajishengallis, George

2009-01-01

LT-IIb-B5, a doughnut-shaped oligomeric protein from enterotoxigenic Escherichia coli, is known to activate the TLR2/TLR1 heterodimer (TLR2/1). We investigated the molecular basis of the LT-IIb-B5 interaction with TLR2/1 in order to define the structure-function relationship of LT-IIb-B5 and, moreover, to gain an insight into how TLR2/1 recognizes large, non-acylated protein ligands that cannot fit within its lipid-binding pockets, as previously shown for the Pam3CSK4 lipopeptide. We first identified four critical residues in the upper region of the LT-IIb-B5 pore: Corresponding point mutants (M69E, A70D, L73E, S74D) were defective in binding TLR2 or TLR1 and could not activate antigen-presenting cells, despite retaining full ganglioside-binding capacity. Point mutations in the TLR2/1 dimer interface, as determined in the crystallographic structure of the TLR2/1-Pam3CSK4 complex, resulted in diminished activation by both Pam3CSK4 and LT-IIb-B5. Docking analysis of the LT-IIb-B5 interaction with this apparently “predominant” activation conformation of TLR2/1 revealed that LT-IIb-B5 may primarily contact the convex surface of the TLR2 central domain. Although the TLR1/LT-IIb-B5 interface is relatively smaller, the leucine-rich repeat motifs 9–12 in the central domain of TLR1 were found to be critical for cooperative TLR2-induced cell activation by LT-IIb-B5. Moreover, the putative LT-IIb-B5 binding site overlaps partially with that of Pam3CSK4; consistent with this, Pam3CSK4 suppressed TLR2 binding of LT-IIb-B5, albeit not as potently as self-competitive inhibition. In conclusion, we identified the upper pore region of LT-IIb-B5 as a TLR2/1 interactive domain, which contacts the heterodimeric receptor at a site that is distinct from, though overlaps with, that of Pam3CSK4. PMID:19234193

Interactions of Notch1 and TLR4 signaling pathways in DRG neurons of in vivo and in vitro models of diabetic neuropathy.

PubMed

Chen, Tianhua; Li, Hao; Yin, Yiting; Zhang, Yuanpin; Liu, Zhen; Liu, Huaxiang

2017-11-02

Understanding the interactions between Notch1 and toll-like receptor 4 (TLR4) signaling pathways in the development of diabetic peripheral neuropathy may lead to interpretation of the mechanisms and novel approaches for preventing diabetic neuropathic pain. In the present study, the interactions between Notch1 and TLR4 signaling pathways were investigated by using dorsal root ganglion (DRG) from diabetic neuropathic pain rats and cultured DRG neurons under high glucose challenge. The results showed that high glucose induced not only Notch1 mRNA, HES1 mRNA, and TLR4 mRNA expression, but also Notch1 intracellular domain (NICD1) and TLR4 protein expression in DRG neurons. The proportion of NICD1-immunoreactive (IR) and TLR4-IR neurons in DRG cultures was also increased after high glucose challenge. The above alterations could be partially reversed by inhibition of either Notch1 or TLR4 signaling pathway. Inhibition of either Notch1 or TLR4 signaling pathway could improve mechanical allodynia and thermal hyperalgesia thresholds. Inhibition of Notch1 or TLR4 signaling also decreased tumor necrosis factor-α (TNF-α) levels in DRG from diabetic neuropathic rats. These data imply that the interaction between Notch1 and TLR4 signaling pathways is one of the important mechanisms in the development or progression of diabetic neuropathy.

Epigallocatechin gallate (EGCG) suppresses lipopolysaccharide-induced Toll-like receptor 4 (TLR4) activity via 67 kDa laminin receptor (67LR) in 3T3-L1 adipocytes.

PubMed

Bao, Suqing; Cao, Yanli; Zhou, Haicheng; Sun, Xin; Shan, Zhongyan; Teng, Weiping

2015-03-18

Obesity-related insulin resistance is associated with chronic systemic low-grade inflammation, and toll-like receptor 4 (TLR4) regulates inflammation. We investigated the pathways involved in epigallocatechin gallate (EGCG) modulation of insulin and TLR4 signaling in adipocytes. Inflammation was induced in adipocytes by lipopolysaccharide (LPS). An antibody against the 67 kDa laminin receptor (67LR, to which EGCG exclusively binds) was used to examine the effect of EGCG on TLR4 signaling, and a TLR4/MD-2 antibody was used to inhibit TLR4 activity and to determine the insulin sensitivity of differentiated 3T3-L1 adipocytes. We found that EGCG dose-dependently inhibited LPS stimulation of adipocyte inflammation by reducing inflammatory mediator and cytokine levels (IKKβ, p-NF-κB, TNF-α, and IL-6). Pretreatment with the 67LR antibody prevented EGCG inhibition of inflammatory cytokines, decreased glucose transporter isoform 4 (GLUT4) expression, and inhibited insulin-stimulated glucose uptake. TLR4 inhibition attenuated inflammatory cytokine levels and increased glucose uptake by reversing GLUT4 levels. These data suggest that EGCG suppresses TLR4 signaling in LPS-stimulated adipocytes via 67LR and attenuates insulin-stimulated glucose uptake associated with decreased GLUT4 expression.

Lipopolysaccharide promotes lipid accumulation in human adventitial fibroblasts via TLR4-NF-κB pathway.

PubMed

Wang, Jun; Si, Yanfang; Wu, Chen; Sun, Lu; Ma, Yudong; Ge, Aili; Li, Baomin

2012-10-17

Atherosclerosis is a chronic degenerative disease of the arteries and is thought to be one of the most common causes of death globally. In recent years, the functions of adventitial fibroblasts in the development of atherosclerosis and tissue repair have gained increased interests. LPS can increase the morbidity and mortality of atherosclerosis-associated cardiovascular disease. Although LPS increases neointimal via TLR4 activation has been reported, how LPS augments atherogenesis through acting on adventitial fibroblasts is still unknown. Here we explored lipid deposition within adventitial fibroblasts mediated by lipopolysaccharide (LPS) to imitate inflammatory conditions. In our study, LPS enhanced lipid deposition by the up-regulated expression of adipose differentiation-related protein (ADRP) as the silencing of ADRP abrogated lipid deposition in LPS-activated adventitial fibroblasts. In addition, pre-treatment with anti-Toll-like receptor 4 (TLR4) antibody diminished the LPS-induced lipid deposition and ADRP expression. Moreover, LPS induced translocation of nuclear factor-κB (NF-κB), which could markedly up-regulate lipid deposition as pre-treatment with the NF-κB inhibitor, PDTC, significantly reduced lipid droplets. In addition, the lowering lipid accumulation was accompanied with the decreased ADRP expression. Furthermore, LPS-induced adventitial fibroblasts secreted more monocyte chemoattractant protein (MCP-1), compared with transforming growth factor-β1 (TGF-β1). Taken together, these results suggest that LPS promotes lipid accumulation via the up-regulation of ADRP expression through TLR4 activated downstream of NF-κB in adventitial fibroblasts. Increased levels of MCP-1 released from LPS-activated adventitial fibroblasts and lipid accumulation may accelerate monocytes recruitment and lipid-laden macrophage foam cells formation. Here, our study provides a new explanation as to how bacterial infection contributes to the pathological process of

Lipopolysaccharide promotes lipid accumulation in human adventitial fibroblasts via TLR4-NF-κB pathway

PubMed Central

2012-01-01

Background Atherosclerosis is a chronic degenerative disease of the arteries and is thought to be one of the most common causes of death globally. In recent years, the functions of adventitial fibroblasts in the development of atherosclerosis and tissue repair have gained increased interests. LPS can increase the morbidity and mortality of atherosclerosis-associated cardiovascular disease. Although LPS increases neointimal via TLR4 activation has been reported, how LPS augments atherogenesis through acting on adventitial fibroblasts is still unknown. Here we explored lipid deposition within adventitial fibroblasts mediated by lipopolysaccharide (LPS) to imitate inflammatory conditions. Results In our study, LPS enhanced lipid deposition by the up-regulated expression of adipose differentiation-related protein (ADRP) as the silencing of ADRP abrogated lipid deposition in LPS-activated adventitial fibroblasts. In addition, pre-treatment with anti-Toll-like receptor 4 (TLR4) antibody diminished the LPS-induced lipid deposition and ADRP expression. Moreover, LPS induced translocation of nuclear factor-κB (NF-κB), which could markedly up-regulate lipid deposition as pre-treatment with the NF-κB inhibitor, PDTC, significantly reduced lipid droplets. In addition, the lowering lipid accumulation was accompanied with the decreased ADRP expression. Furthermore, LPS-induced adventitial fibroblasts secreted more monocyte chemoattractant protein (MCP-1), compared with transforming growth factor-β1 (TGF-β1). Conclusions Taken together, these results suggest that LPS promotes lipid accumulation via the up-regulation of ADRP expression through TLR4 activated downstream of NF-κB in adventitial fibroblasts. Increased levels of MCP-1 released from LPS-activated adventitial fibroblasts and lipid accumulation may accelerate monocytes recruitment and lipid-laden macrophage foam cells formation. Here, our study provides a new explanation as to how bacterial infection contributes to

Eritoran inhibits S100A8-mediated TLR4/MD-2 activation and tumor growth by changing the immune microenvironment.

PubMed

Deguchi, A; Tomita, T; Ohto, U; Takemura, K; Kitao, A; Akashi-Takamura, S; Miyake, K; Maru, Y

2016-03-17

S100A8/A9 is a major component of the acute phase of inflammation, and appears to regulate cell proliferation, redox regulation and chemotaxis. We previously reported that S100A8/S100A9 are upregulated in the premetastatic lung. However, the detailed mechanisms by which S100A8 contributes to tumor progression have not been elucidated. In this study, we investigated the TLR4/MD-2 dependency by S100A8 on tumor progression. We found that S100A8 (2-89) peptide stimulated cell migration in a manner dependent on TLR4, MD-2 and MyD88. The S100A8 (2-89) peptide also activated p38 and NF-κB in TLR4-dependent manner. The peptide induced the upregulation of both IL-6 and Ccl2 in peritoneal macrophages obtained from wild-type mice, but not TLR4-deficient mice. We then investigated the responsible region of S100A8 for TLR4/MD-2 binding by a binding assay, and found that C-terminal region of S100A8 binds to TLR4/MD-2 complex. To further evaluate the TLR4 dependency on tumor microenvironment, Lewis lung carcinoma-bearing mice were treated with Eritoran, an antagonist of TLR4/MD-2 complex. We found that both tumor volume and pulmonary recruitment of myeloid-derived suppressor cells were reduced with the treatment of Eritoran for five consecutive days. Eritoran reduced the development of tumor vasculature, and increased tumor-infiltration of CD8(+) T-cells. Taken together, S100A8 appears to play a crucial role in the activation of the TLR4/MD-2 pathway and the promotion of a tumor growth-enhancing immune microenvironment.

Discovery and Structure-Activity Relationships of the Neoseptins: A New Class of Toll-like Receptor-4 (TLR4) Agonists.

PubMed

Morin, Matthew D; Wang, Ying; Jones, Brian T; Su, Lijing; Surakattula, Murali M R P; Berger, Michael; Huang, Hua; Beutler, Elliot K; Zhang, Hong; Beutler, Bruce; Boger, Dale L

2016-05-26

Herein, we report studies leading to the discovery of the neoseptins and a comprehensive examination of the structure-activity relationships (SARs) of this new class of small-molecule mouse Toll-like receptor 4 (mTLR4) agonists. The compounds in this class, which emerged from screening an α-helix mimetic library, stimulate the immune response, act by a well-defined mechanism (mouse TLR4 agonist), are easy to produce and structurally manipulate, exhibit exquisite SARs, are nontoxic, and elicit improved and qualitatively different responses compared to lipopolysaccharide, even though they share the same receptor.

A sex-specific association of common variants of neuroligin genes (NLGN3 and NLGN4X) with autism spectrum disorders in a Chinese Han cohort

PubMed Central

2011-01-01

Background Synaptic genes, NLGN3 and NLGN4X, two homologous members of the neuroligin family, have been supposed as predisposition loci for autism spectrum disorders (ASDs), and defects of these two genes have been identified in a small fraction of individuals with ASDs. But no such rare variant in these two genes has as yet been adequately replicated in Chinese population and no common variant has been further investigated to be associated with ASDs. Methods 7 known ASDs-related rare variants in NLGN3 and NLGN4X genes were screened for replication of the initial findings and 12 intronic tagging single nucleotide polymorphisms (SNPs) were genotyped for case-control association analysis in a total of 229 ASDs cases and 184 control individuals in a Chinese Han cohort, using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Results We found that a common intronic variant, SNP rs4844285 in NLGN3 gene, and a specific 3-marker haplotype XA-XG-XT (rs11795613-rs4844285-rs4844286) containing this individual SNP were associated with ASDs and showed a male bias, even after correction for multiple testing (SNP allele: P = 0.048, haplotype:P = 0.032). Simultaneously, none of these 7 known rare mutation of NLGN3 and NLGN4X genes was identified, neither in our patients with ASDs nor controls, giving further evidence that these known rare variants might be not enriched in Chinese Han cohort. Conclusion The present study provides initial evidence that a common variant in NLGN3 gene may play a role in the etiology of ASDs among affected males in Chinese Han population, and further supports the hypothesis that defect of synapse might involvement in the pathophysiology of ASDs. PMID:21569590

A sex-specific association of common variants of neuroligin genes (NLGN3 and NLGN4X) with autism spectrum disorders in a Chinese Han cohort.

PubMed

Yu, Jindan; He, Xue; Yao, Dan; Li, Zhongyue; Li, Hui; Zhao, Zhengyan

2011-05-14

Synaptic genes, NLGN3 and NLGN4X, two homologous members of the neuroligin family, have been supposed as predisposition loci for autism spectrum disorders (ASDs), and defects of these two genes have been identified in a small fraction of individuals with ASDs. But no such rare variant in these two genes has as yet been adequately replicated in Chinese population and no common variant has been further investigated to be associated with ASDs. 7 known ASDs-related rare variants in NLGN3 and NLGN4X genes were screened for replication of the initial findings and 12 intronic tagging single nucleotide polymorphisms (SNPs) were genotyped for case-control association analysis in a total of 229 ASDs cases and 184 control individuals in a Chinese Han cohort, using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. We found that a common intronic variant, SNP rs4844285 in NLGN3 gene, and a specific 3-marker haplotype XA-XG-XT (rs11795613-rs4844285-rs4844286) containing this individual SNP were associated with ASDs and showed a male bias, even after correction for multiple testing (SNP allele: P = 0.048, haplotype:P = 0.032). Simultaneously, none of these 7 known rare mutation of NLGN3 and NLGN4X genes was identified, neither in our patients with ASDs nor controls, giving further evidence that these known rare variants might be not enriched in Chinese Han cohort. The present study provides initial evidence that a common variant in NLGN3 gene may play a role in the etiology of ASDs among affected males in Chinese Han population, and further supports the hypothesis that defect of synapse might involvement in the pathophysiology of ASDs.

Mechanism of Activation of Enteric Nociceptive Neurons via Interaction of TLR4 and TRPV1 Receptors.

PubMed

Filippova, L V; Fedorova, A V; Nozdrachev, A D

2018-03-01

Evidence obtained by immunohistochemical double labeling and confocal laser scanning microscopy suggests that capsaicin, a ligand of the TRPV1 nociceptive vanilloid receptor, increases the number of TLR4-positive neurons in the rat colon myenteric plexus. In colitis caused by trinitrobenzene sulfonate, an increase in TRPV1 expression was more significant in both plexuses. Specific inhibitor of the TLR4 (C34) pattern-recognition receptor reduces TRPV1 expression in enteric neurons of both intact rats and rats with induced acute colitis. Thus, stimulation of nociceptive neurons by means of direct activation of their receptors of innate immunity (TLR4) is one of the possible mechanisms underlying the visceral pain in bacterial invasion and inflammatory bowel diseases.

SASH1 is a scaffold molecule in endothelial TLR4 signaling.

PubMed

Dauphinee, Shauna M; Clayton, Ashley; Hussainkhel, Angela; Yang, Cindy; Park, Yoo-Jin; Fuller, Megan E; Blonder, Josip; Veenstra, Timothy D; Karsan, Aly

2013-07-15

Recognition of microbial products by TLRs is critical for mediating innate immune responses to invading pathogens. In this study, we identify a novel scaffold protein in TLR4 signaling called SAM and SH3 domain containing protein 1 (SASH1). Sash1 is expressed across all microvascular beds and functions as a scaffold molecule to independently bind TRAF6, TAK1, IκB kinase α, and IκB kinase β. This interaction fosters ubiquitination of TRAF6 and TAK1 and promotes LPS-induced NF-κB, JNK, and p38 activation, culminating in increased production of proinflammatory cytokines and increased LPS-induced endothelial migration. Our findings suggest that SASH1 acts to assemble a signaling complex downstream of TLR4 to activate early endothelial responses to receptor activation.

Hydroxysafflor Yellow A Attenuates Neuron Damage by Suppressing the Lipopolysaccharide-Induced TLR4 Pathway in Activated Microglial Cells.

PubMed

Lv, Yanni; Qian, Yisong; Ou-Yang, Aijun; Fu, Longsheng

2016-11-01

Microglia activation initiates a neurological deficit cascade that contributes to substantial neuronal damage and impairment following ischemia stroke. Toll-like receptor 4 (TLR4) has been demonstrated to play a critical role in this cascade. In the current study, we tested the hypothesis that hydroxysafflor yellow A (HSYA), an active ingredient extracted from Flos Carthami tinctorii, alleviated inflammatory damage, and mediated neurotrophic effects in neurons by inducing the TLR4 pathway in microglia. A non-contact Transwell co-culture system comprised microglia and neurons was treated with HSYA followed by a 1 mg/mL lipopolysaccharide (LPS) stimulation. The microglia were activated prior to neuronal apoptosis, which were induced by increasing TLR4 expression in the activated microglia. However, HSYA suppressed TLR4 expression in the activated microglia, resulting in less neuronal damage at the early stage of LPS stimulation. Western blot analysis and immunofluorescence indicated that dose-dependently HSYA down-regulated TLR4-induced downstream effectors myeloid differentiation factor 88 (MyD88), nuclear factor kappa b (NF-κB), and the mitogen-activated protein kinases (MAPK)-regulated proteins c-Jun NH2-terminal protein kinase (JNK), protein kinase (ERK) 1/2 (ERK1/2), p38 MAPK (p38), as well as the LPS-induced inflammatory cytokine release. However, HSYA up-regulated brain-derived neurotrophic factor (BDNF) expression. Our data suggest that HSYA could exert neurotrophic and anti-inflammatory functions in response to LPS stimulation by inhibiting TLR4 pathway-mediated signaling.

Molecular characterization of a fish-specific toll-like receptor 22 (TLR22) gene from common carp (Cyprinus carpio L.): Evolutionary relationship and induced expression upon immune stimulants.

PubMed

Li, Hua; Yang, Guiwen; Ma, Fei; Li, Ting; Yang, Huiting; Rombout, Jan H W M; An, Liguo

2017-04-01

In the host innate immune system, various pattern recognition receptors (PRRs) recognize conserved pathogens-associated molecular patterns (PAMPs), and represent an efficient first line of defense against invading pathogens. TLR22 is one of the fish-specific Toll-like receptors (TLRs), identified in a variety of fish species. In this study, we report the cloning and identification of a TLR22 cDNA from the gills of common carp (Cyprinus carpio L.). The full-length CcTLR22 cDNA was 3301 bp long, including a 32 bp 5'-untranslated region (UTR), an open reading frame (ORF) of 2838 bp and a 432 bp 3'-UTR.The CcTLR22 protein was found to comprise a signal peptide, 16 LRR domains, a LRRCT domain in the extracellular region and a TIR domain in the cytoplasmic region, which fits with the characteristic TLR domain architecture. The genomic organization of CcTLR22 was identified, which was encoded by an uninterrupted exon. Sequence alignment and phylogenetic analysis showed that all known teleost TLR22 members were clustered into an independent clade of the TLR22 family, and showed high amino acid identities with other fish TLRs. Real-time PCR assay showed that CcTLR22 mRNA was expressed in almost all tissues examined, while the levels obviously varied among different tissues. When challenged with poly(I:C) (a viral model) or A. hydrophila bacteria, the expression level of CcTLR22 was up-regulated in a variety of common carp tissues. These results indicate that CcTLR22 plays a significant role in systemic as well as mucosal defence after viral or bacterial stimulation or infection. Copyright © 2017 Elsevier Ltd. All rights reserved.

Endotoxaemia-augmented murine venous thrombosis is dependent on TLR-4 and ICAM-1, and potentiated by neutropenia.

PubMed

Obi, Andrea T; Andraska, Elizabeth; Kanthi, Yogendra; Kessinger, Chase W; Elfline, Megan; Luke, Cathy; Siahaan, Teruna J; Jaffer, Farouc A; Wakefield, Thomas W; Henke, Peter K

2017-01-26

Venous thromboembolism is a major cause of death during and immediately post-sepsis. Venous thrombosis (VT) is mediated by cell adhesion molecules and leukocytes, including neutrophil extracellular traps (NETs). Sepsis, or experimentally, endotoxaemia, shares similar characteristics and is modulated via toll like receptor 4 (TLR4). This study was undertaken to determine if endotoxaemia potentiates early stasis thrombogenesis, and secondarily to determine the role of VT TLR4, ICAM-1 and neutrophils (PMNs). Wild-type (WT), ICAM-1 -/- and TLR4 -/- mice underwent treatment with saline or LPS (10 mg/kg i. p.) alone, or followed by inferior vena cava (IVC) ligation to generate stasis VT. In vivo microscopy of leukocyte trafficking was performed in non-thrombosed mice, and tissue and plasma were harvested during early VT formation. Pre-thrombosis, circulating ICAM-1 was elevated and increased leukocyte adhesion and rolling occurred on the IVC of LPS-treated mice. Post-thrombosis, endotoxaemic mice formed larger, platelet-poor thrombi. Endotoxaemic TLR4 -/- mice did not have an augmented thrombotic response and exhibited significantly decreased circulating ICAM-1 compared to endotoxaemic WT controls. Endotoxaemic ICAM-1 -/- mice had significantly smaller thrombi compared to controls. Hypothesising that PMNs localised to the inflamed endothelium were promoting thrombosis, PMN depletion using anti-Ly6G antibody was performed. Paradoxically, VT formed without PMNs was amplified, potentially related to endotoxaemia induced elevation of PAI-1 and circulating FXIII, and decreased uPA. Endotoxaemia enhanced early VT occurs in a TLR-4 and ICAM-1 dependent fashion, and is potentiated by neutropenia. ICAM-1 and/or TLR-4 inhibition may be a unique strategy to prevent sepsis-associated VT.

Lactobacillus amylovorus Inhibits the TLR4 Inflammatory Signaling Triggered by Enterotoxigenic Escherichia coli via Modulation of the Negative Regulators and Involvement of TLR2 in Intestinal Caco-2 Cells and Pig Explants

PubMed Central

Finamore, Alberto; Roselli, Marianna; Imbinto, Ambra; Seeboth, Julie; Oswald, Isabelle P.; Mengheri, Elena

2014-01-01

Inflammation derived from pathogen infection involves the activation of toll-like receptor (TLR) signaling. Despite the established immunomodulatory activities of probiotics, studies relating the ability of such bacteria to inhibit the TLR signaling pathways are limited or controversial. In a previous study we showed that Lactobacillus amylovorus DSM 16698T, a novel lactobacillus isolated from unweaned pigs, protects the intestinal cells from enterotoxigenic Escherichia coli (ETEC) K88 infection through cytokine regulation. In the present study we investigated whether the ability of L. amylovorus to counteract the inflammatory status triggered by ETEC in intestine is elicited through inhibition of the TLR4 signaling pathway. We used the human intestinal Caco-2/TC7 cells and intestinal explants isolated from 5 week-old crossbreed Pietrain/Duroc/Large-White piglets, treated with ETEC, L. amylovorus or L. amylovorus cell free supernatant, either alone or simultaneously with ETEC. Western blot analysis showed that L. amylovorus and its cell free supernatant suppress the activation of the different steps of TLR4 signaling in Caco-2/TC7 cells and pig explants, by inhibiting the ETEC induced increase in the level of TLR4 and MyD88, the phosphorylation of the IKKα, IKKβ, IκBα and NF-κB subunit p65, as well as the over-production of inflammatory cytokines IL-8 and IL-1β. The immunofluorescence analysis confirms the lack of phospho-p65 translocation into the nucleus. These anti-inflammatory effects are achieved through modulation of the negative regulators Tollip and IRAK-M. We also found that L. amylovorus blocks the up-regulation of the extracellular heat shock protein (Hsp)72 and Hsp90, that are critical for TLR4 function. By using anti-TLR2 antibody, we demonstrate that TLR2 is required for the suppression of TLR4 signaling activation. These results may contribute to develop therapeutic interventions using L. amylovorus in intestinal disorders of piglets and humans

Sequence Based Structural Characterization and Genetic Diversity Analysis of Full Length TLR4 CDS in Crossbred and Indigenous Cattle.

PubMed

Mishra, Chinmoy; Kumar, Subodh; Sonwane, Arvind Asaram; Yathish, H M; Chaudhary, Rajni

2017-01-02

The exploration of candidate genes for immune response in cattle may be vital for improving our understanding regarding the species specific response to pathogens. Toll-like receptor 4 (TLR4) is mostly involved in protection against the deleterious effects of Gram negative pathogens. Approximately 2.6 kb long cDNA sequence of TLR4 gene covering the entire coding region was characterized in two Indian milk cattle (Vrindavani and Tharparkar). The phylogenetic analysis confirmed that the bovine TLR4 was apparently evolved from an ancestral form that predated the appearance of vertebrates, and it is grouped with buffalo, yak, and mithun TLR4s. Sequence analysis revealed a 2526-nucleotide long open reading frame (ORF) encoding 841 amino acids, similar to other cattle breeds. The calculated molecular weight of the translated ORF was 96144 and 96040.9 Da; the isoelectric point was 6.35 and 6.42 in Vrindavani and Tharparkar cattle, respectively. The Simple Modular Architecture Research Tool (SMART) analysis identified 14 leucine rich repeats (LRR) motifs in bovine TLR4 protein. The deduced TLR4 amino acid sequence of Tharparkar had 4 different substitutions as compared to Bos taurus, Sahiwal, and Vrindavani. The signal peptide cleavage site predicted to lie between 16th and 17th amino acid of mature peptide. The transmebrane helix was identified between 635-657 amino acids in the mature peptide.

Toll-Like Receptor 4 (TLR4) and Triggering Receptor Expressed on Myeloid Cells-2 (TREM-2) Activation Balance Astrocyte Polarization into a Proinflammatory Phenotype.

PubMed

Rosciszewski, Gerardo; Cadena, Vanesa; Murta, Veronica; Lukin, Jeronimo; Villarreal, Alejandro; Roger, Thierry; Ramos, Alberto Javier

2018-05-01

Astrocytes react to brain injury with a generic response known as reactive gliosis, which involves activation of multiple intracellular pathways including several that may be beneficial for neuronal survival. However, by unknown mechanisms, reactive astrocytes can polarize into a proinflammatory phenotype that induces neurodegeneration. In order to study reactive gliosis and astroglial polarization into a proinflammatory phenotype, we used cortical devascularization-induced brain ischemia in Wistar rats and primary astroglial cell cultures exposed to oxygen-glucose deprivation (OGD). We analyzed the profile of TLR4 expression and the consequences of its activation by gain- and loss-of-function studies, and the effects produced by the activation of triggering receptor expressed on myeloid cells-2 (TREM-2), a negative regulator of TLR4 signaling. Both OGD exposure on primary astroglial cell cultures and cortical devascularization brain ischemia in rats induced TLR4 expression in astrocytes. In vivo, astroglial TLR4 expression was specifically observed in the ischemic penumbra surrounding necrotic core. Functional studies showed that OGD increased the astroglial response to the TLR4 agonist lipopolysaccharide (LPS), and conversely, TLR4 knockout primary astrocytes had impaired nuclear factor kappa-B (NF-κB) activation when exposed to LPS. In gain-of-function studies, plasmid-mediated TLR4 over-expression exacerbated astroglial response to LPS as shown by sustained NF-κB activation and increased expression of proinflammatory cytokines IL-1β and TNFα. TREM-2 expression, although present in naïve primary astrocytes, was induced by OGD, LPS, or high-mobility group box 1 protein (HMGB-1) exposure. TREM-2 activation by antibody cross-linking or the overexpression of TREM-2 intracellular adaptor, DAP12, partially suppressed LPS-induced NF-κB activation in purified astrocytic cultures. In vivo, TREM-2 expression was observed in macrophages and astrocytes located in the

Insights into the species-specific TLR4 signaling mechanism in response to Rhodobacter sphaeroides lipid A detection

NASA Astrophysics Data System (ADS)

Anwar, Muhammad Ayaz; Panneerselvam, Suresh; Shah, Masaud; Choi, Sangdun

2015-01-01

TLR4 in complex with MD2 senses the presence of lipid A (LA) and initiates a signaling cascade that curb the infection. This complex is evolutionarily conserved and can initiate the immune system in response to a variety of LAs. In this study, molecular dynamics simulation (25 ns) was performed to elucidate the differential behavior of TLR4/MD2 complex in response to Rhodobacter sphaeroides lipid A (RsLA). Penta-acyl chain-containing RsLA is at the verge of agonist (6 acyl-chains) and antagonist (4 acyl-chains) structure, and activates the TLR4 pathway in horses and hamsters, while inhibiting in humans and murine. In the time-evolved coordinates, the promising factors that dictated the differential response included the local and global mobility pattern of complexes, solvent-accessible surface area of ligand, and surface charge distributions of TLR4 and MD2. We showed that the GlcN1-GlcN2 backbone acquires agonist (3FXI)-like configurations in horses and hamsters, while acquiring antagonist (2E59)-like configurations in humans and murine systems. Moreover, analysis of F126 behavior in the MD2 F126 loop (amino acids 123-129) and loop EF (81-89) suggested that certain sequence variations also contribute to species-specific response. This study underlines the TLR4 signaling mechanism and provides new therapeutic opportunities.

Cyanidin attenuates Aβ25-35-induced neuroinflammation by suppressing NF-κB activity downstream of TLR4/NOX4 in human neuroblastoma cells.

PubMed

Thummayot, Sarinthorn; Tocharus, Chainarong; Jumnongprakhon, Pichaya; Suksamrarn, Apichart; Tocharus, Jiraporn

2018-04-19

Cyanidin is polyphenolic pigment found in plants. We have previously demonstrated that cyanidin protects nerve cells against Aβ 25-35 -induced toxicity by decreasing oxidative stress and attenuating apoptosis mediated by both the mitochondrial apoptotic pathway and the ER stress pathway. To further elucidate the molecular mechanisms underlying the neuroprotective effects of cyanidin, we investigated the effects of cyanidin on neuroinflammation mediated by the TLR4/NOX4 pathway in Aβ 25-35 -treated human neuroblastoma cell line (SK-N-SH). SK-N-SH cells were exposed to Aβ 25-35 (10 μmol/L) for 24 h. Pretreatment with cyanidin (20 μmol/L) or NAC (20 μmol/L) strongly inhibited the NF-κB signaling pathway in the cells evidenced by suppressing the degradation of IκBα, translocation of the p65 subunit of NF-κB from the cytoplasm to the nucleus, and thereby reducing the expression of iNOS protein and the production of NO. Furthermore, pretreatment with cyanidin greatly promoted the translocation of the Nrf2 protein from the cytoplasm to the nucleus; upregulating cytoprotective enzymes, including HO-1, NQO-1 and GCLC; and increased the activity of SOD enzymes. Pretreatment with cyanidin also decreased the expression of TLR4, directly improved intracellular ROS levels and regulated the activity of inflammation-related downstream pathways including NO production and SOD activity through TLR4/NOX4 signaling. These results demonstrate that TLR4 is a primary receptor in SK-N-SH cells, by which Aβ 25-35 triggers neuroinflammation, and cyanidin attenuates Aβ-induced inflammation and ROS production mediated by the TLR4/NOX4 pathway, suggesting that inhibition of TLR4 by cyanidin could be beneficial in preventing neuronal cell death in the process of Alzheimer's disease.

Evaluation of murine lung epithelial cells (TC-1 JHU-1) line to develop Th2-promoting cytokines IL-25/IL-33/TSLP and genes Tlr2/Tlr4 in response to Aspergillus fumigatus.

PubMed

Khosravi, A R; Shokri, H; Hassan Al-Heidary, S; Ghafarifar, F

2018-03-07

The aims of this study were to determine the role of live and heat-killed Aspergillus fumigatus conidia in releasing interleukin (IL)-25, IL-33 and thymic stromal lymphopoietin (TSLP) and to express Toll-like receptor (Tlr)2 and Tlr4 genes. Murine lung epithelial cells were incubated with live and heat-killed A. fumigatus conidia at 37°C for 6, 24 and 48h. After treatments, ELISA was performed to measure the concentrations of IL-25, IL-33 and TSLP in the supernatants. Quantitative real-time PCR (qPCR) was performed to assess the expression levels of Tlr2 and Tlr4 genes. The concentrations of IL-25 and IL-33 significantly increased after exposure to live and heat-killed conidia for various times when compared with untreated control (P<0.05). The secretion of TSLP at different concentrations of heat-killed conidia was significantly higher than both live conidia and untreated control (P<0.05). qRT-PCR results indicated a up-regulation from 1.08 to 3.60-fold for Tlr2 gene expression and 1.20 to 1.80-fold for Tlr4 gene expression exposed to heat-killed conidia. A. fumigatus has a potential ability to stimulate murine lung epithelial cells to produce IL-25/IL-33/TSLP, as well as to express Tlr2/Tlr4 genes, indicating an important role of lung epithelial cells in innate immune responses to A. fumigatus interaction. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Structural insights into pharmacophore-assisted in silico identification of protein-protein interaction inhibitors for inhibition of human toll-like receptor 4 - myeloid differentiation factor-2 (hTLR4-MD-2) complex.

PubMed

Mishra, Vinita; Pathak, Chandramani

2018-05-29

Toll-like receptor 4 (TLR4) is a member of Toll-Like Receptors (TLRs) family that serves as a receptor for bacterial lipopolysaccharide (LPS). TLR4 alone cannot recognize LPS without aid of co-receptor myeloid differentiation factor-2 (MD-2). Binding of LPS with TLR4 forms a LPS-TLR4-MD-2 complex and directs downstream signaling for activation of immune response, inflammation and NF-κB activation. Activation of TLR4 signaling is associated with various pathophysiological consequences. Therefore, targeting protein-protein interaction (PPI) in TLR4-MD-2 complex formation could be an attractive therapeutic approach for targeting inflammatory disorders. The aim of present study was directed to identify small molecule PPI inhibitors (SMPPIIs) using pharmacophore mapping-based approach of computational drug discovery. Here, we had retrieved the information about the hot spot residues and their pharmacophoric features at both primary (TLR4-MD-2) and dimerization (MD-2-TLR4*) protein-protein interaction interfaces in TLR4-MD-2 homo-dimer complex using in silico methods. Promising candidates were identified after virtual screening, which may restrict TLR4-MD-2 protein-protein interaction. In silico off-target profiling over the virtually screened compounds revealed other possible molecular targets. Two of the virtually screened compounds (C11 and C15) were predicted to have an inhibitory concentration in μM range after HYDE assessment. Molecular dynamics simulation study performed for these two compounds in complex with target protein confirms the stability of the complex. After virtual high throughput screening we found selective hTLR4-MD-2 inhibitors, which may have therapeutic potential to target chronic inflammatory diseases.

Upregulation of TLR4 via PKC activation contributes to impaired wound healing in high-glucose-treated kidney proximal tubular cells.

PubMed

Peng, Jianping; Zheng, Hang; Wang, Xia; Cheng, Zhixiang

2017-01-01

Acute kidney injury (AKI) leads to a worse prognosis in diabetic patients compared with prognoses in non-diabetic patients, but whether and how diabetes affects kidney repair after AKI remains unknown. Here, we used scratch-wound healing and transwell migration models to examine whether and how wound healing is affected by high glucose levels in cultured kidney proximal tubular cells (RPTC). The results show that scratch-wound healing and transwell migration were significantly slower in high-glucose-treated kidney tubular cells (30 mM glucose) than in low-glucose-treated cells (5.5 mM). Toll-like receptor 4 (TLR4), MyD88, phospho-protein kinase C (PKC), phospho-p38 MAPK and monocyte chemoattractant protein-1 (MCP-1) mRNA levels were upregulated after high glucose treatments. Staurosporine, a selective PKC inhibitor, inhibited TLR4, MyD88 and p-p38 upregulation in the high-glucose-treated cells, indicating the involvement of PKC in high-glucose-induced TLR4 upregulation. The pharmacological inhibition of TLR4 or shRNA-mediated TLR4 knockdown improved wound healing and transwell migration in high-glucose-treated RPTC. In contrast, the overexpression of TLR4 in low-glucose-treated RPTC suppressed wound healing, mimicking the effects of high glucose levels. These results suggest that the upregulation of TLR4 expression via PKC activation contributes to defective wound healing in high-glucose-treated kidney tubular cells.

Upregulation of TLR4 via PKC activation contributes to impaired wound healing in high-glucose-treated kidney proximal tubular cells

PubMed Central

Peng, Jianping; Zheng, Hang; Wang, Xia; Cheng, Zhixiang

2017-01-01

Acute kidney injury (AKI) leads to a worse prognosis in diabetic patients compared with prognoses in non-diabetic patients, but whether and how diabetes affects kidney repair after AKI remains unknown. Here, we used scratch-wound healing and transwell migration models to examine whether and how wound healing is affected by high glucose levels in cultured kidney proximal tubular cells (RPTC). The results show that scratch-wound healing and transwell migration were significantly slower in high-glucose-treated kidney tubular cells (30 mM glucose) than in low-glucose-treated cells (5.5 mM). Toll-like receptor 4 (TLR4), MyD88, phospho-protein kinase C (PKC), phospho-p38 MAPK and monocyte chemoattractant protein-1 (MCP-1) mRNA levels were upregulated after high glucose treatments. Staurosporine, a selective PKC inhibitor, inhibited TLR4, MyD88 and p-p38 upregulation in the high-glucose-treated cells, indicating the involvement of PKC in high-glucose-induced TLR4 upregulation. The pharmacological inhibition of TLR4 or shRNA-mediated TLR4 knockdown improved wound healing and transwell migration in high-glucose-treated RPTC. In contrast, the overexpression of TLR4 in low-glucose-treated RPTC suppressed wound healing, mimicking the effects of high glucose levels. These results suggest that the upregulation of TLR4 expression via PKC activation contributes to defective wound healing in high-glucose-treated kidney tubular cells. PMID:28542370

Saturated fatty acids enhance TLR4 immune pathways in human trophoblasts.

PubMed

Yang, Xiaohua; Haghiac, Maricela; Glazebrook, Patricia; Minium, Judi; Catalano, Patrick M; Hauguel-de Mouzon, Sylvie

2015-09-01

What are the effects of fatty acids on placental inflammatory cytokine with respect to toll-like receptor-4/nuclear factor-kappa B (TLR4/NF-kB)? Exogenous fatty acids induce a pro-inflammatory cytokine response in human placental cells in vitro via activation of TLR4 signaling pathways. The placenta is exposed to changes in circulating maternal fatty acid concentrations throughout pregnancy. Fatty acids are master regulators of innate immune pathways through recruitment of toll-like receptors and activation of cytokine synthesis. Trophoblast cells isolated from 14 normal term human placentas were incubated with long chain fatty acids (FA) of different carbon length and degree of saturation. The expression and secretion of interleukin-6 (IL-6), IL-8 and tumor necrosis factor-alpha (TNF-α) were measured by reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay. Antibodies against TLR4 ligand binding domain, downstream signaling and anti-p65 NFkB-inhibitor were used to characterize the pathways of FA action. General approach used primary human term trophoblast cell culture. Methods and end-points used real-time quantitative PCR, cytokine measurements, immunohistochemistry, western blots. The long chain saturated fatty acids, stearic and palmitic (PA), stimulated the synthesis as well as the release of TNF-α, IL-6 and IL-8 by trophoblast cells (2- to 6-fold, P < 0.001). In contrast, the unsaturated (palmitoleic, oleic, linoleic) acids did not modify cytokine expression significantly. Palmitate-induced inflammatory effects were mediated via TLR4 activation, NF-kB phosphorylation and nuclear translocation. TNF-α protein level was close to the limit of detection in the culture medium even when cells were cultured with PA. These mechanisms open the way to a better understanding of how changes in maternal lipid homeostasis may regulate placental inflammatory status. X.Y. was recipient of fellowship award from West China Second University

TMEM126A, a CD137 ligand binding protein, couples with the TLR4 signal transduction pathway in macrophages.

PubMed

Kim, Eun-Cheol; Moon, Ji-Hoi; Kang, Sang W; Kwon, Byungsuk; Lee, Hyeon-Woo

2015-04-01

We showed previously that a novel protein, transmembrane protein 126A (TMEM126A), binds to CD137 ligand (CD137L, 4-1BBL) and couples with its reverse signals in macrophages. Here, we present data showing that TMEM126A relays TLR4 signaling. Thus, up-regulation of CD54 (ICAM-1), MHC II, CD86 and CD40 expression in response to TLR4 activation was diminished in TMEM126A-deficient macrophages. Moreover in TMEM126A-deficient RAW264.7 cells, LPS/TLR4-induced late-phase JNK/SAPK and IRF-3 phosphorylation was abolished. These findings indicate that TMEM126A contributes to the TLR4 signal up-regulating the expression of genes whose products are involved in antigen presentation. Copyright © 2014 Elsevier Ltd. All rights reserved.

SOCS3 promotes TLR4 response in macrophages by feedback inhibiting TGF-beta1/Smad3 signaling.

PubMed

Liu, Xia; Zhang, Yongliang; Yu, Yizhi; Yang, Xiao; Cao, Xuetao

2008-03-01

Endogenous transforming growth factor-beta1 (TGF-beta1) plays an important role in the negative regulation of toll-like receptor (TLR) signaling in a feedback manner. Suppressors of cytokine signaling 3 (SOCS3) has been shown to be induced by TGF-beta1 in osteoclast/macrophage, while the reports on the role of SOCS3 in regulating TLR4 signaling were controversial. The functional relationship between SOCS3 and TGF-beta1/Smad3 pathway in TLR4 response also remains unclear. In this study, we demonstrate that LPS-induced endogenous TGF-beta1 contributes to the inducible SOCS3 expression in macrophages. SOCS3 silencing could markedly decrease the LPS-induced production of TNF-alpha and IL-6 in macrophages. Interestingly, less decrease of LPS-induced TNF-alpha, IL-6 by SOCS3 silencing was observed in Smad3 null macrophages. Furthermore, we found SOCS3 could interact with Smad3, and inhibit Smad3 nuclear translocation and transcriptional activity. Therefore, our data demonstrate that SOCS3 is a positive regulator of TLR4 response by feedback inhibiting endogenous TGF-beta1/Smad3 signaling, thus outlining a new feedback regulatory manner for TLR4 response in macrophages.

Species-Specific Activation of TLR4 by Hypoacylated Endotoxins Governed by Residues 82 and 122 of MD-2

PubMed Central

Oblak, Alja; Jerala, Roman

2014-01-01

The Toll-like receptor 4/MD-2 receptor complex recognizes endotoxin, a Gram-negative bacterial cell envelope component. Recognition of the most potent hexaacylated form of endotoxin is mediated by the sixth acyl chain that protrudes from the MD-2 hydrophobic pocket and bridges TLR4/MD-2 to the neighboring TLR4 ectodomain, driving receptor dimerization via hydrophobic interactions. In hypoacylated endotoxins all acyl chains could be accommodated within the binding pocket of the human hMD-2. Nevertheless, tetra- and pentaacylated endotoxins activate the TLR4/MD-2 receptor of several species. We observed that amino acid residues 82 and 122, located at the entrance to the endotoxin binding site of MD-2, have major influence on the species-specific endotoxin recognition. We show that substitution of hMD-2 residue V82 with an amino acid residue with a bulkier hydrophobic side chain enables activation of TLR4/MD-2 by pentaacylated and tetraacylated endotoxins. Interaction of the lipid A phosphate group with the amino acid residue 122 of MD-2 facilitates the appropriate positioning of the hypoacylated endotoxin. Moreover, mouse TLR4 contributes to the agonistic effect of pentaacylated msbB endotoxin. We propose a molecular model that explains how the molecular differences between the murine or equine MD-2, which both have sufficiently large hydrophobic pockets to accommodate all five or four acyl chains, influence the positioning of endotoxin so that one of the acyl chains remains outside the pocket and enables hydrophobic interactions with TLR4, leading to receptor activation. PMID:25203747

Species-specific activation of TLR4 by hypoacylated endotoxins governed by residues 82 and 122 of MD-2.

PubMed

Oblak, Alja; Jerala, Roman

2014-01-01

The Toll-like receptor 4/MD-2 receptor complex recognizes endotoxin, a Gram-negative bacterial cell envelope component. Recognition of the most potent hexaacylated form of endotoxin is mediated by the sixth acyl chain that protrudes from the MD-2 hydrophobic pocket and bridges TLR4/MD-2 to the neighboring TLR4 ectodomain, driving receptor dimerization via hydrophobic interactions. In hypoacylated endotoxins all acyl chains could be accommodated within the binding pocket of the human hMD-2. Nevertheless, tetra- and pentaacylated endotoxins activate the TLR4/MD-2 receptor of several species. We observed that amino acid residues 82 and 122, located at the entrance to the endotoxin binding site of MD-2, have major influence on the species-specific endotoxin recognition. We show that substitution of hMD-2 residue V82 with an amino acid residue with a bulkier hydrophobic side chain enables activation of TLR4/MD-2 by pentaacylated and tetraacylated endotoxins. Interaction of the lipid A phosphate group with the amino acid residue 122 of MD-2 facilitates the appropriate positioning of the hypoacylated endotoxin. Moreover, mouse TLR4 contributes to the agonistic effect of pentaacylated msbB endotoxin. We propose a molecular model that explains how the molecular differences between the murine or equine MD-2, which both have sufficiently large hydrophobic pockets to accommodate all five or four acyl chains, influence the positioning of endotoxin so that one of the acyl chains remains outside the pocket and enables hydrophobic interactions with TLR4, leading to receptor activation.

Boxb mediate BALB/c mice corneal inflammation through a TLR4/MyD88-dependent signaling pathway in Aspergillus fumigatus keratitis.

PubMed

Liu, Min; Li, Cui; Zhao, Gui-Qiu; Lin, Jing; Che, Cheng-Ye; Xu, Qiang; Wang, Qian; Xu, Rui; Niu, Ya-Wen

2018-01-01

To investigate whether high-mobility group box 1 (HMGB1) Boxb exacerbates BALB/c mice corneal immune responses and inflammatory through the Toll-like receptor 4 (TLR4)/myeloid differentiation primary response 88 (MyD88)-dependent signaling pathway in Aspergillus fumigatus ( A. fumigatus ) keratitis. The mice corneas were pretreated with phosphate buffer saline (PBS), Boxb before A. fumigatus infection. The abdominal cavity extracted macrophages were pretreated with PBS, Boxb, TLR4 inhibitor (CLI-095), Dimethyl sulfoxide (DMSO) separately before A. fumigatus hyphae stimulation. HMGB1 was detected in normal and infected mice corneas and macrophages by real-time reverse transcriptase polymerase chain reaction (RT-PCR), the TLR4, MyD88, interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) were detected by Western blot and PCR. In BALB/c mice corneas, the expressions of TLR4, HMGB1, IL-1β, TNF-α were increased after A. fumigatus infection. While pretreatment with Boxb significantly increased the expressions of TLR4, HMGB1, MyD88, IL-1β, TNF-α compared with PBS control after infection. In BALB/c mice abdominal cavity extracted macrophages, pretreatment with Boxb increased the expressions of TLR4, HMGB1, MyD88, IL-1β, TNF-α, while pretreatment with CLI-095 and Boxb significantly decreased the expressions of TLR4, HMGB1, MyD88, IL-1β, TNF-α. In A. fumigatus keratitis, Boxb play a pro-inflammatory role in corneal anti-fungi immune response through the HMGB1-TLR4-MyD88 signal pathway.

Structure-Based Rational Design of a Toll-like Receptor 4 (TLR4) Decoy Receptor with High Binding Affinity for a Target Protein

PubMed Central

Lee, Sang-Chul; Hong, Seungpyo; Park, Keunwan; Jeon, Young Ho; Kim, Dongsup; Cheong, Hae-Kap; Kim, Hak-Sung

2012-01-01

Repeat proteins are increasingly attracting much attention as alternative scaffolds to immunoglobulin antibodies due to their unique structural features. Nonetheless, engineering interaction interface and understanding molecular basis for affinity maturation of repeat proteins still remain a challenge. Here, we present a structure-based rational design of a repeat protein with high binding affinity for a target protein. As a model repeat protein, a Toll-like receptor4 (TLR4) decoy receptor composed of leucine-rich repeat (LRR) modules was used, and its interaction interface was rationally engineered to increase the binding affinity for myeloid differentiation protein 2 (MD2). Based on the complex crystal structure of the decoy receptor with MD2, we first designed single amino acid substitutions in the decoy receptor, and obtained three variants showing a binding affinity (KD) one-order of magnitude higher than the wild-type decoy receptor. The interacting modes and contributions of individual residues were elucidated by analyzing the crystal structures of the single variants. To further increase the binding affinity, single positive mutations were combined, and two double mutants were shown to have about 3000- and 565-fold higher binding affinities than the wild-type decoy receptor. Molecular dynamics simulations and energetic analysis indicate that an additive effect by two mutations occurring at nearby modules was the major contributor to the remarkable increase in the binding affinities. PMID:22363519

Relation of genomic variants for Alzheimer disease dementia to common neuropathologies

PubMed Central

Yu, Lei; Buchman, Aron S.; Schneider, Julie A.; De Jager, Philip L.; Bennett, David A.

2016-01-01

Objective: To investigate the associations of previously reported Alzheimer disease (AD) dementia genomic variants with common neuropathologies. Methods: This is a postmortem study including 1,017 autopsied participants from 2 clinicopathologic cohorts. Analyses focused on 22 genomic variants associated with AD dementia in large-scale case-control genome-wide association study (GWAS) meta-analyses. The neuropathologic traits of interest were a pathologic diagnosis of AD according to NIA-Reagan criteria, macroscopic and microscopic infarcts, Lewy bodies (LB), and hippocampal sclerosis. For each variant, multiple logistic regression was used to investigate its association with neuropathologic traits, adjusting for age, sex, and subpopulation structure. We also conducted power analyses to estimate the sample sizes required to detect genome-wide significance (p < 5 × 10−8) for pathologic AD for all variants. Results: APOE ε4 allele was associated with greater odds of pathologic AD (odds ratio [OR] 3.82, 95% confidence interval [CI] 2.67–5.46, p = 1.9 × 10−13), while ε2 allele was associated with lower odds of pathologic AD (OR 0.42, 95% CI 0.30–0.61, p = 3.1 × 10−6). Four additional genomic variants including rs6656401 (CR1), rs1476679 (ZCWPW1), rs35349669 (INPP5D), and rs17125944 (FERMT2) had p values less than 0.05. Remarkably, half of the previously reported AD dementia variants are not likely to be detected for association with pathologic AD with a sample size in excess of the largest GWAS meta-analyses of AD dementia. Conclusions: Many recently discovered genomic variants for AD dementia are not associated with the pathology of AD. Some genomic variants for AD dementia appear to be associated with other common neuropathologies. PMID:27371493

Relation of genomic variants for Alzheimer disease dementia to common neuropathologies.

PubMed

Farfel, Jose M; Yu, Lei; Buchman, Aron S; Schneider, Julie A; De Jager, Philip L; Bennett, David A

2016-08-02

To investigate the associations of previously reported Alzheimer disease (AD) dementia genomic variants with common neuropathologies. This is a postmortem study including 1,017 autopsied participants from 2 clinicopathologic cohorts. Analyses focused on 22 genomic variants associated with AD dementia in large-scale case-control genome-wide association study (GWAS) meta-analyses. The neuropathologic traits of interest were a pathologic diagnosis of AD according to NIA-Reagan criteria, macroscopic and microscopic infarcts, Lewy bodies (LB), and hippocampal sclerosis. For each variant, multiple logistic regression was used to investigate its association with neuropathologic traits, adjusting for age, sex, and subpopulation structure. We also conducted power analyses to estimate the sample sizes required to detect genome-wide significance (p < 5 × 10(-8)) for pathologic AD for all variants. APOE ε4 allele was associated with greater odds of pathologic AD (odds ratio [OR] 3.82, 95% confidence interval [CI] 2.67-5.46, p = 1.9 × 10(-13)), while ε2 allele was associated with lower odds of pathologic AD (OR 0.42, 95% CI 0.30-0.61, p = 3.1 × 10(-6)). Four additional genomic variants including rs6656401 (CR1), rs1476679 (ZCWPW1), rs35349669 (INPP5D), and rs17125944 (FERMT2) had p values less than 0.05. Remarkably, half of the previously reported AD dementia variants are not likely to be detected for association with pathologic AD with a sample size in excess of the largest GWAS meta-analyses of AD dementia. Many recently discovered genomic variants for AD dementia are not associated with the pathology of AD. Some genomic variants for AD dementia appear to be associated with other common neuropathologies. © 2016 American Academy of Neurology.

Dectin-1 Regulates Hepatic Fibrosis and Hepatocarcinogenesis by Suppressing TLR4 Signaling Pathways.

PubMed

Seifert, Lena; Deutsch, Michael; Alothman, Sara; Alqunaibit, Dalia; Werba, Gregor; Pansari, Mridul; Pergamo, Matthew; Ochi, Atsuo; Torres-Hernandez, Alejandro; Levie, Elliot; Tippens, Daniel; Greco, Stephanie H; Tiwari, Shaun; Ly, Nancy Ngoc Giao; Eisenthal, Andrew; van Heerden, Eliza; Avanzi, Antonina; Barilla, Rocky; Zambirinis, Constantinos P; Rendon, Mauricio; Daley, Donnele; Pachter, H Leon; Hajdu, Cristina; Miller, George

2015-12-01

Dectin-1 is a C-type lectin receptor critical in anti-fungal immunity, but Dectin-1 has not been linked to regulation of sterile inflammation or oncogenesis. We found that Dectin-1 expression is upregulated in hepatic fibrosis and liver cancer. However, Dectin-1 deletion exacerbates liver fibro-inflammatory disease and accelerates hepatocarcinogenesis. Mechanistically, we found that Dectin-1 protects against chronic liver disease by suppressing TLR4 signaling in hepatic inflammatory and stellate cells. Accordingly, Dectin-1(-/-) mice exhibited augmented cytokine production and reduced survival in lipopolysaccharide (LPS)-mediated sepsis, whereas Dectin-1 activation was protective. We showed that Dectin-1 inhibits TLR4 signaling by mitigating TLR4 and CD14 expression, which are regulated by Dectin-1-dependent macrophage colony stimulating factor (M-CSF) expression. Our study suggests that Dectin-1 is an attractive target for experimental therapeutics in hepatic fibrosis and neoplastic transformation. More broadly, our work deciphers critical cross-talk between pattern recognition receptors and implicates a role for Dectin-1 in suppression of sterile inflammation, inflammation-induced oncogenesis, and LPS-mediated sepsis. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Common genetic variants in fatty acid-binding protein-4 (FABP4) and clinical diabetes risk in the Women's Health Initiative Observational Study.

PubMed

Chan, Kei-Hang K; Song, Yiqing; Hsu, Yi-Hsiang; You, Nai-Chieh Y; F Tinker, Lesley; Liu, Simin

2010-09-01

Adipocypte fatty acid-binding protein-4 (FABP4/adipocyte P2) may play a central role in energy metabolism and inflammation. In animal models, defects of the aP2 gene (aP2(-/-)) partially protected against the development of obesity-related insulin resistance, dyslipidemia, and atherosclerosis. However, it is unclear whether common genetic variation in FABP4 gene contributes to risk of type 2 diabetes (T2D) or diabetes-related metabolic traits in humans. We comprehensively assess the genetic associations of variants in the FABP4 gene with T2D risk and diabetes-associated biomarkers in a prospective study of 1,529 cases and 2,147 controls among postmenopausal women aged 50-79 years who enrolled in the Women's Health Initiative Observational Study (WHI-OS). We selected and genotyped a total of 11 haplotype-tagging single-nucleotide polymorphisms (tSNPs) spanning 41.3 kb across FABP4 in all samples. None of the SNPs and their derived haplotypes showed significant association with T2D risk. There were no significant associations between SNPs and plasma levels of inflammatory and endothelial biomarkers, including C-reactive protein, tumor necrosis factor (TNF), interleukin-6 (IL-6), E-selectin, and intercellular adhesion molecule (ICAM-1). Among African-American women, several SNPs were significantly associated with lower levels of vascular cell adhesion molecule-1 (VCAM-1), especially among those with incident T2D. On average, plasma levels of VCAM-1 were significantly lower among carriers of each minor allele at rs1486004(C/T; -1.08 ng/ml, P = 0.01), rs7017115(A/G; -1.07 ng/ml, P = 0.02), and rs2290201(C/T; -1.12 ng/ml, P = 0.002) as compared with the homozygotes of the common allele, respectively. After adjusting for multiple testing, carriers of the rs2290201 minor allele remained significantly associated with decreasing levels of plasma VCAM-1 in these women (P = 0.02). In conclusion, our finding from a multiethnic cohort of postmenopausal women did not support the

Neonatal Plasma Polarizes TLR4-Mediated Cytokine Responses towards Low IL-12p70 and High IL-10 Production via Distinct Factors

PubMed Central

Belderbos, Mirjam E.; Levy, Ofer; Stalpers, Femke; Kimpen, Jan L.; Meyaard, Linde; Bont, Louis

2012-01-01

Human neonates are highly susceptible to infection, which may be due in part to impaired innate immune function. Neonatal Toll-like receptor (TLR) responses are biased against the generation of pro-inflammatory/Th1-polarizing cytokines, yet the underlying mechanisms are incompletely defined. Here, we demonstrate that neonatal plasma polarizes TLR4-mediated cytokine production. When exposed to cord blood plasma, mononuclear cells (MCs) produced significantly lower TLR4-mediated IL-12p70 and higher IL-10 compared to MC exposed to adult plasma. Suppression by neonatal plasma of TLR4-mediated IL-12p70 production, but not induction of TLR4-mediated IL-10 production, was maintained up to the age of 1 month. Cord blood plasma conferred a similar pattern of MC cytokine responses to TLR3 and TLR8 agonists, demonstrating activity towards both MyD88-dependent and MyD88-independent agonists. The factor causing increased TLR4-mediated IL-10 production by cord blood plasma was heat-labile, lost after protein depletion and independent of lipoprotein binding protein (LBP) or soluble CD14 (sCD14). The factor causing inhibition of TLR4-mediated IL-12p70 production by cord blood plasma was resistant to heat inactivation or protein depletion and was independent of IL-10, vitamin D and prostaglandin E2. In conclusion, human neonatal plasma contains at least two distinct factors that suppress TLR4-mediated IL-12p70 production or induce IL-10 or production. Further identification of these factors will provide insight into the ontogeny of innate immune development and might identify novel targets for the prevention and treatment of neonatal infection. PMID:22442690

Bardet-Biedl syndrome gene variants are associated with both childhood and adult common obesity in French Caucasians.

PubMed

Benzinou, Michael; Walley, Andrew; Lobbens, Stephan; Charles, Marie-Aline; Jouret, Béatrice; Fumeron, Frédéric; Balkau, Beverley; Meyre, David; Froguel, Philippe

2006-10-01

Bardet-Biedl syndrome (BBS) is a rare developmental disorder with the cardinal features of abdominal obesity, retinopathy, polydactyly, cognitive impairment, renal and cardiac anomalies, hypertension, and diabetes. BBS is genetically heterogeneous, with nine genes identified to date and evidence for additional loci. In this study, we performed mutation analysis of the coding and conserved regions of BBS1, BBS2, BBS4, and BBS6 in 48 French Caucasian individuals. Among the 36 variants identified, 12 were selected and genotyped in 1,943 French-Caucasian case subjects and 1,299 French-Caucasian nonobese nondiabetic control subjects. Variants in BBS2, BBS4, and BBS6 showed evidence of association with common obesity in an age-dependent manner, the BBS2 single nucleotide polymorphism (SNP) being associated with common adult obesity (P = 0.0005) and the BBS4 and BBS6 SNPs being associated with common early-onset childhood obesity (P = 0.0003) and common adult morbid obesity (0.0003 < P < 0.007). The association of the BBS4 rs7178130 variant was found to be supported by transmission disequilibrium testing (P = 0.006). The BBS6 variants also showed nominal evidence of association with quantitative components of the metabolic syndrome (e.g., dyslipidemia, hyperglycemia), a complication previously described in BBS patients. In summary, our preliminary data suggest that variations at BBS genes are associated with risk of common obesity.

Molecular cloning, characterization and expression analysis of TLR9, MyD88 and TRAF6 genes in common carp (Cyprinus carpio).

PubMed

Kongchum, Pawapol; Hallerman, Eric M; Hulata, Gideon; David, Lior; Palti, Yniv

2011-01-01

Induction of innate immune pathways is critical for early host defense, but there is limited understanding of how teleost fishes recognize pathogen molecules and activate these pathways. In mammals, cells of the innate immune system detect pathogenic molecular structures using pattern recognition receptors (PRRs). TLR9 functions as a PRR that recognizes CpG motifs in bacterial and viral DNA and requires adaptor molecules MyD88 and TRAF6 for signal transduction. Here we report full-length cDNA isolation, structural characterization and tissue mRNA expression analysis of the common carp (cc) TLR9, MyD88 and TRAF6 gene orthologs. The ccTLR9 open-reading frame (ORF) is predicted to encode a 1064-amino acid (aa) protein. We found that MyD88 and TRAF6 genes are duplicated in common carp. This is the first report of TRAF6 duplication in a vertebrate genome and stronger evidence in support of MyD88 duplication is provided. The ccMyD88a and b ORFs are predicted to encode 288-aa and 284-aa peptides, respectively. They share 91% aa sequence identity between paralogs. The ccTRAF6a and b ORFs are both predicted to encode 543-aa peptides sharing 95% aa sequence identity between paralogs. The ccTLR9 gene is contained in a single large exon. The ccMyD88a and ccMyD88b coding sequences span five exons. The TRAF6b gene spans six exons. PCR amplification to obtain the entire coding sequence of ccTRAF6a gene was not successful. The 2104-bp fragment amplified covers the 3' end of the gene and it contains a partial sequence of one exon and three complete exons. The predicated protein domains of the ccTLR9, ccMyD88 and ccTRAF6 are conserved and resemble orthologs from other vertebrates. Real-time quantitative PCR assays of the ccTLR9, MyD88a and b, and TRAF6a and b gene transcripts in healthy common carp indicated that mRNA expression varied between tissues. Differential expression of duplicate copies were found for ccMyD88 and ccTRAF6 in white and red muscle tissues, suggesting that

Activation requirements and responses to TLR ligands in human CD4+ T cells: comparison of two T cell isolation techniques.

PubMed

Lancioni, Christina L; Thomas, Jeremy J; Rojas, Roxana E

2009-05-15

Direct regulation of T cell function by microbial ligands through Toll-like receptors (TLR) is an emerging area of T cell biology. Currently either immunomagnetic cell sorting (IMACS) or fluorescence-activated cell sorting (FACS), are utilized to isolate T-cell subsets for such studies. However, it is unknown to what extent differences in T cell purity between these isolation techniques influence T cell functional assays. We compared the purity, response to mitogen, activation requirements, and response to TLR ligands between human CD4(+) T cells isolated either by IMACS (IMACS-CD4(+)) or by IMACS followed by FACS (IMACS/FACS-CD4(+)). As expected, IMACS-CD4(+) were less pure than IMACS/FACS-CD4(+) (92.5%+/-1.4% versus 99.7%+/-0.2%, respectively). Consequently, IMACS-CD4(+) proliferated and produced cytokines in response to mitogen alone and had lower activation requirements compared to IMACS/FACS-CD4(+). In addition IMACS-CD4(+) but not IMACS/FACS-CD4(+) responses were upregulated by the TLR-4 ligand lipopolysaccharide (LPS). On the other hand, TLR-2 and TLR-5 engagement induced costimulation in both IMACS-CD4(+) and highly purified IMACS-/FACS-CD4(+). Altogether these results indicate that small differences in cell purity can significantly alter T cell responses to TLR ligands. This study stresses the importance of a stringent purification method when investigating the role of microbial ligands in T cell function.

Role of acute ethanol exposure and TLR4 in early events of sepsis in a mouse model

PubMed Central

Bhatty, Minny; Jan, Basit L; Tan, Wei; Pruett, Stephen B; Nanduri, Bindu

2011-01-01

Sepsis is a major cause of death worldwide. The associated risks and mortality are known to significantly increase on exposure to alcohol (chronic or acute). The underlying mechanisms of the association of acute ethanol ingestion and poor prognosis of sepsis are largely unknown. The study described here was designed to determine in detail the role of ethanol and TLR4 in the pathogenesis of the sepsis syndrome. The effects of acute ethanol exposure and TLR4 on bacterial clearance, spleen cell numbers, peritoneal macrophage numbers, and cytokine production were evaluated using wild type and TLR4 hypo-responsive mice treated with ethanol and then challenged with a non pathogenic strain of Escherichia. coli (E. coli). Ethanol treated mice exhibited a decreased clearance of bacteria and produced lesser amounts of most pro-inflammatory cytokines in both strains of mice at two hours after challenge. Neither ethanol treatment nor a hypo-responsive TLR4 had significant effects on the cell numbers in the peritoneal cavity and spleen 2 hours post infection. The suppressive effect of acute ethanol exposure on cytokine and chemokine production was more pronounced in the wild type mice, but the untreated hyporesponsive mice produced less of most cytokines than untreated wild type mice. The major conclusion of this study is that acute ethanol exposure suppresses pro-inflammatory cytokine production and that a hypo-responsive TLR4 (in C3H/HeJ mice) decreases pro-inflammatory cytokine levels but the cytokines and other mediators induced through other receptors are sufficient to ultimately clear the infection but not enough to induce lethal septic shock. In addition, results reported here demonstrate previously unknown effects of acute ethanol exposure on LIF (leukemia inhibitory factor) and eotaxin and provide the first evidence that IL-9 is induced through TLR4 in vivo. PMID:21872420

The TLR4 agonist fibronectin extra domain A is cryptic, exposed by elastase-2; use in a fibrin matrix cancer vaccine

DOE PAGES

Julier, Ziad; Martino, Mikaël M.; de Titta, Alexandre; ...

2015-02-24

Fibronectin (FN) is an extracellular matrix (ECM) protein including numerous fibronectin type III (FNIII) repeats with different functions. The alternatively spliced FN variant containing the extra domain A (FNIII EDA), located between FNIII 11 and FNIII 12, is expressed in sites of injury, chronic inflammation, and solid tumors. Although its function is not well understood, FNIII EDA is known to agonize Toll-like receptor 4 (TLR4). Here, by producing various FN fragments containing FNIII EDA, we found that FNIII EDA's immunological activity depends upon its local intramolecular context within the FN chain. N-terminal extension of the isolated FNIII EDA with itsmore » neighboring FNIII repeats (FNIII 9-10-11) enhanced its activity in agonizing TLR4, while C-terminal extension with the native FNIII 12-13-14 heparin-binding domain abrogated it. We reveal that an elastase 2 cleavage site is present between FNIII EDA and FNIII 12. Activity of the C-terminally extended FNIII EDA could be restored after cleavage of the FNIII 12-13-14 domain by elastase 2. FN being naturally bound to the ECM, we immobilized FNIII EDA-containing FN fragments within a fibrin matrix model along with antigenic peptides. Such matrices were shown to stimulate cytotoxic CD8 + T cell responses in two murine cancer models.« less

TLR4 response mediates ethanol-induced neurodevelopment alterations in a model of fetal alcohol spectrum disorders.

PubMed

Pascual, María; Montesinos, Jorge; Montagud-Romero, Sandra; Forteza, Jerónimo; Rodríguez-Arias, Marta; Miñarro, José; Guerri, Consuelo

2017-07-24

Inflammation during brain development participates in the pathogenesis of early brain injury and cognitive dysfunctions. Prenatal ethanol exposure affects the developing brain and causes neural impairment, cognitive and behavioral effects, collectively known as fetal alcohol spectrum disorders (FASD). Our previous studies demonstrate that ethanol activates the innate immune response and TLR4 receptor and causes neuroinflammation, brain damage, and cognitive defects in the developmental brain stage of adolescents. We hypothesize that by activating the TLR4 response, maternal alcohol consumption during pregnancy triggers the release of cytokines and chemokines in both the maternal sera and brains of fetuses/offspring, which impairs brain ontogeny and causes cognitive dysfunction. WT and TLR4-KO female mice treated with or without 10% ethanol in the drinking water during gestation and lactation were used. Cytokine/chemokine levels were determined by ELISA in the amniotic fluid, maternal serum, and cerebral cortex, as well as in the offspring cerebral cortex. Microglial and neuronal markers (evaluated by western blotting), myelin proteins (immunohistochemical and western blotting) and synaptic parameters (western blotting and electron microscopy) were assessed in the cortices of the WT and TLR4-KO pups on PND 0, 20, and 66. Behavioral tests (elevated plus maze and passive avoidance) were performed in the WT and TLR4-KO mice on PND 66 exposed or not to ethanol. We show that alcohol intake during gestation and lactation increases the levels of several cytokines/chemokines (IL-1β, IL-17, MIP-1α, and fractalkine) in the maternal sera, amniotic fluid, and brains of fetuses and offspring. The upregulation of cytokines/chemokines is associated with an increase in activated microglia markers (CD11b and MHC-II), and with a reduction in some synaptic (synaptotagmin, synapsin IIa) and myelin (MBP, PLP) proteins in the brains of offspring on days 0, 20, and 66 (long-term effects

Analysis of binding site for the novel small-molecule TLR4 signal transduction inhibitor TAK-242 and its therapeutic effect on mouse sepsis model

PubMed Central

Takashima, K; Matsunaga, N; Yoshimatsu, M; Hazeki, K; Kaisho, T; Uekata, M; Hazeki, O; Akira, S; Iizawa, Y; Ii, M

2009-01-01

Background and purpose: TAK-242, a novel synthetic small-molecule, suppresses production of multiple cytokines by inhibiting Toll-like receptor (TLR) 4 signalling. In this study, we investigated the target molecule of TAK-242 and examined its therapeutic effect in a mouse sepsis model. Experimental approach: Binding assay with [3H]-TAK-242 and nuclear factor-κB reporter assay were used to identify the target molecule and binding site of TAK-242. Bacillus calmette guerin (BCG)-primed mouse sepsis model using live Escherichia coli was used to estimate the efficacy of TAK-242 in sepsis. Key results: TAK-242 strongly bound to TLR4, but binding to TLR2, 3, 5, 9, TLR-related adaptor molecules and MD-2 was either not observed or marginal. Mutational analysis using TLR4 mutants indicated that TAK-242 inhibits TLR4 signalling by binding to Cys747 in the intracellular domain of TLR4. TAK-242 inhibited MyD88-independent pathway as well as MyD88-dependent pathway and its inhibitory effect was largely unaffected by lipopolysaccharide (LPS) concentration and types of TLR4 ligands. TAK-242 had no effect on the LPS-induced conformational change of TLR4-MD-2 and TLR4 homodimerization. In mouse sepsis model, although TAK-242 alone did not affect bacterial counts in blood, if co-administered with ceftazidime it inhibited the increases in serum cytokine levels and improved survival of mice. Conclusions and implications: TAK-242 suppressed TLR4 signalling by binding directly to a specific amino acid Cys747 in the intracellular domain of TLR4. When co-administered with antibiotics, TAK-242 showed potent therapeutic effects in an E. coli-induced sepsis model using BCG-primed mice. Thus, TAK-242 may be a promising therapeutic agent for sepsis. PMID:19563534

Common variants at the CHEK2 gene locus and risk of epithelial ovarian cancer

PubMed Central

Lawrenson, Kate; Iversen, Edwin S.; Tyrer, Jonathan; Weber, Rachel Palmieri; Concannon, Patrick; Hazelett, Dennis J.; Li, Qiyuan; Marks, Jeffrey R.; Berchuck, Andrew; Lee, Janet M.; Aben, Katja K.H.; Anton-Culver, Hoda; Antonenkova, Natalia; Bandera, Elisa V.; Bean, Yukie; Beckmann, Matthias W.; Bisogna, Maria; Bjorge, Line; Bogdanova, Natalia; Brinton, Louise A.; Brooks-Wilson, Angela; Bruinsma, Fiona; Butzow, Ralf; Campbell, Ian G.; Carty, Karen; Chang-Claude, Jenny; Chenevix-Trench, Georgia; Chen, Ann; Chen, Zhihua; Cook, Linda S.; Cramer, Daniel W.; Cunningham, Julie M.; Cybulski, Cezary; Plisiecka-Halasa, Joanna; Dennis, Joe; Dicks, Ed; Doherty, Jennifer A.; Dörk, Thilo; du Bois, Andreas; Eccles, Diana; Easton, Douglas T.; Edwards, Robert P.; Eilber, Ursula; Ekici, Arif B.; Fasching, Peter A.; Fridley, Brooke L.; Gao, Yu-Tang; Gentry-Maharaj, Aleksandra; Giles, Graham G.; Glasspool, Rosalind; Goode, Ellen L.; Goodman, Marc T.; Gronwald, Jacek; Harter, Philipp; Hasmad, Hanis Nazihah; Hein, Alexander; Heitz, Florian; Hildebrandt, Michelle A.T.; Hillemanns, Peter; Hogdall, Estrid; Hogdall, Claus; Hosono, Satoyo; Jakubowska, Anna; Paul, James; Jensen, Allan; Karlan, Beth Y.; Kjaer, Susanne Kruger; Kelemen, Linda E.; Kellar, Melissa; Kelley, Joseph L.; Kiemeney, Lambertus A.; Krakstad, Camilla; Lambrechts, Diether; Lambrechts, Sandrina; Le, Nhu D.; Lee, Alice W.; Cannioto, Rikki; Leminen, Arto; Lester, Jenny; Levine, Douglas A.; Liang, Dong; Lissowska, Jolanta; Lu, Karen; Lubinski, Jan; Lundvall, Lene; Massuger, Leon F.A.G.; Matsuo, Keitaro; McGuire, Valerie; McLaughlin, John R.; Nevanlinna, Heli; McNeish, Iain; Menon, Usha; Modugno, Francesmary; Moysich, Kirsten B.; Narod, Steven A.; Nedergaard, Lotte; Ness, Roberta B.; Noor Azmi, Mat Adenan; Odunsi, Kunle; Olson, Sara H.; Orlow, Irene; Orsulic, Sandra; Pearce, Celeste L.; Pejovic, Tanja; Pelttari, Liisa M.; Permuth-Wey, Jennifer; Phelan, Catherine M.; Pike, Malcolm C.; Poole, Elizabeth M.; Ramus, Susan J.; Risch, Harvey A.; Rosen, Barry; Rossing, Mary Anne; Rothstein, Joseph H.; Rudolph, Anja; Runnebaum, Ingo B.; Rzepecka, Iwona K.; Salvesen, Helga B.; Budzilowska, Agnieszka; Sellers, Thomas A.; Shu, Xiao-Ou; Shvetsov, Yurii B.; Siddiqui, Nadeem; Sieh, Weiva; Song, Honglin; Southey, Melissa C.; Sucheston, Lara; Tangen, Ingvild L.; Teo, Soo-Hwang; Terry, Kathryn L.; Thompson, Pamela J.; Timorek, Agnieszka; Tworoger, Shelley S.; Nieuwenhuysen, Els Van; Vergote, Ignace; Vierkant, Robert A.; Wang-Gohrke, Shan; Walsh, Christine; Wentzensen, Nicolas; Whittemore, Alice S.; Wicklund, Kristine G.; Wilkens, Lynne R.; Woo, Yin-Ling; Wu, Xifeng; Wu, Anna H.; Yang, Hannah; Zheng, Wei; Ziogas, Argyrios; Coetzee, Gerhard A.; Freedman, Matthew L.; Monteiro, Alvaro N.A.; Moes-Sosnowska, Joanna; Kupryjanczyk, Jolanta; Pharoah, Paul D.; Gayther, Simon A.; Schildkraut, Joellen M.

2015-01-01

Genome-wide association studies have identified 20 genomic regions associated with risk of epithelial ovarian cancer (EOC), but many additional risk variants may exist. Here, we evaluated associations between common genetic variants [single nucleotide polymorphisms (SNPs) and indels] in DNA repair genes and EOC risk. We genotyped 2896 common variants at 143 gene loci in DNA samples from 15 397 patients with invasive EOC and controls. We found evidence of associations with EOC risk for variants at FANCA, EXO1, E2F4, E2F2, CREB5 and CHEK2 genes (P ≤ 0.001). The strongest risk association was for CHEK2 SNP rs17507066 with serous EOC (P = 4.74 x 10–7). Additional genotyping and imputation of genotypes from the 1000 genomes project identified a slightly more significant association for CHEK2 SNP rs6005807 (r 2 with rs17507066 = 0.84, odds ratio (OR) 1.17, 95% CI 1.11–1.24, P = 1.1×10−7). We identified 293 variants in the region with likelihood ratios of less than 1:100 for representing the causal variant. Functional annotation identified 25 candidate SNPs that alter transcription factor binding sites within regulatory elements active in EOC precursor tissues. In The Cancer Genome Atlas dataset, CHEK2 gene expression was significantly higher in primary EOCs compared to normal fallopian tube tissues (P = 3.72×10−8). We also identified an association between genotypes of the candidate causal SNP rs12166475 (r 2 = 0.99 with rs6005807) and CHEK2 expression (P = 2.70×10-8). These data suggest that common variants at 22q12.1 are associated with risk of serous EOC and CHEK2 as a plausible target susceptibility gene. PMID:26424751

Common variants at the CHEK2 gene locus and risk of epithelial ovarian cancer.

PubMed

Lawrenson, Kate; Iversen, Edwin S; Tyrer, Jonathan; Weber, Rachel Palmieri; Concannon, Patrick; Hazelett, Dennis J; Li, Qiyuan; Marks, Jeffrey R; Berchuck, Andrew; Lee, Janet M; Aben, Katja K H; Anton-Culver, Hoda; Antonenkova, Natalia; Bandera, Elisa V; Bean, Yukie; Beckmann, Matthias W; Bisogna, Maria; Bjorge, Line; Bogdanova, Natalia; Brinton, Louise A; Brooks-Wilson, Angela; Bruinsma, Fiona; Butzow, Ralf; Campbell, Ian G; Carty, Karen; Chang-Claude, Jenny; Chenevix-Trench, Georgia; Chen, Ann; Chen, Zhihua; Cook, Linda S; Cramer, Daniel W; Cunningham, Julie M; Cybulski, Cezary; Plisiecka-Halasa, Joanna; Dennis, Joe; Dicks, Ed; Doherty, Jennifer A; Dörk, Thilo; du Bois, Andreas; Eccles, Diana; Easton, Douglas T; Edwards, Robert P; Eilber, Ursula; Ekici, Arif B; Fasching, Peter A; Fridley, Brooke L; Gao, Yu-Tang; Gentry-Maharaj, Aleksandra; Giles, Graham G; Glasspool, Rosalind; Goode, Ellen L; Goodman, Marc T; Gronwald, Jacek; Harter, Philipp; Hasmad, Hanis Nazihah; Hein, Alexander; Heitz, Florian; Hildebrandt, Michelle A T; Hillemanns, Peter; Hogdall, Estrid; Hogdall, Claus; Hosono, Satoyo; Jakubowska, Anna; Paul, James; Jensen, Allan; Karlan, Beth Y; Kjaer, Susanne Kruger; Kelemen, Linda E; Kellar, Melissa; Kelley, Joseph L; Kiemeney, Lambertus A; Krakstad, Camilla; Lambrechts, Diether; Lambrechts, Sandrina; Le, Nhu D; Lee, Alice W; Cannioto, Rikki; Leminen, Arto; Lester, Jenny; Levine, Douglas A; Liang, Dong; Lissowska, Jolanta; Lu, Karen; Lubinski, Jan; Lundvall, Lene; Massuger, Leon F A G; Matsuo, Keitaro; McGuire, Valerie; McLaughlin, John R; Nevanlinna, Heli; McNeish, Iain; Menon, Usha; Modugno, Francesmary; Moysich, Kirsten B; Narod, Steven A; Nedergaard, Lotte; Ness, Roberta B; Noor Azmi, Mat Adenan; Odunsi, Kunle; Olson, Sara H; Orlow, Irene; Orsulic, Sandra; Pearce, Celeste L; Pejovic, Tanja; Pelttari, Liisa M; Permuth-Wey, Jennifer; Phelan, Catherine M; Pike, Malcolm C; Poole, Elizabeth M; Ramus, Susan J; Risch, Harvey A; Rosen, Barry; Rossing, Mary Anne; Rothstein, Joseph H; Rudolph, Anja; Runnebaum, Ingo B; Rzepecka, Iwona K; Salvesen, Helga B; Budzilowska, Agnieszka; Sellers, Thomas A; Shu, Xiao-Ou; Shvetsov, Yurii B; Siddiqui, Nadeem; Sieh, Weiva; Song, Honglin; Southey, Melissa C; Sucheston, Lara; Tangen, Ingvild L; Teo, Soo-Hwang; Terry, Kathryn L; Thompson, Pamela J; Timorek, Agnieszka; Tworoger, Shelley S; Van Nieuwenhuysen, Els; Vergote, Ignace; Vierkant, Robert A; Wang-Gohrke, Shan; Walsh, Christine; Wentzensen, Nicolas; Whittemore, Alice S; Wicklund, Kristine G; Wilkens, Lynne R; Woo, Yin-Ling; Wu, Xifeng; Wu, Anna H; Yang, Hannah; Zheng, Wei; Ziogas, Argyrios; Coetzee, Gerhard A; Freedman, Matthew L; Monteiro, Alvaro N A; Moes-Sosnowska, Joanna; Kupryjanczyk, Jolanta; Pharoah, Paul D; Gayther, Simon A; Schildkraut, Joellen M

2015-11-01

Genome-wide association studies have identified 20 genomic regions associated with risk of epithelial ovarian cancer (EOC), but many additional risk variants may exist. Here, we evaluated associations between common genetic variants [single nucleotide polymorphisms (SNPs) and indels] in DNA repair genes and EOC risk. We genotyped 2896 common variants at 143 gene loci in DNA samples from 15 397 patients with invasive EOC and controls. We found evidence of associations with EOC risk for variants at FANCA, EXO1, E2F4, E2F2, CREB5 and CHEK2 genes (P ≤ 0.001). The strongest risk association was for CHEK2 SNP rs17507066 with serous EOC (P = 4.74 x 10(-7)). Additional genotyping and imputation of genotypes from the 1000 genomes project identified a slightly more significant association for CHEK2 SNP rs6005807 (r (2) with rs17507066 = 0.84, odds ratio (OR) 1.17, 95% CI 1.11-1.24, P = 1.1×10(-7)). We identified 293 variants in the region with likelihood ratios of less than 1:100 for representing the causal variant. Functional annotation identified 25 candidate SNPs that alter transcription factor binding sites within regulatory elements active in EOC precursor tissues. In The Cancer Genome Atlas dataset, CHEK2 gene expression was significantly higher in primary EOCs compared to normal fallopian tube tissues (P = 3.72×10(-8)). We also identified an association between genotypes of the candidate causal SNP rs12166475 (r (2) = 0.99 with rs6005807) and CHEK2 expression (P = 2.70×10(-8)). These data suggest that common variants at 22q12.1 are associated with risk of serous EOC and CHEK2 as a plausible target susceptibility gene. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Curcumin attenuates acute inflammatory injury by inhibiting the TLR4/MyD88/NF-κB signaling pathway in experimental traumatic brain injury

PubMed Central

2014-01-01

Background Traumatic brain injury (TBI) initiates a neuroinflammatory cascade that contributes to substantial neuronal damage and behavioral impairment, and Toll-like receptor 4 (TLR4) is an important mediator of thiscascade. In the current study, we tested the hypothesis that curcumin, a phytochemical compound with potent anti-inflammatory properties that is extracted from the rhizome Curcuma longa, alleviates acute inflammatory injury mediated by TLR4 following TBI. Methods Neurological function, brain water content and cytokine levels were tested in TLR4-/- mice subjected to weight-drop contusion injury. Wild-type (WT) mice were injected intraperitoneally with different concentrations of curcumin or vehicle 15 minutes after TBI. At 24 hours post-injury, the activation of microglia/macrophages and TLR4 was detected by immunohistochemistry; neuronal apoptosis was measured by FJB and TUNEL staining; cytokines were assayed by ELISA; and TLR4, MyD88 and NF-κB levels were measured by Western blotting. In vitro, a co-culture system comprised of microglia and neurons was treated with curcumin following lipopolysaccharide (LPS) stimulation. TLR4 expression and morphological activation in microglia and morphological damage to neurons were detected by immunohistochemistry 24 hours post-stimulation. Results The protein expression of TLR4 in pericontusional tissue reached a maximum at 24 hours post-TBI. Compared with WT mice, TLR4-/- mice showed attenuated functional impairment, brain edema and cytokine release post-TBI. In addition to improvement in the above aspects, 100 mg/kg curcumin treatment post-TBI significantly reduced the number of TLR4-positive microglia/macrophages as well as inflammatory mediator release and neuronal apoptosis in WT mice. Furthermore, Western blot analysis indicated that the levels of TLR4 and its known downstream effectors (MyD88, and NF-κB) were also decreased after curcumin treatment. Similar outcomes were observed in the microglia and

Lipopolysaccharide-induced inhibition of transcription of tlr4 in vitro is reversed by dexamethasone and correlates with presence of conserved NFκB binding sites

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

Bonin, Camila P., E-mail: mila_bonin@yahoo.com.br; Baccarin, Raquel Y.A., E-mail: baccarin@usp.br; Nostell, Katarina, E-mail: katarina.nostell@slu.se

2013-03-08

Highlights: ► Chimpanzees, horses and humans have regions of similarity on TLR4 and MD2 promoters. ► Rodents have few regions of similarity on TLR4 promoter when compared to primates. ► Conserved NFkB binding sites were found in the promoters of TLR4 and MD2. ► LPS-induced inhibition of TLR4 transcription is reversed by dexamethasone. ► LPS-induced transcription of MD2 is inhibited by dexamethasone. -- Abstract: Engagement of Toll-like receptor 4 (TLR4) by lipopolysaccharide (LPS) is a master trigger of the deleterious effects of septic shock. Horses and humans are considered the most sensitive species to septic shock, but the mechanisms explainingmore » these phenomena remain elusive. Analysis of tlr4 promoters revealed high similarity among LPS-sensitive species (human, chimpanzee, and horse) and low similarity with LPS-resistant species (mouse and rat). Four conserved nuclear factor kappa B (NFκB) binding sites were found in the tlr4 promoter and two in the md2 promoter sequences that are likely to be targets for dexamethasone regulation. In vitro treatment of equine peripheral blood mononuclear cells (eqPBMC) with LPS decreased transcripts of tlr4 and increased transcription of md2 (myeloid differentiation factor 2) and cd14 (cluster of differentiation 14). Treatment with dexamethasone rescued transcription of tlr4 after LPS inhibition. LPS-induced transcription of md2 was inhibited in the presence of dexamethasone. Dexamethasone alone did not affect transcription of tlr4 and md2.« less

Tissue factor and Toll-like receptor (TLR)4 in hyperglycaemia-hyperinsulinaemia. Effects in healthy subjects, and type 1 and type 2 diabetes mellitus.

PubMed

Singh, Anamika; Boden, Guenther; Rao, A Koneti

2015-04-01

Diabetes mellitus (DM) patients have an increased incidence of cardiovascular events. Blood tissue factor-procoagulant activity (TF-PCA), the initiating mechanism for blood coagulation, is elevated in DM. We have shown that hyperglycaemia (HG), hyperinsulinaemia (HI) and combined HG+HI (induced using 24-hour infusion clamps) increases TF-PCA in healthy and type 2 DM (T2DM) subjects, but not in type 1 DM (T1DM) subjects. The mechanisms for this are unknown. DM patients have elevated plasma lipopolysaccharide (LPS), a toll-like receptor (TLR) 4 ligand. We postulated that TLR4 plays a role in modulating TF levels. We studied the effect of HG+HI on TLR4 and TF-PCA in vivo during 24-hour HG+HI infusion clamps in healthy subjects, and T1DM and T2DM subjects, and in vitro in blood. In vivo, in healthy subjects, 24-hour HG + HI infusion increased TLR4 six-fold, which correlated with TF-PCA (r= 0.91, p<0.0001). T2DM patients showed smaller increases in both. In T1DM subjects, TLR4 declined (50%, p<0.05) and correlated with TF-PCA (r=0.55; p<0.05). In vitro, HG (200 mg/dl added glucose) and HI (1-100 nM added insulin) increased TF-PCA in healthy subjects (~2-fold, 2-4 hours). Insulin inhibited by ~30% LPS-induced increase in TF-PCA and high glucose reversed it. TLR4 levels paralleled TF-PCA (r=0.71, p<0.0001); HG and HI increased TLR4 and insulin inhibited LPS-induced TLR4 increase. This is first evidence that even in healthy subjects, HG of short duration increases TLR4 and TF-PCA, key players in inflammation and thrombosis. TLR4-TF interplay is strikingly different in non-diabetic, T1DM and T2DM subjects.

Tissue Factor and Toll Like Receptor (TLR)4 in Hyperglycemia-Hyperinsulinemia: Effects in Healthy Subjects, and Type 1 and Type 2 Diabetes Mellitus

PubMed Central

Singh, Anamika; Boden, Guenther; Rao, A. Koneti

2015-01-01

SUMMARY Background Diabetes mellitus (DM) patients have increased cardiovascular events. Blood tissue factor-procoagulant activity (TF-PCA), the initiating mechanism for blood coagulation, is elevated in DM. We have shown that hyperglycemia (HG), hyperinsulinemia (HI) and combined HG+HI (induced using 24 hr infusion clamps) increases TF-PCA in healthy and T2DM subjects, but not in T1DM subjects. The mechanisms for this are unknown. DM patients have elevated plasma lipopolysaccharide (LPS), a toll-like receptor (TLR) 4 ligand. We postulated that TLR4 plays a role in modulating TF levels. Objectives and Methods We studied the effect of HG+HI on TLR4 and TF-PCA in vivo during 24 hr HG+HI infusion clamps in healthy subjects, and T1DM and T2DM subjects, and in vitro in blood. Results In vivo, in healthy subjects, 24 hr HG + HI infusion increased TLR4 6-fold, which correlated with TF-PCA (r= 0.91, p<0.0001). T2DM patients showed smaller increases in both. In T1DM subjects, TLR4 declined (50%, p<0.05) and correlated with TF-PCA (r=0.55; p<0.05). In vitro, HG (200 mg/dl added glucose) and HI (1-100 nM added insulin) increased TF-PCA in healthy subjects (~2-fold, 2-4 hr). Insulin inhibited by ~30% LPS-induced increase in TF-PCA and high glucose reversed it. TLR4 levels paralleled TF-PCA (r=0.71, p<0.0001); HG and HI increased TLR4 and insulin inhibited LPS-induced TLR4 increase. Conclusions This is first evidence that even in healthy subjects, HG of short duration increases TLR4 and TF-PCA, key players in inflammation and thrombosis. TLR4-TF interplay is strikingly different in non-diabetic, T1DM and T2DM subjects. PMID:25653143

Lipopolysaccharide Clearance, Bacterial Clearance, and Systemic Inflammatory Responses Are Regulated by Cell Type–Specific Functions of TLR4 during Sepsis

PubMed Central

Deng, Meihong; Loughran, Patricia; Gibson, Gregory; Sodhi, Chhinder; Watkins, Simon; Hackam, David

2013-01-01

The morbidity associated with bacterial sepsis is the result of host immune responses to pathogens, which are dependent on pathogen recognition by pattern recognition receptors, such as TLR4. TLR4 is expressed on a range of cell types, yet the mechanisms by which cell-specific functions of TLR4 lead to an integrated sepsis response are poorly understood. To address this, we generated mice in which TLR4 was specifically deleted from myeloid cells (LysMTLR4KO) or hepatocytes (HCTLR4KO) and then determined survival, bacterial counts, host inflammatory responses, and organ injury in a model of cecal ligation and puncture (CLP), with or without antibiotics. LysM-TLR4 was required for phagocytosis and efficient bacterial clearance in the absence of antibiotics. Survival, the magnitude of the systemic and local inflammatory responses, and liver damage were associated with bacterial levels. HCTLR4 was required for efficient LPS clearance from the circulation, and deletion of HCTLR4 was associated with enhanced macrophage phagocytosis, lower bacterial levels, and improved survival in CLP without antibiotics. Antibiotic administration during CLP revealed an important role for hepatocyte LPS clearance in limiting sepsis-induced inflammation and organ injury. Our work defines cell type–selective roles for TLR4 in coordinating complex immune responses to bacterial sepsis and suggests that future strategies for modulating microbial molecule recognition should account for varying roles of pattern recognition receptors in multiple cell populations. PMID:23562812

SNP-SNP Interaction between TLR4 and MyD88 in Susceptibility to Coronary Artery Disease in the Chinese Han Population.

PubMed

Sun, Dandan; Sun, Liping; Xu, Qian; Gong, Yuehua; Wang, Honghu; Yang, Jun; Yuan, Yuan

2016-03-04

The toll-like receptor 4 (TLR4)-myeloid differentiation factor 88 (MyD88)-dependent signaling pathway plays a role in the initiation and progression of coronary artery disease (CAD). We investigated SNP-SNP interactions between the TLR4 and MyD88 genes in CAD susceptibility and assessed whether the effects of such interactions were modified by confounding risk factors (hyperglycemia, hyperlipidemia and Helicobacter pylori (H. pylori) infection). Participants with CAD (n = 424) and controls (n = 424) without CAD were enrolled. Polymerase chain restriction-restriction fragment length polymorphism was performed on genomic DNA to detect polymorphisms in TLR4 (rs10116253, rs10983755, and rs11536889) and MyD88 (rs7744). H. pylori infections were evaluated by enzyme-linked immunosorbent assays, and the cardiovascular risk factors for each subject were evaluated clinically. The significant interaction between TLR4 rs11536889 and MyD88 rs7744 was associated with an increased CAD risk (p value for interaction = 0.024). In conditions of hyperglycemia, the interaction effect was strengthened between TLR4 rs11536889 and MyD88 rs7744 (p value for interaction = 0.004). In hyperlipidemic participants, the interaction strength was also enhanced for TLR4 rs11536889 and MyD88 rs7744 (p value for interaction = 0.006). Thus, the novel interaction between TLR4 rs11536889 and MyD88 rs7744 was related with an increased risk of CAD, that could be strengthened by the presence of hyperglycemia or hyperlipidemia.

Berberine ameliorates diabetic nephropathy by inhibiting TLR4/NF-κB pathway.

PubMed

Zhu, Liping; Han, Jiakai; Yuan, Rongrong; Xue, Lei; Pang, Wuyan

2018-03-31

Diabetic nephropathy (DN) is the leading cause of end-stage renal failure, contributing to severe morbidity and mortality in diabetic patients. Berberine (BBR) has been well characterized to exert renoprotective effects in DN progression. However, the action mechanism of BBR in DN remains to be fully understood. The DN rat model was generated by intraperitoneal injection of streptozotocin (STZ, 65 mg/kg body weight) while 30 mM high glucose (HG)-treated podocytes were used as an in vitro DN model. The fasting blood glucose level and ratio of kidney weight to body weight were measured after BBR treatment (50, 100, or 200 mg/kg) in STZ-induced DN rats. The renal injury parameters including 24-h urinary protein, blood urea nitrogen and serum creatinine were assessed. qRT-PCR was performed to detect the transcript amounts of inflammatory factors. The concentrations of inflammatory factors were evaluated by ELISA kits. Western blot analysis was conducted to measure the amounts of TLR4/NF-κB-related proteins. The apoptotic rate of podocytes was analyzed by flow cytometry using Annexin V/propidium iodide. Berberine reduced renal injury in STZ-induced DN rat model, as evidenced by the decrease in fasting blood glucose, ratio of kidney weight to body weight, 24-h urinary protein, serum creatinine, and blood urine nitrogen. BBR attenuated the systemic and renal cortex inflammatory response and inhibited TLR4/NF-κB pathway in STZ-induced DN rats and HG-induced podocytes. Also, HG-induced apoptosis of podocytes was lowered by BBR administration. Furthermore, blockade of TLR4/NF-κB pathway by resatorvid (TAK-242) or pyrrolidine dithiocarbamate aggravated the inhibitory effect of BBR on HG-induced inflammatory response and apoptosis in podocytes. Berberine ameliorated DN through relieving STZ-induced renal injury, inflammatory response, and podocyte HG-induced apoptosis via inactivating TLR4/NF-κB pathway.

NOS2A, TLR4, and IFNGR1 interactions influence pulmonary tuberculosis susceptibility in African-Americans

PubMed Central

Velez, Digna Rosa; Hulme, William F.; Myers, Jamie L.; Weinberg, J. Brice; Levesque, Marc C.; Stryjewski, Martin E.; Abbate, Eduardo; Estevan, Rosa; Patillo, Sara G.; Gilbert, John R; Hamilton, Carol D.; Scott, William K.

2010-01-01

Tuberculosis (TB) has substantial mortality worldwide with 5-10% of those exposed progressing to active TB disease. Studies in mice and humans indicate that the inducible nitric oxide synthase (iNOS) molecule plays an important role in immune response to TB. A mixed case-control association study of individuals with TB, relatives, or close contact controls was performed in 726 individuals (279 case and 166 control African-Americans; 198 case and 123 control Caucasians). Thirty-nine single nucleotide polymorphisms (SNPs) were selected from the NOS2A gene for single SNP, haplotype, and multilocus interaction analyses with other typed candidate genes using generalized estimating equations. In African-Americans, ten NOS2A SNPs were associated with TB. The strongest associations were observed at rs2274894 (odds ratio (OR) = 1.84, 95% confidence interval (CI) [1.23-2.77], p = 0.003) and rs7215373 (OR 1.67, 95% CI [1.17-2.37], p = 0.004), both of which passed a false discovery rate (FDR) correction for multiple comparisons (q*=0.20). The strongest gene-gene interactions were observed between NOS2A rs2248814 and IFNGR1 rs1327474 (p = 0.0004) and NOS2A rs944722 and IFNGR1 rs1327474 (p = 0.0006). Three other SNPs in NOS2A interacted with TLR4 rs5030729 and five other NOS2A SNPs interacted with IFNGR1 rs1327474. No significant associations were observed in Caucasians. These results suggest that NOS2A variants may contribute to TB susceptibility, particularly in individuals of African descent, and may act synergistically with SNPs in TLR4 and IFNGR1. PMID:19575238

PKC-epsilon and TLR4 synergistically regulate resistin-mediated inflammation in human macrophages.

PubMed

Zuniga, Mary C; Raghuraman, Gayatri; Hitchner, Elizabeth; Weyand, Cornelia; Robinson, William; Zhou, Wei

2017-04-01

Resistin has been associated with atherosclerotic inflammation and cardiovascular complications. We and others have previously shown that PKC-epsilon (PKCε) is involved in resistin-induced smooth muscle cell (VSMC) dysfunction at a high pathological concentration. This study aimed to evaluate the role and potential pathways of resistin at a physiological concentration, in atherosclerosis-related inflammation. Plasma from patients with atherosclerosis was analyzed for resistin concentration. Patients were divided into tertiles based on resistin levels and cytokines were compared between tertiles. Macrophages were then treated with resistin in the presence or absence of PKCε inhibitor and/or TLR4 blocking-antibody, and their inflammatory state was evaluated with ELISA, RT-PCR, immunocytochemistry, and Western blot. We observed significant associations between plasma resistin levels and TNF-α, IL-6, IL-12, MIP-1α, MIP-1β, and CD40L. Our in vitro analyses revealed that resistin activated PKCε via TLR4. This was followed by NF-kB activation and induction of a pro-inflammatory phenotype in macrophages, significantly upregulating CD40, downregulating CD206 and stimulating gene expression and secretion of the inflammatory cytokines, for which we found association in our plasma analysis. Resistin also induced persistent TRAM and CD40L upregulation up to 36 h after resistin treatment. PKCε and TLR4 inhibitors suppressed gene expression to levels similar to control, especially when used in combination. Resistin, at a physiological concentration, exacerbates the inflammatory response of macrophages. PKCε is a key upstream mediator in resistin-induced inflammation that may interact synergistically with TLR4 to promote NF-kB activation, while TRAM is an important signal. PKCε and TRAM may represent novel molecular targets for resistin-associated chronic atherosclerotic inflammation. Copyright © 2017 Elsevier B.V. All rights reserved.

Lipid-induced insulin resistance mediated by the proinflammatory receptor TLR4 requires saturated fatty acid-induced ceramide biosynthesis in mice.

PubMed

Holland, William L; Bikman, Benjamin T; Wang, Li-Ping; Yuguang, Guan; Sargent, Katherine M; Bulchand, Sarada; Knotts, Trina A; Shui, Guanghou; Clegg, Deborah J; Wenk, Markus R; Pagliassotti, Michael J; Scherer, Philipp E; Summers, Scott A

2011-05-01

Obesity is associated with an enhanced inflammatory response that exacerbates insulin resistance and contributes to diabetes, atherosclerosis, and cardiovascular disease. One mechanism accounting for the increased inflammation associated with obesity is activation of the innate immune signaling pathway triggered by TLR4 recognition of saturated fatty acids, an event that is essential for lipid-induced insulin resistance. Using in vitro and in vivo systems to model lipid induction of TLR4-dependent inflammatory events in rodents, we show here that TLR4 is an upstream signaling component required for saturated fatty acid-induced ceramide biosynthesis. This increase in ceramide production was associated with the upregulation of genes driving ceramide biosynthesis, an event dependent of the activity of the proinflammatory kinase IKKβ. Importantly, increased ceramide production was not required for TLR4-dependent induction of inflammatory cytokines, but it was essential for TLR4-dependent insulin resistance. These findings suggest that sphingolipids such as ceramide might be key components of the signaling networks that link lipid-induced inflammatory pathways to the antagonism of insulin action that contributes to diabetes.

Lipid-induced insulin resistance mediated by the proinflammatory receptor TLR4 requires saturated fatty acid–induced ceramide biosynthesis in mice

PubMed Central

Holland, William L.; Bikman, Benjamin T.; Wang, Li-Ping; Yuguang, Guan; Sargent, Katherine M.; Bulchand, Sarada; Knotts, Trina A.; Shui, Guanghou; Clegg, Deborah J.; Wenk, Markus R.; Pagliassotti, Michael J.; Scherer, Philipp E.; Summers, Scott A.

2011-01-01

Obesity is associated with an enhanced inflammatory response that exacerbates insulin resistance and contributes to diabetes, atherosclerosis, and cardiovascular disease. One mechanism accounting for the increased inflammation associated with obesity is activation of the innate immune signaling pathway triggered by TLR4 recognition of saturated fatty acids, an event that is essential for lipid-induced insulin resistance. Using in vitro and in vivo systems to model lipid induction of TLR4-dependent inflammatory events in rodents, we show here that TLR4 is an upstream signaling component required for saturated fatty acid–induced ceramide biosynthesis. This increase in ceramide production was associated with the upregulation of genes driving ceramide biosynthesis, an event dependent of the activity of the proinflammatory kinase IKKβ. Importantly, increased ceramide production was not required for TLR4-dependent induction of inflammatory cytokines, but it was essential for TLR4-dependent insulin resistance. These findings suggest that sphingolipids such as ceramide might be key components of the signaling networks that link lipid-induced inflammatory pathways to the antagonism of insulin action that contributes to diabetes. PMID:21490391

Prevalence of common hemoglobin variants in an afro-descendent Ecuadorian population

PubMed Central

2013-01-01

Background Hemoglobinopathies are among the most studied and frequent pathologies. These genetic disorders are considered a very important health care threat in many tropical countries. Ecuador is a tropical Latin-American country with an important presence of afro-descendants (7.2%). Afro-descendants are among the ethnic groups with higher frequency of hemoglobinopathies reported. Ambuqui is a region within the Imbabura province with an important presence of afro-descendants (>50%). The present study analyzed the frequency of the most common hemoglobin variants in an asymptomatic afro-descendent population using capillary electrophoresis. Findings From 114 individuals, 25 (22%) reported a hemoglobin variant. All individuals that presented hemoglobin variants were heterozygotes (asymptomatic). Hemoglobin S (sickle cell trait) was the most frequent variant found (14%), followed by hemoglobin E (4.4%), Fetal (2.6%) and C (1%). Conclusion Prevalence of hemoglobin S was consistent with populations from other countries, but it was lower than other Ecuadorian afro-descendent populations. Frequency of hemoglobin C was lower than other afro-descendent populations. This data suggests the possibility of gene flow from Native American individuals to the Ambuqui population there by lowering the frequency of their hemoglobin variants compared with other afro-descendant populations. Evaluating the frequency of hemoglobinopathies in Ecuadorian populations is essential. Despite the high frequency of these disorders, very few health care facilities implement hemoglobinopathies tests as a routine practice. PMID:23557107

Association Between Antibiotic Exposure, Bronchiolitis, and TLR4 (rs1927911) Polymorphisms in Childhood Asthma

PubMed Central

Lee, Eun; Kwon, Ji-Won; Kim, Hyo-Bin; Yu, Ho-Sung; Kang, Mi-Jin; Hong, Kyungmo; Yang, Song I; Jung, Young Ho; Lee, Seung-Hwa; Choi, Kil Young; Shin, Hye Lim; Hong, Seo Ah; Kim, Hyung Young; Seo, Ju-Hee; Kim, Byoung-Ju; Lee, So Yeon; Song, Dae Jin; Kim, Woo-Kyung; Jang, Gwang Cheon; Shim, Jung Yeon

2015-01-01

Purpose The complex interplay between environmental and genetic factors plays an important role in the development of asthma. Several studies have yielded conflicting results regarding the 2 asthma-related risk factors: antibiotic usage during infancy and/or a history of bronchiolitis during early life and the development of asthma. In addition to these risk factors, we also explored the effects of Toll-like receptor 4 (TLR4) polymorphism on the development of childhood asthma. Methods This cross-sectional study involved 7,389 middle school students who were from 8 areas of Seoul, Korea, and completed the International Study of Asthma and Allergies in Childhood questionnaire. The TLR4 polymorphism rs1927911 was genotyped in 1,395 middle school students from two areas using the TaqMan assay. Results Bronchiolitis in the first 2 years of life, antibiotic exposure during the first year of life, and parental history of asthma were independent risk factors for the development of asthma. When combined, antibiotic use and a history of bronchiolitis increased the risk of asthma (adjusted odds ratio [aOR]: 4.64, 95% confidence interval [CI]: 3.09-6.97, P value for interaction=0.02). In subjects with CC genotype of TLR4, antibiotic exposure and a history of bronchiolitis during infancy, the risk of asthma was increased, compared to subjects without these risk factors (aOR: 5.72, 95% CI: 1.74-18.87). Conclusions Early-life antibiotic exposures and a history of bronchiolitis are risk factors for asthma in young adolescents. Polymorphisms of TLR4 modified the influence of these environmental factors. Reducing antibiotic exposure and preventing bronchiolitis during infancy may prevent the development of asthma, especially in genetically susceptible subjects. PMID:25729624

Pro-Inflammatory and Pro-Oxidant Status of Pancreatic Islet In Vitro Is Controlled by TLR-4 and HO-1 Pathways

PubMed Central

Vivot, Kevin; Langlois, Allan; Bietiger, William; Dal, Stéphanie; Seyfritz, Elodie; Pinget, Michel; Jeandidier, Nathalie; Maillard, Elisa; Gies, Jean-Pierre; Sigrist, Séverine

2014-01-01

Since their isolation until implantation, pancreatic islets suffer a major stress leading to the activation of inflammatory reactions. The maintenance of controlled inflammation is essential to preserve survival and function of the graft. Identification and targeting of pathway(s) implicated in post-transplant detrimental inflammatory events, is mandatory to improve islet transplantation success. We sought to characterize the expression of the pro-inflammatory and pro-oxidant mediators during islet culture with a focus on Heme oxygenase (HO-1) and Toll-like receptors-4 signaling pathways. Rat pancreatic islets were isolated and pro-inflammatory and pro-oxidant status were evaluated after 0, 12, 24 and 48 hours of culture through TLR-4, HO-1 and cyclooxygenase-2 (COX-2) expression, CCL-2 and IL-6 secretion, ROS (Reactive Oxygen Species) production (Dihydroethidine staining, DHE) and macrophages migration. To identify the therapeutic target, TLR4 inhibition (CLI-095) and HO-1 activation (cobalt protoporphyrin,CoPP) was performed. Activation of NFκB signaling pathway was also investigated. After isolation and during culture, pancreatic islet exhibited a proinflammatory and prooxidant status (increase levels of TLR-4, COX-2, CCL-2, IL-6, and ROS). Activation of HO-1 or inhibition of TLR-4 decreased inflammatory status and oxidative stress of islets. Moreover, the overexpression of HO-1 induced NFκB phosphorylation while the inhibition of TLR-4 had no effect NFκB activation. Finally, inhibition of pro-inflammatory pathway induced a reduction of macrophages migration. These data demonstrated that the TLR-4 signaling pathway is implicated in early inflammatory events leading to a pro-inflammatory and pro-oxidant status of islets in vitro. Moreover, these results provide the mechanism whereby the benefits of HO-1 target in TLR-4 signaling pathway. HO-1 could be then an interesting target to protect islets before transplantation. PMID:25343247

Common Genetic Variants in Fatty Acid–Binding Protein-4 (FABP4) and Clinical Diabetes Risk in the Women's Health Initiative Observational study

PubMed Central

Chan, Kei-Hang K.; Song, Yiqing; Hsu, Yi-Hsiang; You, Nai-chieh Y.; Tinker, Lesley F.; Liu, Simin

2011-01-01

Adipocypte fatty acid–binding protein-4 (FABP4/adipocyte P2) may play a central role in energy metabolism and inflammation. In animal models, defects of the aP2 gene (aP2–/–) partially protected against the development of obesity-related insulin resistance, dyslipidemia, and atherosclerosis. However, it is unclear whether common genetic variation in FABP4 gene contributes to risk of type 2 diabetes (T2D) or diabetes-related metabolic traits in humans. We comprehensively assess the genetic associations of variants in the FABP4 gene with T2D risk and diabetes-associated biomarkers in a prospective study of 1,529 cases and 2,147 controls among postmenopausal women aged 50–79 years who enrolled in the Women's Health Initiative Observational Study (WHI-OS). We selected and genotyped a total of 11 haplotype-tagging single-nucleotide polymorphisms (tSNPs) spanning 41.3 kb across FABP4 in all samples. None of the SNPs and their derived haplotypes showed significant association with T2D risk. There were no significant associations between SNPs and plasma levels of inflammatory and endothelial biomarkers, including C-reactive protein, tumor necrosis factor (TNF), interleukin-6 (IL-6), E-selectin, and intercellular adhesion molecule (ICAM-1). Among African-American women, several SNPs were significantly associated with lower levels of vascular cell adhesion molecule-1 (VCAM-1), especially among those with incident T2D. On average, plasma levels of VCAM-1 were significantly lower among carriers of each minor allele at rs1486004(C/T; −1.08 ng/ml, P = 0.01), rs7017115(A/G; −1.07 ng/ml, P = 0.02), and rs2290201(C/T; −1.12 ng/ml, P = 0.002) as compared with the homozygotes of the common allele, respectively. After adjusting for multiple testing, carriers of the rs2290201 minor allele remained significantly associated with decreasing levels of plasma VCAM-1 in these women (P = 0.02). In conclusion, our finding from a multiethnic cohort of postmenopausal women did not

Toll-like receptor (TLR)-4 mediates anti-β2GPI/β2GPI-induced tissue factor expression in THP-1 cells

PubMed Central

Zhou, H; Yan, Y; Xu, G; Zhou, B; Wen, H; Guo, D; Zhou, F; Wang, H

2011-01-01

Our previous study demonstrated that annexin A2 (ANX2) on cell surface could function as a mediator and stimulate tissue factor (TF) expression of monocytes by anti-β2-glycoprotein I/β2-glycoprotein I complex (anti-β2GPI/β2GPI). However, ANX2 is not a transmembrane protein and lacks the intracellular signal transduction pathway. Growing evidence suggests that Toll-like receptor 4 (TLR-4) might act as an ‘adaptor’ for intracellular signal transduction in anti-β2GPI/β2GPI-induced TF expressing cells. In the current study, we investigated the roles of TLR-4 and its related molecules, myeloid differentiation protein 2 (MD-2) and myeloid differentiation factor 88 (MyD88), in anti-β2GPI/β2GPI-induced TF expressing human monocytic-derived THP-1 (human acute monocytic leukaemia) cells. The relationship of TLR-4 and ANX2 in this process was also explored. Along with TF, expression of TLR-4, MD-2 and MyD88 in THP-1 cells increased significantly when treated by anti-β2GPI (10 µg/ml)/β2GPI (100 µg/ml) complex. The addition of paclitaxel, which competes with the MD-2 ligand, could inhibit the effects of anti-β2GPI/β2GPI on TLR-4, MD-2, MyD88 and TF expression. Both ANX2 and TLR-4 in THP-1 cell lysates could bind to β2GPI that had been conjugated to a column (β2GPI-Affi-Gel). Furthermore, TLR-4, MD-2, MyD88 and TF expression was remarkably diminished in THP-1 cells infected with ANX2-specific RNA interference (RNAi) lentivirus (LV-RNAi-ANX2), in spite of treatment with a similar concentration of anti-β2GPI/β2GPI complex. These results indicate that TLR-4 and its signal transduction pathway contribute to anti-β2GPI/β2GPI-induced TF expression in THP-1 cells, and the effects of TLR-4 with ANX2 are tightly co-operative. PMID:21091668

Antitumor Activity of Portulaca Oleracea L. Polysaccharide on HeLa Cells Through Inducing TLR4/NF-κB Signaling.

PubMed

Zhao, Rui; Zhang, Tao; Ma, Baoling; Li, Xing

2017-01-01

Abstarct We have previously shown that Portulaca oleracea L. polysaccharide (POL-P3b) possesses the ability to inhibit cervical cancer cell growth in vitro and in vivo. In this study, we explored how toll-like receptor 4 (TLR4) signaling correlated with the antitumor mechanism of POL-P3b. Western blotting was utilized to detect the expression of TLR4 and the downstream signaling pathway. The level of inflammatory mediator was quantified using enzyme-linked immunosorbent assay (ELISA) kits. The effects of POL-P3b on the proliferation and apoptosis in HeLa cells were determined by WST-8 assay and Hoechst 33342/propidium iodide (PI) assay. Our results demonstrated that lipopolysaccharide (LPS) binding to TLR4 on tumor cells could enhance HeLa cell proliferation and increase the expression of TLR4 and the downstream molecules. Treating HeLa cells with POL-P3b could decrease the proliferation of HeLa cells, and upregulate Bax level and downregulate Bcl-2 level in a concentration-dependent manner. In addition, POL-P3b inhibited the protein expression levels of TLR4, MyD88, TRAF6, Activator Protein-1 (AP-1) and nuclear factor-κB (NF-κB) subunit P65 in HeLa cells. Furthermore, POL-P3b also reduced the production of cytokine/chemokine. Taken together, the present work suggested the antitumor mechanism of POL-P3b by downregulating TLR4 downstream signaling pathway and inducing cell apoptosis. Our results may provide direct evidence to suggest that POL-P3b should be considered as a potent nutrient supplement for oncotherapy.

Matrine pretreatment improves cardiac function in rats with diabetic cardiomyopathy via suppressing ROS/TLR-4 signaling pathway.

PubMed

Liu, Zhong-wei; Wang, Jun-kui; Qiu, Chuan; Guan, Gong-chang; Liu, Xin-hong; Li, Shang-jian; Deng, Zheng-rong

2015-03-01

Matrine is an alkaloid from Sophora alopecuroides L, which has shown a variety of pharmacological activities and potential therapeutic value in cardiovascular diseases. In this study we examined the protective effects of matrine against diabetic cardiomyopathy (DCM) in rats. Male SD rats were injected with streptozotocin (STZ) to induce DCM. One group of DCM rats was pretreated with matrine (200 mg·kg(-1)·d(-1), po) for 10 consecutive days before STZ injection. Left ventricular function was evaluated using invasive hemodynamic examination, and myocardiac apoptosis was assessed. Primary rat myocytes were used for in vitro experiments. Intracellular ROS generation, MDA content and GPx activity were determined. Real-time PCR and Western blotting were performed to detect the expression of relevant mRNAs and proteins. DCM rats exhibited abnormally elevated non-fasting blood glucose levels at 4 weeks after STZ injection, and LV function impairment at 16 weeks. The cardiac tissues of DCM rats showed markedly increased apoptosis, excessive ROS production, and activation of TLR-4/MyD-88/caspase-8/caspase-3 signaling. Pretreatment with matrine significantly decreased non-fasting blood glucose levels and improved LV function in DCM rats, which were associated with reducing apoptosis and ROS production, and suppressing TLR-4/MyD-88/caspase-8/caspase-3 signaling in cardiac tissues. Incubation in a high-glucose medium induced oxidative stress and activation of TLR-4/MyD-88 signaling in cultured myocytes in vitro, which were significantly attenuated by pretreatment with N-acetylcysteine. Excessive ROS production in DCM activates the TLR-4/MyD-88 signaling, resulting in cardiomyocyte apoptosis, whereas pretreatment with matrine improves cardiac function via suppressing ROS/TLR-4 signaling pathway.

Encryption of agonistic motifs for TLR4 into artificial antigens augmented the maturation of antigen-presenting cells.

PubMed

Ito, Masaki; Hayashi, Kazumi; Minamisawa, Tamiko; Homma, Sadamu; Koido, Shigeo; Shiba, Kiyotaka

2017-01-01

Adjuvants are indispensable for achieving a sufficient immune response from vaccinations. From a functional viewpoint, adjuvants are classified into two categories: "physical adjuvants" increase the efficacy of antigen presentation by antigen-presenting cells (APC) and "signal adjuvants" induce the maturation of APC. Our previous study has demonstrated that a physical adjuvant can be encrypted into proteinous antigens by creating artificial proteins from combinatorial assemblages of epitope peptides and those peptide sequences having propensities to form certain protein structures (motif programming). However, the artificial antigens still require a signal adjuvant to maturate the APC; for example, co-administration of the Toll-like receptor 4 (TLR4) agonist monophosphoryl lipid A (MPLA) was required to induce an in vivo immunoreaction. In this study, we further modified the previous artificial antigens by appending the peptide motifs, which have been reported to have agonistic activity for TLR4, to create "adjuvant-free" antigens. The created antigens with triple TLR4 agonistic motifs in their C-terminus have activated NF-κB signaling pathways through TLR4. These proteins also induced the production of the inflammatory cytokine TNF-α, and the expression of the co-stimulatory molecule CD40 in APC, supporting the maturation of APC in vitro. Unexpectedly, these signal adjuvant-encrypted proteins have lost their ability to be physical adjuvants because they did not induce cytotoxic T lymphocytes (CTL) in vivo, while the parental proteins induced CTL. These results confirmed that the manifestation of a motif's function is context-dependent and simple addition does not always work for motif-programing. Further optimization of the molecular context of the TLR4 agonistic motifs in antigens should be required to create adjuvant-free antigens.

Polymorphisms in NFKB1 and TLR4 and Interaction with Dietary and Life Style Factors in Relation to Colorectal Cancer in a Danish Prospective Case-Cohort Study

PubMed Central

Kopp, Tine Iskov; Andersen, Vibeke; Tjonneland, Anne; Vogel, Ulla

2015-01-01

Maintenance of a balance between commensal bacteria and the mucosal immune system is crucial and intestinal dysbiosis may be a key event in the pathogenesis of colorectal cancer (CRC). The toll-like receptor 4 (TLR4) is an important pattern-recognition receptor that regulates inflammation and barrier function in the gut by a mechanism that involves activation of the nuclear factor–κB (NF-κB) transcription factor. Dietary and life style factors may impact these functions. We therefore used a Danish prospective case-cohort study of 1010 CRC cases and 1829 randomly selected participants from the Danish Diet, Cancer and Health cohort to investigate three polymorphisms in NFKB1 and TLR4 and their possible interactions with diet and life style factors in relation to risk of CRC. Homozygous carriage of the variant allele of the TLR4/rs5030728 polymorphism was associated with increased risk of CRC (incidence rate ratio (IRR) = 1.30; 95% confidence interval (CI): 1.05–1.60; P = 0.02 (gene-dose model); IRR = 1.24; 95%CI: 1.01–1.51; P = 0.04 (recessive model)). Del-carriers of the NFKB1/rs28362491 polymorphism had a 17% (95%CI: 1.03–1.34; P = 0.02) increased risk of CRC compared to homozygous carriers of the ins-allele. However, none of these risk estimates withstood adjustment for multiple comparisons. We found no strong gene-environment interactions between the examined polymorphism and diet and life style factors in relation to CRC risk. PMID:25705893

Modulation of endotoxicity of Shigella generalized modules for membrane antigens (GMMA) by genetic lipid A modifications: relative activation of TLR4 and TLR2 pathways in different mutants.

PubMed

Rossi, Omar; Pesce, Isabella; Giannelli, Carlo; Aprea, Susanna; Caboni, Mariaelena; Citiulo, Francesco; Valentini, Sara; Ferlenghi, Ilaria; MacLennan, Calman Alexander; D'Oro, Ugo; Saul, Allan; Gerke, Christiane

2014-09-05

Outer membrane particles from Gram-negative bacteria are attractive vaccine candidates as they present surface antigens in their natural context. We previously developed a high yield production process for genetically derived particles, called generalized modules for membrane antigens (GMMA), from Shigella. As GMMA are derived from the outer membrane, they contain immunostimulatory components, especially lipopolysaccharide (LPS). We examined ways of reducing their reactogenicity by modifying lipid A, the endotoxic part of LPS, through deletion of late acyltransferase genes, msbB or htrB, in GMMA-producing Shigella sonnei and Shigella flexneri strains. GMMA with resulting penta-acylated lipid A from the msbB mutants showed a 600-fold reduced ability, and GMMA from the S. sonnei ΔhtrB mutant showed a 60,000-fold reduced ability compared with GMMA with wild-type lipid A to stimulate human Toll-like receptor 4 (TLR4) in a reporter cell line. In human peripheral blood mononuclear cells, GMMA with penta-acylated lipid A showed a marked reduction in induction of inflammatory cytokines (S. sonnei ΔhtrB, 800-fold; ΔmsbB mutants, 300-fold). We found that the residual activity of these GMMA is largely due to non-lipid A-related TLR2 activation. In contrast, in the S. flexneri ΔhtrB mutant, a compensatory lipid A palmitoleoylation resulted in GMMA with hexa-acylated lipid A with ∼10-fold higher activity to stimulate peripheral blood mononuclear cells than GMMA with penta-acylated lipid A, mostly due to retained TLR4 activity. Thus, for use as vaccines, GMMA will likely require lipid A penta-acylation. The results identify the relative contributions of TLR4 and TLR2 activation by GMMA, which need to be taken into consideration for GMMA vaccine development. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Effect of rottlerin, a PKC-{delta} inhibitor, on TLR-4-dependent activation of murine microglia

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

Kim, Dong-Chan; Division of Research and Development, Neuronex, Inc., San31, Hyoja-dong, Nam-gu, Pohang 790-784; Kim, Sun-Hee

2005-11-11

In microglia, Toll-like receptors have been shown to recognize pathogen-associated molecular patterns and initiate innate immune responses upon interaction with infectious agents. The effect of rottlerin, a PKC-{delta} specific inhibitor, on TLR-4-mediated signaling was investigated in murine microglia stimulated with lipopolysaccharide and taxol. Pretreatment of microglia cells with rottlerin decreased LPS- and taxol-induced nitric oxide production in a concentration-dependent manner (IC{sub 50} = 99.1 {+-} 1.5 nM). Through MTT and FACS analysis, we found that the inhibition effect of rottlerin was not due to microglial cell death. Rottlerin pretreatment also attenuated LPS-induced phosphorylation of I{kappa}B-{alpha}, nuclear translocation of NF-{kappa}B, andmore » expression of type II nitric oxide synthase. In addition, microglial phagocytosis in response to TLR-4 activation was diminished in which rottlerin was pretreated. Together, these data raise the possibility that certain PKC-{delta} specific inhibitors can modulate TLR-4-derived signaling and inflammatory target gene expression, and can alter susceptibility to microbial infection and chronic inflammatory diseases in central nervous system.« less

Vaccine-Mediated Activation of Human TLR4 Is Affected by Modulation of Culture Conditions during Whole-Cell Pertussis Vaccine Preparation

PubMed Central

Hoonakker, Marieke E.; Verhagen, Lisa M.; Pupo, Elder; de Haan, Alex; Metz, Bernard; Hendriksen, Coenraad F. M.; Han, Wanda G. H.; Sloots, Arjen

2016-01-01

The potency of whole-cell pertussis (wP) vaccines is still determined by an intracerebral mouse protection test. To allow development of suitable in vitro alternatives to this test, insight into relevant parameters to monitor the consistency of vaccine quality is essential. To this end, a panel of experimental wP vaccines of varying quality was prepared by sulfate-mediated suppression of the BvgASR master virulence regulatory system of Bordetella pertussis during cultivation. This system regulates the transcription of a range of virulence proteins, many of which are considered important for the induction of effective host immunity. The protein compositions and in vivo potencies of the vaccines were BvgASR dependent, with the vaccine containing the highest amount of virulence proteins having the highest in vivo potency. Here, the capacities of these vaccines to stimulate human Toll-like receptors (hTLR) 2 and 4 and the role these receptors play in wP vaccine-mediated activation of antigen-presenting cells in vitro were studied. Prolonged BvgASR suppression was associated with a decreased capacity of vaccines to activate hTLR4. In contrast, no significant differences in hTLR2 activation were observed. Similarly, vaccine-induced activation of MonoMac-6 and monocyte-derived dendritic cells was strongest with the highest potency vaccine. Blocking of TLR2 and TLR4 showed that differences in antigen-presenting cell activation could be largely attributed to vaccine-dependent variation in hTLR4 signalling. Interestingly, this BvgASR-dependent decrease in hTLR4 activation coincided with a reduction in GlcN-modified lipopolysaccharides in these vaccines. Accordingly, expression of the lgmA-C genes, required for this glucosamine modification, was significantly reduced in bacteria exposed to sulfate. Together, these findings demonstrate that the BvgASR status of bacteria during wP vaccine preparation is critical for their hTLR4 activation capacity and suggest that including

[Effect of resveratrol on expression of TLR4 and inflammatory factors in gingival epithelial cells under high glucose environment].

PubMed

Lv, Jia-Shu; Jiang, Xue-Wei; Zhang, Yan; Zhen, Lei

2017-02-01

Through a study of the molecular mechanism of the effect of resveratrol(RSV) on expression of TLR4 and inflammatory factors in gingival epithelial cells under high glucose environment, the therapeutic effect and molecular mechanism of resveratrol on periodontitis in patients with diabetes mellitus was investigated. Gingival epithelial cells were cultured in vitro; according to the way of action, the cultured cells were divided into control group, high glucose group(HG) and HG+RSV group. The mRNA expression of TLR4 was detected by PCR; The third generation of gingival epithelial cells were pre-treated with or without RSV for 24 h under high glucose conditions, and subsequently treated with LPS at 100 ng/mL for 2 h. ELISA was used to detect the secretion of IL-1 beta, IL-6, IL-8 and TNF- alpha; the activation of TLR4 downstream signaling molecules NF-κB p65, p38 MAPK, and STAT3 was determined by Western blot. SPSS17.0 software package was used for statistical analysis. RSV could reverse the increase of TLR4 level in gingival epithelial cells in high glucose medium.LPS markedly increased the expression and secretion of IL-1β, IL-6, IL-8, and TNF-α in GECs cultured in high glucose medium, which was partly blocked in the presence of RSV. Furthermore, Western blot results showed that RSV significantly suppressed the phosphorylation of TLR4 downstream factors NF-κB p65, p38MAPK, and STAT3. RSV reduces inflammatory cytokine secretion in gingival epithelial cells, through negative regulation of TLR4 signaling pathway.

Investigation of the role of TCF4 rare sequence variants in schizophrenia.

PubMed

Basmanav, F Buket; Forstner, Andreas J; Fier, Heide; Herms, Stefan; Meier, Sandra; Degenhardt, Franziska; Hoffmann, Per; Barth, Sandra; Fricker, Nadine; Strohmaier, Jana; Witt, Stephanie H; Ludwig, Michael; Schmael, Christine; Moebus, Susanne; Maier, Wolfgang; Mössner, Rainald; Rujescu, Dan; Rietschel, Marcella; Lange, Christoph; Nöthen, Markus M; Cichon, Sven

2015-07-01

Transcription factor 4 (TCF4) is one of the most robust of all reported schizophrenia risk loci and is supported by several genetic and functional lines of evidence. While numerous studies have implicated common genetic variation at TCF4 in schizophrenia risk, the role of rare, small-sized variants at this locus-such as single nucleotide variants and short indels which are below the resolution of chip-based arrays requires further exploration. The aim of the present study was to investigate the association between rare TCF4 sequence variants and schizophrenia. Exon-targeted resequencing was performed in 190 German schizophrenia patients. Six rare variants at the coding exons and flanking sequences of the TCF4 gene were identified, including two missense variants and one splice site variant. These six variants were then pooled with nine additional rare variants identified in 379 European participants of the 1000 Genomes Project, and all 15 variants were genotyped in an independent German sample (n = 1,808 patients; n = 2,261 controls). These data were then analyzed using six statistical methods developed for the association analysis of rare variants. No significant association (P < 0.05) was found. However, the results from our association and power analyses suggest that further research into the possible involvement of rare TCF4 sequence variants in schizophrenia risk is warranted by the assessment of larger cohorts with higher statistical power to identify rare variant associations. © 2015 Wiley Periodicals, Inc.

TLR4 induces CREB-mediated IL-6 production via upregulation of F-spondin to promote vascular smooth muscle cell migration

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

Lee, Guan-Lin; Graduate Institutes of Life Sciences, National Defense Medical Center, Taipei, Taiwan; Wu, Jing-Yiing

Toll-like receptor 4 (TLR4) is important in promoting inflammation and vascular smooth muscle cell (VSMC) migration, both of which contribute to atherosclerosis development and progression. But the mechanism underlying the regulation of TLR4 in VSMC migration remains unclear. Stimulation of VSMCs with LPS increased the cellular level of F-spondin which is associated with the regulation of proinflammatory cytokine production. The LPS-induced F-spondin expression depended on TLR4-mediated PI3K/Akt pathway. Suppression of F-spondin level by siRNA inhibited not only F-spondin expression but also LPS-induced phosphorylation of cAMP response element binding protein (CREB) and IL-6 expression, VSMC migration and proliferation as well asmore » MMP9 expression. Moreover, suppression of CREB level by siRNA inhibited TLR4-induced IL-6 production and VSMC migration. Inhibition of F-spondin siRNA on LPS-induced migration was restored by addition of exogenous recombinant mouse IL-6. We conclude that upon ligand binding, TLR4 activates PI3K/Akt signaling to induce F-spondin expression, subsequently control CREB-mediated IL-6 production to promote VSMC migration. These findings provide vital insights into the essential role of F-spondin in VSMC function and will be valuable for developing new therapeutic strategies against atherosclerosis. -- Highlights: •LPS-induced F-spondin expression of VSMCs is via a TLR4/PI3K/Akt signaling. •F-spondin is pivotal for LPS-induced CREB-mediated IL-6 production. •F-spondin is required for LPS-induced VSMC migration and proliferation.« less

MiR-27-3p regulates TLR2/4-dependent mouse alveolar macrophage activation by targetting PPARγ.

PubMed

Wang, Dan; He, Sirong; Liu, Bicui; Liu, Chuntao

2018-05-16

Activation of alveolar macrophages (AMs) and the release of cytokines play critical roles in the pathogenesis of chronic obstructive pulmonary disease (COPD). However, little is known about the mechanisms of AM activation. miRNAs have recently emerged as key regulators of inflammation and as mediators of macrophage activation and polarization. We identified potential miRNAs related to AM activation using miRNA microarray analysis, which showed that miR-27-3p expression was up-regulated in AMs and the lung tissues of mice exposed to cigarette smoke (CS)/lipopolysaccharide (LPS), and found that miR-27-3p regulated proinflammatory cytokine production and AM polarization depending on TLR2/4 intracellular signaling in AMs. We also found that miR-27-3p controlled TLR2/4 signaling in AMs via targetting the 3'-UTR sequences of peroxisome proliferator-activated receptor γ (PPARγ) and inhibiting PPARγ activation. Moreover, we found that PPARγ activation not only inhibited CS/LPS-induced TLR2/4 expression and miR-27-3p -mediated TLR2/4 signaling cascades involving the nuclear factor-κB (NF-κB), c-Jun NH 2 -terminal kinase (JNK)/p38, and Janus kinase-signal transducer and activator of transcription (JAK/STAT) pathways in AMs but also ameliorated CS/LPS-induced AM activation and pulmonary inflammation. Our study revealed that miR-27-3p mediated AM activation by the inhibition of PPARγ activation and sensitization of TLR signaling. © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

RETRACTED: Sophocarpine displays anti-inflammatory effect via inhibiting TLR4 and TLR4 downstream pathways on LPS-induced mastitis in the mammary gland of mice.

PubMed

Wang, Dehai; Xu, Niannian; Zhang, Zhenbiao; Yang, Shijin; Qiu, Changwei; Li, Chengye; Deng, Ganzhen; Guo, Mengyao

2016-06-01

Mastitis is defined as the inflammation of the mammary gland. LPS, which is widely used to induce mastitis models for the study of this disease, triggers similar inflammation as Escherichia coli. Sophocarpine, isolated from Sophora alopecuroides L., exhibits multiple biological properties. The aim of the present study was to determine the anti-inflammatory effect and mechanism of action of sophocarpine on mastitis within an LPS-induced mouse model. ELISA and western blotting were performed to detect protein levels. The qPCR was performed to detect mRNA levels. The ELISA and qRT-PCR results showed that sophocarpine inhibited the expression of TNF-α, IL-1β and IL-6 in a dose-dependent manner. However, sophocarpine suppressed TLR4 expression. Further study showed that sophocarpine could suppress the phosphorylation of IκBα, p65 and p38. These results confirm that sophocarpine played an anti-inflammatory role in LPS-induced mastitis by regulating TLR4 and the NF-κB and MAPK signaling pathways in mammary gland tissues. Therefore, sophocarpine may be a potential therapeutic drug for the treatment of mastitis. Copyright © 2016 Elsevier B.V. All rights reserved.

Gadolinium compounds signaling through TLR4 and TLR7 in normal human macrophages: establishment of a proinflammatory phenotype and implications for the pathogenesis of nephrogenic systemic fibrosis.

PubMed

Wermuth, Peter J; Jimenez, Sergio A

2012-07-01

Nephrogenic systemic sibrosis is a progressive disorder occurring in some renal insufficiency patients exposed to gadolinium-based contrast agents (GdBCA). Previous studies demonstrated that the GdBCA Omniscan upregulated several innate immunity pathways in normal differentiated human macrophages, induced rapid nuclear localization of the transcription factor NF-κB, and increased the expression and production of numerous profibrotic/proinflammatory cytokines, chemokines, and growth factors. To further examine GdBCA stimulation of the innate immune system, cultured human embryonic kidney 293 cells expressing one of seven different human TLRs or one of two human nucleotide-binding oligomerization domain-like receptors were exposed in vitro for 24 h to various GdBCA. The signaling activity of each compound was evaluated by its ability to activate an NF-κB-inducible reporter gene. Omniscan and gadodiamide induced strong TLR4- and TLR7-mediated reporter gene activation. The other Gd compounds examined failed to induce reporter gene activation. TLR pathway inhibition using chloroquine or an inhibitor of IL-1R-associated kinases 1 and 4 in normal differentiated human macrophages abrogated Omniscan-induced gene expression. Omniscan and gadodiamide signaling via TLRs 4 and 7 resulted in increased production and expression of numerous proinflammatory/profibrotic cytokines, chemokines, and growth factors, including CXCL10, CCL2, CCL8, CXCL12, IL-4, IL-6, TGF-β, and vascular endothelial growth factor. These observations suggest that TLR activation by environmental stimuli may participate in the pathogenesis of nephrogenic systemic fibrosis and of other fibrotic disorders including systemic sclerosis.

Fisetin Alleviates Lipopolysaccharide-Induced Acute Lung Injury via TLR4-Mediated NF-κB Signaling Pathway in Rats.

PubMed

Feng, Guang; Jiang, Ze-Yu; Sun, Bo; Fu, Jie; Li, Tian-Zuo

2016-02-01

Acute lung injury (ALI), a common component of systemic inflammatory disease, is a life-threatening condition without many effective treatments. Fisetin, a natural flavonoid from fruits and vegetables, was reported to have wide pharmacological properties such as anti-inflammatory, antioxidant, and anticancer activities. The aim of this study was to detect the effects of fisetin on lipopolysaccharide (LPS)-induced acute lung injury and investigate the potential mechanism. Fisetin was injected (1, 2, and 4 mg/kg, i.v.) 30 min before LPS administration (5 mg/kg, i.v.). Our results showed that fisetin effectively reduced the inflammatory cytokine release and total protein in bronchoalveolar lavage fluids (BALF), decreased the lung wet/dry ratios, and obviously improved the pulmonary histology in LPS-induced ALI. Furthermore, fisetin inhibited LPS-induced increases of neutrophils and macrophage infiltration and attenuated MPO activity in lung tissues. Additionally, fisetin could significantly inhibit the Toll-like receptor 4 (TLR4) expression and the activation of NF-κB in lung tissues. Our data indicates that fisetin has a protective effect against LPS-induced ALI via suppression of TLR4-mediated NF-κB signaling pathways, and fisetin may be a promising candidate for LPS-induced ALI treatment.

Rational design of novel TLR4 ligands by in silico screening and their functional and structural characterization in vitro.

PubMed

Honegr, Jan; Malinak, David; Dolezal, Rafael; Soukup, Ondrej; Benkova, Marketa; Hroch, Lukas; Benek, Ondrej; Janockova, Jana; Kuca, Kamil; Prymula, Roman

2018-02-25

The purpose of this study was to identify new small molecules that possess activity on human toll-like receptor 4 associated with the myeloid differentiation protein 2 (hTLR4/MD2). Following current rational drug design principles, we firstly performed a ligand and structure based virtual screening of more than 130 000 compounds to discover until now unknown class of hTLR4/MD2 modulators that could be used as novel type of immunologic adjuvants. The core of the in silico study was molecular docking of flexible ligands in a partially flexible hTLR4/MD2 receptor model using a peta-flops-scale supercomputer. The most promising substances resulting from this study, related to anthracene-succimide hybrids, were synthesized and tested. The best prepared candidate exhibited 80% of Monophosphoryl Lipid A in vitro agonistic activity in cell lines expressing hTLR4/MD2. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Evolution of the Bovine TLR Gene Family and Member Associations with Mycobacterium avium Subspecies paratuberculosis Infection

PubMed Central

Fisher, Colleen A.; Bhattarai, Eric K.; Osterstock, Jason B.; Dowd, Scot E.; Seabury, Paul M.; Vikram, Meenu; Whitlock, Robert H.; Schukken, Ynte H.; Schnabel, Robert D.; Taylor, Jeremy F.; Womack, James E.; Seabury, Christopher M.

2011-01-01

Members of the Toll-like receptor (TLR) gene family occupy key roles in the mammalian innate immune system by functioning as sentries for the detection of invading pathogens, thereafter provoking host innate immune responses. We utilized a custom next-generation sequencing approach and allele-specific genotyping assays to detect and validate 280 biallelic variants across all 10 bovine TLR genes, including 71 nonsynonymous single nucleotide polymorphisms (SNPs) and one putative nonsense SNP. Bayesian haplotype reconstructions and median joining networks revealed haplotype sharing between Bos taurus taurus and Bos taurus indicus breeds at every locus, and specialized beef and dairy breeds could not be differentiated despite an average polymorphism density of 1 marker/158 bp. Collectively, 160 tagSNPs and two tag insertion-deletion mutations (indels) were sufficient to predict 100% of the variation at 280 variable sites for both Bos subspecies and their hybrids, whereas 118 tagSNPs and 1 tagIndel predictively captured 100% of the variation at 235 variable sites for B. t. taurus. Polyphen and SIFT analyses of amino acid (AA) replacements encoded by bovine TLR SNPs indicated that up to 32% of the AA substitutions were expected to impact protein function. Classical and newly developed tests of diversity provide strong support for balancing selection operating on TLR3 and TLR8, and purifying selection acting on TLR10. An investigation of the persistence and continuity of linkage disequilibrium (r2≥0.50) between adjacent variable sites also supported the presence of selection acting on TLR3 and TLR8. A case-control study employing validated variants from bovine TLR genes recognizing bacterial ligands revealed six SNPs potentially eliciting small effects on susceptibility to Mycobacterium avium spp paratuberculosis infection in dairy cattle. The results of this study will broadly impact domestic cattle research by providing the necessary foundation to explore several

Initiation of the TLR4 signal transduction network : deeper understanding for better therapeutics.

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

Branda, Steven S.; Hayden, Carl C.; Sherman, Michael Y.

2010-09-01

The innate immune system represents our first line of defense against microbial pathogens, and in many cases is activated by recognition of pathogen cellular components (dsRNA, flagella, LPS, etc.) by cell surface membrane proteins known as toll-like receptors (TLRs). As the initial trigger for innate immune response activation, TLRs also represent a means by which we can effectively control or modulate inflammatory responses. This proposal focused on TLR4, which is the cell-surface receptor primarily responsible for initiating the innate immune response to lipopolysaccharide (LPS), a major component of the outer membrane envelope of gram-negative bacteria. The goal was to bettermore » understand TLR4 activation and associated membrane proximal events, in order to enhance the design of small molecule therapeutics to modulate immune activation. Our approach was to reconstitute the receptor in biomimetic systems in-vitro to allow study of the structure and dynamics with biophysical methods. Structural studies were initiated in the first year but were halted after the crystal structure of the dimerized receptor was published early in the second year of the program. Methods were developed to determine the association constant for oligomerization of the soluble receptor. LPS-induced oligomerization was observed to be a strong function of buffer conditions. In 20 mM Tris pH 8.0 with 200 mM NaCl, the onset of receptor oligomerization occurred at 0.2 uM TLR4/MD2 with E coli LPS Ra mutant in excess. However, in the presence of 0.5 uM CD14 and 0.5 uM LBP, the onset of receptor oligomerization was observed to be less than 10 nM TLR4/MD2. Several methods were pursued to study LPS-induced oligomerization of the membrane-bound receptor, including CryoEM, FRET, colocalization and codiffusion followed by TIRF, and fluorescence correlation spectroscopy. However, there approaches met with only limited success.« less

Aging and amyloid β oligomers enhance TLR4 expression, LPS-induced Ca2+ responses, and neuron cell death in cultured rat hippocampal neurons.

PubMed

Calvo-Rodríguez, María; de la Fuente, Carmen; García-Durillo, Mónica; García-Rodríguez, Carmen; Villalobos, Carlos; Núñez, Lucía

2017-01-31

Toll-like receptors (TLRs) are transmembrane pattern-recognition receptors of the innate immune system recognizing diverse pathogen-derived and tissue damage-related ligands. It has been suggested that TLR signaling contributes to the pathogenesis of age-related, neurodegenerative diseases, including Alzheimer's disease (AD). AD is associated to oligomers of the amyloid β peptide (Aβo) that cause intracellular Ca 2+ dishomeostasis and neuron cell death in rat hippocampal neurons. Here we assessed the interplay between inflammation and Aβo in long-term cultures of rat hippocampal neurons, an in vitro model of neuron aging and/or senescence. Ca 2+ imaging and immunofluorescence against annexin V and TLR4 were applied in short- and long-term cultures of rat hippocampal neurons to test the effects of TLR4-agonist LPS and Aβo on cytosolic [Ca 2+ ] and on apoptosis as well as on expression of TLR4. LPS increases cytosolic [Ca 2+ ] and promotes apoptosis in rat hippocampal neurons in long-term culture considered aged and/or senescent neurons, but not in short-term cultured neurons considered young neurons. TLR4 antagonist CAY10614 prevents both effects. TLR4 expression in rat hippocampal neurons is significantly larger in aged hippocampal cultures. Treatment of aged hippocampal cultures with Aβo increases TLR4 expression and enhances LPS-induced Ca 2+ responses and neuron cell death. Aging and amyloid β oligomers, the neurotoxin involved in Alzheimer's disease, enhance TLR4 expression as well as LPS-induced Ca 2+ responses and neuron cell death in rat hippocampal neurons aged in vitro.

Encryption of agonistic motifs for TLR4 into artificial antigens augmented the maturation of antigen-presenting cells

PubMed Central

Hayashi, Kazumi; Minamisawa, Tamiko; Homma, Sadamu; Koido, Shigeo; Shiba, Kiyotaka

2017-01-01

Adjuvants are indispensable for achieving a sufficient immune response from vaccinations. From a functional viewpoint, adjuvants are classified into two categories: “physical adjuvants” increase the efficacy of antigen presentation by antigen-presenting cells (APC) and “signal adjuvants” induce the maturation of APC. Our previous study has demonstrated that a physical adjuvant can be encrypted into proteinous antigens by creating artificial proteins from combinatorial assemblages of epitope peptides and those peptide sequences having propensities to form certain protein structures (motif programming). However, the artificial antigens still require a signal adjuvant to maturate the APC; for example, co-administration of the Toll-like receptor 4 (TLR4) agonist monophosphoryl lipid A (MPLA) was required to induce an in vivo immunoreaction. In this study, we further modified the previous artificial antigens by appending the peptide motifs, which have been reported to have agonistic activity for TLR4, to create “adjuvant-free” antigens. The created antigens with triple TLR4 agonistic motifs in their C-terminus have activated NF-κB signaling pathways through TLR4. These proteins also induced the production of the inflammatory cytokine TNF-α, and the expression of the co-stimulatory molecule CD40 in APC, supporting the maturation of APC in vitro. Unexpectedly, these signal adjuvant-encrypted proteins have lost their ability to be physical adjuvants because they did not induce cytotoxic T lymphocytes (CTL) in vivo, while the parental proteins induced CTL. These results confirmed that the manifestation of a motif’s function is context-dependent and simple addition does not always work for motif-programing. Further optimization of the molecular context of the TLR4 agonistic motifs in antigens should be required to create adjuvant-free antigens. PMID:29190754

Association of TLR4 polymorphisms with subclinical mastitis in Brazilian holsteins

PubMed Central

de Mesquita, Adriano Queiroz; e Rezende, Cintia Silva Minafra; de Mesquita, Albenones José; Jardim, Eurione Antonio Garcia da Veiga; Kipnis, Ana Paula Junqueira

2012-01-01

The identification of dairy cows with greater or lower potential to develop mastits has been pursued for many years among different segments of the milk industry, including governmental organizations. Genomic studies have suggested that Single Nucleotide Polymorphisms (SNPs) within the pattern recognition receptors (PRR) could lead to different responses to pathogens, and consequently result in mastitis resistance or susceptibility. To investigate whether toll like receptor 4 (TLR4) gene is associated with subclinical mastitis in Holstein cows from a property in the state of Goiás, Brazil, TaqMan allelic discrimination and somatic cell count were performed. One hundred and fifty milk samples were analyzed for SCC and centesimal composition. Twenty percent of those samples with SCC above 200,000 (n=13) were screened for real-time PCR identification of microorganisms and blood samples were genotyped for TLR4 SNPs. There was a higher prevalence of Gram-positive bacteria in the analyzed samples (88.9%) and animals that had the combined genotypes AACCCC, GGTCGG and GACCGC presented the lowest somatic cell scores, and consequently those genotypes have the potential to be applied as molecular markers for assisted animal selection to improve milk quality. PMID:24031881

Decoy peptide targeted to Toll-IL-1R domain inhibits LPS and TLR4-active metabolite morphine-3 glucuronide sensitization of sensory neurons.

PubMed

Allette, Yohance M; Kim, Youngsook; Randolph, Aaron L; Smith, Jared A; Ripsch, Matthew S; White, Fletcher A

2017-06-16

Accumulating evidence indicates that Toll-like receptor (TLR) signaling adapter protein interactions with Toll/Interleukin-1 Receptor (TIR) domains present in sensory neurons may modulate neuropathic pain states. Following ligand interaction with TLRs, TIR serves to both initiate intracellular signaling and facilitate recruitment of signaling adapter proteins to the intracytoplasmic domain. Although TLR TIR is central to a number of TLR signaling cascades, its role in sensory neurons is poorly understood. In this study we investigated the degree to which TLR TIR decoy peptide modified to include a TAT sequence (Trans-Activator of Transcription gene in HIV; TAT-4BB) affected LPS-induced intracellular calcium flux and excitation in sensory neurons, and behavioral changes due to TLR4 active metabolite, morphine-3-glucuronide (M3G) exposure in vivo. TAT-4BB inhibited LPS-induced calcium changes in a majority of sensory neurons and decreased LPS-dependent neuronal excitability in small diameter neurons. Acute systemic administration of the TAT-4BB reversed M3G-induced tactile allodynia in a dose-dependent manner but did not affect motor activity, anxiety or responses to noxious thermal stimulus. These data suggest that targeting TLR TIR domains may provide novel pharmacological targets to reduce or reverse TLR4-dependent pain behavior in the rodent.

From Common to Rare Variants: The Genetic Component of Alzheimer Disease.

PubMed

Nicolas, Gaël; Charbonnier, Camille; Campion, Dominique

2016-01-01

Alzheimer disease (AD) is a remarkable example of genetic heterogeneity. Extremely rare variants in the APP, PSEN1, or PSEN2 genes, or duplications of the APP gene cause autosomal dominant forms, generally with complete penetrance by the age of 65 years. Nonautosomal dominant forms are considered as a complex disorder with a high genetic component, whatever the age of onset. Although genetically heterogeneous, AD is defined by the same neuropathological criteria in all configurations. According to the amyloid cascade hypothesis, the Aβ peptide, which aggregates in AD brains, is a key player. APP, PSEN1, or PSEN2 gene mutations increase the production of more aggregation-prone forms of the Aβ peptide, triggering the pathological process. Several risk factors identified in association studies hit genes involved in Aβ production/secretion, aggregation, clearance, or toxicity. Among them, the APOE ε4 allele is a rare example of a common allele with a large effect size, the ORs ranging from 4 to 11-14 for heterozygous and homozygous carriers, respectively. In addition, genome-wide association studies have identified more than two dozen loci with a weak but significant association, the OR of the at-risk allele ranging from 1.08 to 1.30. Recently, the use of massive parallel sequencing has enabled the analysis of rare variants in a genome-wide manner. Two rare variants have been nominally associated with AD risk or protection (TREM2 p.R47H, MAF approximately 0.002, OR approximately 4 and APP p.A673T, MAF approximately 0.0005, OR approximately 0.2). Association analyses at the gene level identified rare loss-of-function and missense, predicted damaging, variants (MAF <0.01) in the SORL1 and ABCA7 genes associated with a moderate relative risk (OR approximately 5 and approximately 2.8, respectively). Although the latter analyses revealed association signals with moderately rare variants by collapsing them, the power to detect genes hit by extremely rare variants is

Rare, low frequency, and common coding variants in CHRNA5 and their contribution to nicotine dependence in European and African Americans

PubMed Central

Olfson, Emily; Saccone, Nancy L.; Johnson, Eric O.; Chen, Li-Shiun; Culverhouse, Robert; Doheny, Kimberly; Foltz, Steven M.; Fox, Louis; Gogarten, Stephanie M.; Hartz, Sarah; Hetrick, Kurt; Laurie, Cathy C.; Marosy, Beth; Amin, Najaf; Arnett, Donna; Barr, R. Graham; Bartz, Traci M.; Bertelsen, Sarah; Borecki, Ingrid B.; Brown, Michael R.; Chasman, Daniel I.; van Duijn, Cornelia M.; Feitosa, Mary F.; Fox, Ervin R.; Franceschini, Nora; Franco, Oscar H.; Grove, Megan L.; Guo, Xiuqing; Hofman, Albert; Kardia, Sharon L.R.; Morrison, Alanna C.; Musani, Solomon K.; Psaty, Bruce M.; Rao, D.C.; Reiner, Alex P.; Rice, Kenneth; Ridker, Paul M.; Rose, Lynda M.; Schick, Ursula M.; Schwander, Karen; Uitterlinden, Andre G.; Vojinovic, Dina; Wang, Jen-Chyong; Ware, Erin B.; Wilson, Gregory; Yao, Jie; Zhao, Wei; Breslau, Naomi; Hatsukami, Dorothy; Stitzel, Jerry A.; Rice, John; Goate, Alison; Bierut, Laura J.

2015-01-01

The common nonsynonymous variant rs16969968 in the α5 nicotinic receptor subunit gene (CHRNA5) is the strongest genetic risk factor for nicotine dependence in European Americans and contributes to risk in African Americans. To comprehensively examine whether other CHRNA5 coding variation influences nicotine dependence risk, we performed targeted sequencing on 1582 nicotine dependent cases (Fagerström Test for Nicotine Dependence score≥4) and 1238 non-dependent controls, with independent replication of common and low frequency variants using 12 studies with exome chip data. Nicotine dependence was examined using logistic regression with individual common variants (MAF≥0.05), aggregate low frequency variants (0.05>MAF≥0.005), and aggregate rare variants (MAF<0.005). Meta-analysis of primary results was performed with replication studies containing 12 174 heavy and 11 290 light smokers. Next-generation sequencing with 180X coverage identified 24 nonsynonymous variants and 2 frameshift deletions in CHRNA5, including 9 novel variants in the 2820 subjects. Meta-analysis confirmed the risk effect of the only common variant (rs16969968, European ancestry: OR=1.3, p=3.5×10−11; African ancestry: OR=1.3, p=0.01) and demonstrated that 3 low frequency variants contributed an independent risk (aggregate term, European ancestry: OR=1.3, p=0.005; African ancestry: OR=1.4, p=0.0006). The remaining 22 rare coding variants were associated with increased risk of nicotine dependence in the European American primary sample (OR=12.9, p=0.01) and in the same risk direction in African Americans (OR=1.5, p=0.37). Our results indicate that common, low frequency and rare CHRNA5 coding variants are independently associated with nicotine dependence risk. These newly identified variants likely influence risk for smoking-related diseases such as lung cancer. PMID:26239294

Rare, low frequency and common coding variants in CHRNA5 and their contribution to nicotine dependence in European and African Americans.

PubMed

Olfson, E; Saccone, N L; Johnson, E O; Chen, L-S; Culverhouse, R; Doheny, K; Foltz, S M; Fox, L; Gogarten, S M; Hartz, S; Hetrick, K; Laurie, C C; Marosy, B; Amin, N; Arnett, D; Barr, R G; Bartz, T M; Bertelsen, S; Borecki, I B; Brown, M R; Chasman, D I; van Duijn, C M; Feitosa, M F; Fox, E R; Franceschini, N; Franco, O H; Grove, M L; Guo, X; Hofman, A; Kardia, S L R; Morrison, A C; Musani, S K; Psaty, B M; Rao, D C; Reiner, A P; Rice, K; Ridker, P M; Rose, L M; Schick, U M; Schwander, K; Uitterlinden, A G; Vojinovic, D; Wang, J-C; Ware, E B; Wilson, G; Yao, J; Zhao, W; Breslau, N; Hatsukami, D; Stitzel, J A; Rice, J; Goate, A; Bierut, L J

2016-05-01

The common nonsynonymous variant rs16969968 in the α5 nicotinic receptor subunit gene (CHRNA5) is the strongest genetic risk factor for nicotine dependence in European Americans and contributes to risk in African Americans. To comprehensively examine whether other CHRNA5 coding variation influences nicotine dependence risk, we performed targeted sequencing on 1582 nicotine-dependent cases (Fagerström Test for Nicotine Dependence score⩾4) and 1238 non-dependent controls, with independent replication of common and low frequency variants using 12 studies with exome chip data. Nicotine dependence was examined using logistic regression with individual common variants (minor allele frequency (MAF)⩾0.05), aggregate low frequency variants (0.05>MAF⩾0.005) and aggregate rare variants (MAF<0.005). Meta-analysis of primary results was performed with replication studies containing 12 174 heavy and 11 290 light smokers. Next-generation sequencing with 180 × coverage identified 24 nonsynonymous variants and 2 frameshift deletions in CHRNA5, including 9 novel variants in the 2820 subjects. Meta-analysis confirmed the risk effect of the only common variant (rs16969968, European ancestry: odds ratio (OR)=1.3, P=3.5 × 10(-11); African ancestry: OR=1.3, P=0.01) and demonstrated that three low frequency variants contributed an independent risk (aggregate term, European ancestry: OR=1.3, P=0.005; African ancestry: OR=1.4, P=0.0006). The remaining 22 rare coding variants were associated with increased risk of nicotine dependence in the European American primary sample (OR=12.9, P=0.01) and in the same risk direction in African Americans (OR=1.5, P=0.37). Our results indicate that common, low frequency and rare CHRNA5 coding variants are independently associated with nicotine dependence risk. These newly identified variants likely influence the risk for smoking-related diseases such as lung cancer.

Toll receptor 4 Asp299Gly polymorphism and its association with preterm birth and premature rupture of membranes in a South American population

PubMed Central

Rey, G.; Skowronek, F.; Alciaturi, J.; Alonso, J.; Bertoni, B.; Sapiro, R.

2008-01-01

Preterm birth (PTB) is a worldwide health problem and remains the leading cause of perinatal morbidity and mortality. Systemic and local intrauterine infections have been implicated in the pathogenesis of preterm labor and delivery. Common pathways between PTB, premature rupture of ovular membranes (PROM) and altered molecular routes of inflammation have been proposed. There is evidence to support a genetic component in these conditions. Lipopolysaccharide (LPS), a component of the cell wall of Gram-negative bacteria, is thought to play a key role in eliciting an inflammatory response. LPS is recognized by proteins of the innate immune system, including Toll-like receptor 4 (TLR4). Individuals from some European countries carrying the variant alleles resulting in an amino acid substitution (Asp299Gly) are at increased risk of Gram-negative infections and premature birth. The objective of this study was to determine if preterm newborns have different allele frequency of the Asp299Gly TLR4 variant from healthy term neonates in Uruguay. The impact of PROM was also examined. There was an increase in the risk for fetuses carrying the Asp299Gly substitution in TLR4 of being severely premature (<33 weeks) and to present PROM at the same time. PMID:18723631

S100A8 contributes to postoperative cognitive dysfunction in mice undergoing tibial fracture surgery by activating the TLR4/MyD88 pathway.

PubMed

Lu, Shun-Mei; Yu, Chan-Juan; Liu, Ya-Hua; Dong, Hong-Quan; Zhang, Xiang; Zhang, Su-Su; Hu, Liu-Qing; Zhang, Feng; Qian, Yan-Ning; Gui, Bo

2015-02-01

Neuro-inflammation plays a key role in the occurrence and development of postoperative cognitive dysfunction (POCD). Although S100A8 and Toll-like receptor 4 (TLR4) have been increasingly recognized to contribute to neuro-inflammation, little is known about the interaction between S100A8 and TLR4/MyD88 signaling in the process of systemic inflammation that leads to neuro-inflammation. Firstly, we demonstrated that C57BL/6 wide-type mice exhibit cognitive deficit 24h after the tibial fracture surgery. Subsequently, increased S100A8 and S100A9 expression was found in the peripheral blood mononuclear cells (PBMCs), spleen, and hippocampus of C57BL/6 wide-type mice within 48h after the surgery. Pre-operative administration of S100A8 antibody significantly inhibited hippocampal microgliosis and improved cognitive function 24h after the surgery. Secondly, we also observed TLR4/MyD88 activation in the PBMCs, spleen, and hippocampus after the surgery. Compared with those in their corresponding wide-type mice, TLR4(-/-) and MyD88(-/-) mice showed lower immunoreactive area of microglia in the hippocampal CA3 region after operation. TLR4 deficiency also led to reduction of CD45(hi)CD11b(+) cells in the brain and better performance in both Y maze and open field test after surgery, suggesting a new regulatory mechanism of TLR4-dependent POCD. At last, the co-location of S100A8 and TLR4 expression in spleen after operation suggested a close relationship between them. On the one hand, S100A8 could induce TLR4 activation of CD11b(+) cells in the blood and hippocampus via intraperitoneal or intracerebroventricular injection. On the other hand, TLR4 deficiency conversely alleviated S100A8 protein-induced hippocampal microgliosis. Furthermore, the increased expression of S100A8 protein in the hippocampus induced by surgery sharply decreased in both TLR4 and MyD88 genetically deficient mice. Taken together, these data suggest that S100A8 exerts pro-inflammatory effect on the

Lipopolysaccharide regulation of intestinal tight junction permeability is mediated by TLR-4 signal transduction pathway activation of FAK and MyD88

PubMed Central

Guo, Shuhong; Nighot, Meghali; Al-Sadi, Rana; Alhmoud, Tarik; Nighot, Prashant; Ma, Thomas Y.

2015-01-01

Gut-derived bacterial lipopolysaccharides (LPS) play an essential role in inducing intestinal and systemic inflammatory responses and have been implicated as a pathogenic factor of necrotizing enterocolitis (NEC) and inflammatory bowel disease (IBD). The defective intestinal tight junction (TJ) barrier has been shown to be an important factor contributing to the development of intestinal inflammation. LPS, at physiological concentrations, cause an increase in intestinal tight junction permeability (TJP) via a TLR-4 dependent process; however the intracellular mechanisms that mediate LPS regulation of intestinal TJP remain unclear. The aim of this study was to investigate the adaptor proteins and the signaling interactions that mediate LPS modulation of intestinal TJ barrier using an in-vitro and in-vivo model system. LPS caused a TLR-4 dependent activation of membrane-associated adaptor protein FAK in Caco-2 monolayers. LPS caused an activation of both MyD88-dependent and –independent pathways. SiRNA silencing of MyD88 prevented LPS-induced increase in TJP. LPS caused a MyD88-dependent activation of IRAK4. TLR-4, FAK and MyD88 were co-localized. SiRNA silencing of TLR-4 inhibited TLR-4 associated FAK activation; and FAK knockdown prevented MyD88 activation. In-vivo studies also confirmed that LPS-induced increase in mouse intestinal permeability was associated with FAK and MyD88 activation; knockdown of intestinal epithelial FAK prevented LPS-induced increase in intestinal permeability. Additionally, high dose LPS-induced intestinal inflammation was also dependent on TLR-4/FAK/MyD88 signal-transduction axis. Our data show for the first time that LPS-induced increase in intestinal TJP and intestinal inflammation was regulated by TLR-4 dependent activation of FAK-MyD88-IRAK4 signaling pathway. PMID:26466961

Whole-exome sequencing identifies common and rare variant metabolic QTLs in a Middle Eastern population.

PubMed

Yousri, Noha A; Fakhro, Khalid A; Robay, Amal; Rodriguez-Flores, Juan L; Mohney, Robert P; Zeriri, Hassina; Odeh, Tala; Kader, Sara Abdul; Aldous, Eman K; Thareja, Gaurav; Kumar, Manish; Al-Shakaki, Alya; Chidiac, Omar M; Mohamoud, Yasmin A; Mezey, Jason G; Malek, Joel A; Crystal, Ronald G; Suhre, Karsten

2018-01-23

Metabolomics-genome-wide association studies (mGWAS) have uncovered many metabolic quantitative trait loci (mQTLs) influencing human metabolic individuality, though predominantly in European cohorts. By combining whole-exome sequencing with a high-resolution metabolomics profiling for a highly consanguineous Middle Eastern population, we discover 21 common variant and 12 functional rare variant mQTLs, of which 45% are novel altogether. We fine-map 10 common variant mQTLs to new metabolite ratio associations, and 11 common variant mQTLs to putative protein-altering variants. This is the first work to report common and rare variant mQTLs linked to diseases and/or pharmacological targets in a consanguineous Arab cohort, with wide implications for precision medicine in the Middle East.

Inherited human IRAK-1 deficiency selectively impairs TLR signaling in fibroblasts.

PubMed

Della Mina, Erika; Borghesi, Alessandro; Zhou, Hao; Bougarn, Salim; Boughorbel, Sabri; Israel, Laura; Meloni, Ilaria; Chrabieh, Maya; Ling, Yun; Itan, Yuval; Renieri, Alessandra; Mazzucchelli, Iolanda; Basso, Sabrina; Pavone, Piero; Falsaperla, Raffaele; Ciccone, Roberto; Cerbo, Rosa Maria; Stronati, Mauro; Picard, Capucine; Zuffardi, Orsetta; Abel, Laurent; Chaussabel, Damien; Marr, Nico; Li, Xiaoxia; Casanova, Jean-Laurent; Puel, Anne

2017-01-24

Most members of the Toll-like receptor (TLR) and interleukin-1 receptor (IL-1R) families transduce signals via a canonical pathway involving the MyD88 adapter and the interleukin-1 receptor-associated kinase (IRAK) complex. This complex contains four molecules, including at least two (IRAK-1 and IRAK-4) active kinases. In mice and humans, deficiencies of IRAK-4 or MyD88 abolish most TLR (except for TLR3 and some TLR4) and IL-1R signaling in both leukocytes and fibroblasts. TLR and IL-1R responses are weak but not abolished in mice lacking IRAK-1, whereas the role of IRAK-1 in humans remains unclear. We describe here a boy with X-linked MECP2 deficiency-related syndrome due to a large de novo Xq28 chromosomal deletion encompassing both MECP2 and IRAK1 Like many boys with MECP2 null mutations, this child died very early, at the age of 7 mo. Unlike most IRAK-4- or MyD88-deficient patients, he did not suffer from invasive bacterial diseases during his short life. The IRAK-1 protein was completely absent from the patient's fibroblasts, which responded very poorly to all TLR2/6 (PAM 2 CSK 4 , LTA, FSL-1), TLR1/2 (PAM 3 CSK 4 ), and TLR4 (LPS, MPLA) agonists tested but had almost unimpaired responses to IL-1β. By contrast, the patient's peripheral blood mononuclear cells responded normally to all TLR1/2, TLR2/6, TLR4, TLR7, and TLR8 (R848) agonists tested, and to IL-1β. The death of this child precluded long-term evaluations of the clinical consequences of inherited IRAK-1 deficiency. However, these findings suggest that human IRAK-1 is essential downstream from TLRs but not IL-1Rs in fibroblasts, whereas it plays a redundant role downstream from both TLRs and IL-1Rs in leukocytes.

Polysaccharide from black currant (Ribes nigrum L.) stimulates dendritic cells through TLR4 signaling.

PubMed

Ashigai, Hiroshi; Komano, Yuta; Wang, Guanying; Kawachi, Yasuji; Sunaga, Kazuko; Yamamoto, Reiko; Takata, Ryoji; Miyake, Mika; Yanai, Takaaki

2017-01-01

Black currant ( Ribes nigrum ) has various beneficial properties for human health. In particular, polysaccharide from black currant was found to be an immunostimulating food ingredient and was reported to have antitumor activity in a mouse model. We named it cassis polysaccharide (CAPS). In a previous study, CAPS administration caused tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) production in vitro and in vivo , but the immunological mechanism of CAPS was not demonstrated. In this study, we revealed the CAPS immunostimulating mechanism in vitro . First, we found that CAPS activated dendritic cells (DCs). Second, we investigated whether it depends on Toll-like receptor 4 (TLR4) and myeloid differentiation primary response (Myd). We concluded that CAPS stimulates DCs through Myd88 depending TLR4 signaling and activates Th1-type cytokine release.

The Effect of Elevated Intra-Abdominal Pressure on TLR4 Signaling in Intestinal Mucosa and on Intestinal Bacterial Translocation in a Rat.

PubMed

Strier, Adam; Kravarusic, Dragan; Coran, Arnold G; Srugo, Isaac; Bitterman, Nir; Dorfman, Tatiana; Pollak, Yulia; Matter, Ibrahim; Sukhotnik, Igor

2017-02-01

Recent evidence suggests that elevated intra-abdominal pressure (IAP) may adversely affect the intestinal barrier function. Toll-like receptor 4 (TLR-4) is responsible for the recognition of bacterial endotoxin or lipopolysaccharide and for initiation of the Gram-negative septic shock syndrome. The objective of the current study was to determine the effects of elevated IAP on intestinal bacterial translocation (BT) and TLR-4 signaling in intestinal mucosa in a rat model. Male Sprague-Dawley rats were randomly assigned to one of two experimental groups: sham animals (Sham) and IAP animals who were subjected to a 15 mmHg pressure pneumoperitoneum for 30 minutes. Rats were sacrificed 24 hours later. BT to mesenteric lymph nodes, liver, portal vein blood, and peripheral blood was determined at sacrifice. TLR4-related gene and protein expression (TLR-4; myeloid differentiation factor 88 [Myd88] and TNF-α receptor-associated factor 6 [TRAF6]) expression were determined using real-time PCR, western blotting, and immunohistochemistry. Thirty percent of sham rats developed BT in the mesenteric lymph nodes (level I) and 20% of control rats developed BT in the liver and portal vein (level II). abdominal compartment syndrome (ACS) rats demonstrated an 80% BT in the lymph nodes (Level I) and 40% BT in the liver and portal vein (Level II). Elevated BT was accompanied by a significant increase in TLR-4 immunostaining in jejunum (51%) and ileum (35.9%), and in a number of TRAF6-positive cells in jejunum (2.1%) and ileum (24.01%) compared to control animals. ACS rats demonstrated a significant increase in TLR4 and MYD88 protein levels compared to control animals. Twenty-four hours after the induction of elevated IAP in a rat model, increased BT rates were associated with increased TLR4 signaling in intestinal mucosa.

Magnolol inhibits lipopolysaccharide-induced inflammatory response by interfering with TLR4 mediated NF-κB and MAPKs signaling pathways.

PubMed

Fu, Yunhe; Liu, Bo; Zhang, Naisheng; Liu, Zhicheng; Liang, Dejie; Li, Fengyang; Cao, Yongguo; Feng, Xiaosheng; Zhang, Xichen; Yang, Zhengtao

2013-01-09

Magnolia officinalis as a traditional Chinese herb has long been used for the treatment of anxiety, cough, headache and allergic diseases, and also have been used in traditional Chinese medicine to treat a variety of mental disorders including depression. Magnolol, a hydroxylated biphenyl compound isolated from Magnolia officinalis, has been reported to have anti-inflammatory properties. However, the underlying molecular mechanisms are not well understood. The aim of this study was to investigate the molecular mechanism of magnolol in modifying lipopolysaccharide (LPS)-induced signal pathways in RAW264.7 cells. The purity of magnolol was determined by high performance liquid chromatography. RAW264.7 cells were stimulated with LPS in the presence or absence of magnolol. The expression of proinflammatory cytokines were determined by ELISA and reverse transcription-PCR. Nuclear factor-κB (NF-κB), inhibitory kappa B (IκBα) protein, p38, extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and Toll-like receptor 4 (TLR4) were determined by Western blot. Further analyses were performed on mTLR4 and mMD2 co-transfected HEK293 cells. The result showed that the purity of magnolol used in this study was 100%. Magnolol inhibited the expression of TNF-α, IL-6 and IL-1β in LPS-stimulated RAW264.7 cells in a dose-dependent manner. Western blot analysis showed that magnolol suppressed LPS-induced NF-κB activation, IκBα degradation, phosphorylation of ERK, JNK and P38. Magnolol could significantly down-regulated the expression of TLR4 stimulating by LPS. Furthermore, magnolol suppressed LPS-induced IL-8 production in HEK293-mTLR4/MD-2 cells. Our results suggest that magnolol exerts an anti-inflammatory property by down-regulated the expression of TLR4 up-regulated by LPS, thereby attenuating TLR4 mediated the activation of NF-κB and MAPK signaling and the release of pro-inflammatory cytokines. These findings suggest that magnolol may be a

Atorvastatin attenuates experimental contrast-induced acute kidney injury: a role for TLR4/MyD88 signaling pathway.

PubMed

Yue, Rongzheng; Zuo, Chuan; Zeng, Jing; Su, Baihai; Tao, Ye; Huang, Songmin; Zeng, Rui

2017-11-01

To investigate the protective effect of different atorvastatin doses on contrast-induced acute kidney injury and the related mechanism. Healthy male Sprague-Dawley (SD) rats were randomly divided into the blank control group, experimental control group and different-dose atorvastatin groups. A rat model of contrast-induced acute kidney injury was established. We detected changes in serum creatinine (Scr) and blood urea nitrogen (BUN) before and after model establishment, observed and scored renal tubular injury, analyzed rat renal cell apoptosis, and measure the expression of signal pathway proteins and downstream inflammatory factors. After contrast agent injection, the Scr and BUN levels of the experimental control group were significantly increased, the different doses applied in the atorvastatin group significantly reduced the Scr and BUN levels (p < .05) and ameliorated the contrast-induced acute kidney injury (p < .05) and significantly reduced Toll-like receptor 4 (TLR4), Myeloid differentiation factor 88 (Myd88), and Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) protein expression and relative mRNA expression levels (p < .05) and significantly decreased expression levels of downstream inflammatory factors (p < .05). Different atorvastatin doses have protective effects on contrast-induced acute renal tubular injury in rats, possibly by targeting TLR4, suppressing TLR4 expression, regulating the TLR4/Myd88 signaling pathway, and inhibiting the expression of downstream inflammatory factors.

Dectin-1/TLR2 and NOD2 Agonists Render Dendritic Cells Susceptible to Infection by X4-Using HIV-1 and Promote cis-Infection of CD4+ T Cells

PubMed Central

Tremblay, Michel J.

2013-01-01

HIV-1 pathogenesis is intimately linked with microbial infections and innate immunity during all stages of the disease. While the impact of microbial-derived products in transmission of R5-using virus to CD4+ T cells by dendritic cells (DCs) has been addressed before, very limited data are available on the effect of such compounds on DC-mediated dissemination of X4-tropic variant. Here, we provide evidence that treatment of DCs with dectin-1/TLR2 and NOD2 ligands increases cis-infection of autologous CD4+ T cells by X4-using virus. This phenomenon is most likely associated with an enhanced permissiveness of DCs to productive infection with X4 virus, which is linked to increased surface expression of CXCR4 and the acquisition of a maturation profile by DCs. The ensuing DC maturation enhances susceptibility of CD4+ T cells to productive infection with HIV-1. This study highlights the crucial role of DCs at different stages of HIV-1 infection and particularly in spreading of viral strains displaying a X4 phenotype. PMID:23844079

TLR4 Gene Expression and Pro-Inflammatory Cytokines in Alzheimer's Disease and in Response to Hippocampal Deafferentation in Rodents.

PubMed

Miron, Justin; Picard, Cynthia; Frappier, Josée; Dea, Doris; Théroux, Louise; Poirier, Judes

2018-01-01

One important aspect in Alzheimer's disease pathology is the presence of chronic inflammation. Considering its role as a key receptor in the microglial innate immune system, TLR4 was shown to regulate the binding and phagocytosis of amyloid plaques by microglia in several mouse models of amyloidosis, as well as the production of pro-inflammatory cytokines. To our knowledge, TLR4 and its association with cytokines have not been thoroughly examined in the brains of subjects affected with Alzheimer's disease. Using quantitative reverse transcription polymerase chain reaction (qRT-PCR) in postmortem human brains, we observed increased expression for the TLR4 and TNF genes (p = 0.001 and p = 0.025, respectively), as well as a trend for higher IL6 gene expression in the frontal cortex of AD subjects when compared to age-matched controls. Similarly, using a mouse model of hippocampal deafferentation without amyloidosis, (i.e., the entorhinal cortex lesioned mouse), we observed significant increases in the expression of both the Tlr4 (p = 0.0367 and p = 0.0193 compared to sham-lesioned mice or to the contralateral side, respectively) and Il1b (p = 0.0055 and p = 0.0066 compared to sham-lesioned mice or to the contralateral side, respectively) genes in the deafferentation phase, but not during the ensuing reinnervation process. In conclusion, we suggest that the modulation of cytokines by TLR4 is differentially regulated whether by the presence of amyloid plaques or by the ongoing deafferentation process.

Oxymatrine Inhibits Influenza A Virus Replication and Inflammation via TLR4, p38 MAPK and NF-κB Pathways.

PubMed

Dai, Jian-Ping; Wang, Qian-Wen; Su, Yun; Gu, Li-Ming; Deng, Hui-Xiong; Chen, Xiao-Xuan; Li, Wei-Zhong; Li, Kang-Sheng

2018-03-23

Oxymatrine (OMT) is a strong immunosuppressive agent that has been used in the clinic for many years. In the present study, by using plaque inhibition, luciferase reporter plasmids, qRT-PCR, western blotting, and ELISA assays, we have investigated the effect and mechanism of OMT on influenza A virus (IAV) replication and IAV-induced inflammation in vitro and in vivo. The results showed that OMT had excellent anti-IAV activity on eight IAV strains in vitro. OMT could significantly decrease the promoter activity of TLR3, TLR4, TLR7, MyD88, and TRAF6 genes, inhibit IAV-induced activations of Akt, ERK1/2, p38 MAPK, and NF-κB pathways, and suppress the expressions of inflammatory cytokines and MMP-2/-9. Activators of TLR4, p38 MAPK and NF-κB pathways could significantly antagonize the anti-IAV activity of OMT in vitro, including IAV replication and IAV-induced cytopathogenic effect (CPE). Furthermore, OMT could reduce the loss of body weight, significantly increase the survival rate of IAV-infected mice, decrease the lung index, pulmonary inflammation and lung viral titter, and improve pulmonary histopathological changes. In conclusion, OMT possesses anti-IAV and anti-inflammatory activities, the mechanism of action may be linked to its ability to inhibit IAV-induced activations of TLR4, p38 MAPK, and NF-κB pathways.

TLR9 agonist protects mice from radiation-induced gastrointestinal syndrome.

PubMed

Saha, Subhrajit; Bhanja, Payel; Liu, Laibin; Alfieri, Alan A; Yu, Dong; Kandimalla, Ekambar R; Agrawal, Sudhir; Guha, Chandan

2012-01-01

Radiation-induced gastrointestinal syndrome (RIGS) is due to the clonogenic loss of crypt cells and villi depopulation, resulting in disruption of mucosal barrier, bacterial invasion, inflammation and sepsis. Intestinal macrophages could recognize invading bacterial DNA via TLR9 receptors and transmit regenerative signals to the neighboring crypt. We therefore investigated whether systemic administration of designer TLR9 agonist could ameliorate RIGS by activating TLR9. Male C57Bl6 mice were distributed in four experimental cohorts, whole body irradiation (WBI) (8.4-10.4 Gy), TLR9 agonist (1 mg/kg s.c.), 1 h pre- or post-WBI and TLR9 agonist+WBI+iMyd88 (pretreatment with inhibitory peptide against Myd88). Animals were observed for survival and intestine was harvested for histological analysis. BALB/c mice with CT26 colon tumors in abdominal wall were irradiated with 14 Gy single dose of whole abdominal irradiation (AIR) for tumor growth study. Mice receiving pre-WBI TLR9 agonist demonstrated improvement of survival after 10.4 Gy (p<0.03), 9.4 Gy (p<0.008) and 8.4 Gy (p<0.002) of WBI, compared to untreated or iMyd88-treated controls. Post-WBI TLR9 agonist mitigates up to 8.4 Gy WBI (p<0.01). Histological analysis and xylose absorption test demonstrated significant structural and functional restitution of the intestine in WBI+TLR9 agonist cohorts. Although, AIR reduced tumor growth, all animals died within 12 days from RIGS. TLR9 agonist improved the survival of mice beyond 28 days post-AIR (p<0.008) with significant reduction of tumor growth (p<0.0001). TLR9 agonist treatment could serve both as a prophylactic or mitigating agent against acute radiation syndrome and also as an adjuvant therapy to increase the therapeutic ratio of abdominal Radiation Therapy for Gastro Intestinal malignancies.

Identification, characterization and genetic mapping of TLR7, TLR8a1 and TLR8a2 genes in rainbow trout (Oncorhynchus mykiss)

USGS Publications Warehouse

Palti, Yniv; Gahr, Scott A.; Purcell, Maureen K.; Hadidi, Sima; Rexroad, Caird E.; Wiens, Gregory A.

2010-01-01

Induction of the innate immune pathways is critical for early anti-viral defense but there is limited understanding of how teleost fish recognize viral molecules and activate these pathways. In mammals, Toll-like receptors (TLR) 7 and 8 bind single-stranded RNA of viral origin and are activated by synthetic anti-viral imidazoquinoline compounds. Herein, we identify and describe the rainbow trout (Oncorhynchus mykiss) TLR7 and TLR8 gene orthologs and their mRNA expression. Two TLR7/8 loci were identified from a rainbow trout bacterial artificial chromosome (BAC) library using DNA fingerprinting and genetic linkage analyses. Direct sequencing of two representative BACs revealed intact omTLR7 and omTLR8a1 open reading frames (ORFs) located on chromosome 3 and a second locus on chromosome 22 that contains an omTLR8a2 ORF and a putative TLR7 pseudogene. We used the omTLR8a1/2 nomenclature for the two trout TLR8 genes as phylogenetic analysis revealed that they and all the other teleost TLR8 genes sequenced to date are similar to the zebrafish TLR8a, but are distinct from the zebrafish TLR8b. The duplicated trout loci exhibit conserved synteny with other fish genomes extending beyond the tandem of TLR7/8 genes. The trout TLR7 and 8a1/2 genes are composed of a single large exon similar to all other described TLR7/8 genes. The omTLR7 ORF is predicted to encode a 1049 amino acid (aa) protein with 84% similarity to the Fugu TLR7 and a conserved pattern of predicted leucine-rich repeats (LRR). The omTLR8a1 and omTLR8a2 are predicted to encode 1035- and 1034-aa proteins, respectively, and have 86% similarity to each other. omTLR8a1 is likely the ortholog of the only Atlantic salmon TLR8 gene described to date as they have 95% aa sequence similarity. The tissue expression profiles of omTLR7, omTLR8a1 and omTLR8a2 in healthy trout were highest in spleen tissue followed by anterior and then posterior kidney tissues. Rainbow trout anterior kidney leukocytes produced elevated

Necroptosis in microglia contributes to neuroinflammation and retinal degeneration through TLR4 activation.

PubMed

Huang, Zijing; Zhou, Tian; Sun, Xiaowei; Zheng, Yingfeng; Cheng, Bing; Li, Mei; Liu, Xialin; He, Chang

2018-01-01

Inflammation has emerged to be a critical mechanism responsible for neural damage and neurodegenerative diseases. Microglia, the resident innate immune cells in retina, are implicated as principal components of the immunological insult to retinal neural cells. The involvement of microglia in retinal inflammation is complex and here we propose for the first time that necroptosis in microglia triggers neuroinflammation and exacerbates retinal neural damage and degeneration. We found microglia experienced receptor-interacting protein kinase 1 (RIP1)- and RIP3-dependent necroptosis not only in the retinal degenerative rd1 mice, but also in the acute retinal neural injury mice. The necroptotic microglia released various pro-inflammatory cytokines and chemokines, such as tumor necrosis factor-α and chemokine (C-C motif) ligand 2, which orchestrated the retinal inflammation. Importantly, necroptosis blockade using necrostatin-1 could suppress microglia-mediated inflammation, rescue retinal degeneration or prevent neural injury in vivo. Meanwhile, cultured microglia underwent RIP1/3-mediated necroptosis and the necroptotic microglia produced large amounts of pro-inflammatory cytokines in response to lipopolysaccharide or oxidative stress in vitro. Mechanically, TLR4 deficiency ameliorated microglia necroptosis with decreased expression levels of machinery molecules RIP1 and RIP3, and suppressed retinal inflammation, suggesting that TLR4 signaling was required in microglia necroptosis-mediated inflammation. Thus, we proposed that microglia experienced necroptosis through TLR4 activation, promoting an inflammatory response that serves to exacerbate considerable neural damage and degeneration. Necroptosis blockade therefore emerged as a novel therapeutic strategy for tempering microglia-mediated neuroinflammation and ameliorating neural injury and neurodegenerative diseases.

Necroptosis in microglia contributes to neuroinflammation and retinal degeneration through TLR4 activation

PubMed Central

Huang, Zijing; Zhou, Tian; Sun, Xiaowei; Zheng, Yingfeng; Cheng, Bing; Li, Mei; Liu, Xialin; He, Chang

2018-01-01

Inflammation has emerged to be a critical mechanism responsible for neural damage and neurodegenerative diseases. Microglia, the resident innate immune cells in retina, are implicated as principal components of the immunological insult to retinal neural cells. The involvement of microglia in retinal inflammation is complex and here we propose for the first time that necroptosis in microglia triggers neuroinflammation and exacerbates retinal neural damage and degeneration. We found microglia experienced receptor-interacting protein kinase 1 (RIP1)- and RIP3-dependent necroptosis not only in the retinal degenerative rd1 mice, but also in the acute retinal neural injury mice. The necroptotic microglia released various pro-inflammatory cytokines and chemokines, such as tumor necrosis factor-α and chemokine (C-C motif) ligand 2, which orchestrated the retinal inflammation. Importantly, necroptosis blockade using necrostatin-1 could suppress microglia-mediated inflammation, rescue retinal degeneration or prevent neural injury in vivo. Meanwhile, cultured microglia underwent RIP1/3-mediated necroptosis and the necroptotic microglia produced large amounts of pro-inflammatory cytokines in response to lipopolysaccharide or oxidative stress in vitro. Mechanically, TLR4 deficiency ameliorated microglia necroptosis with decreased expression levels of machinery molecules RIP1 and RIP3, and suppressed retinal inflammation, suggesting that TLR4 signaling was required in microglia necroptosis-mediated inflammation. Thus, we proposed that microglia experienced necroptosis through TLR4 activation, promoting an inflammatory response that serves to exacerbate considerable neural damage and degeneration. Necroptosis blockade therefore emerged as a novel therapeutic strategy for tempering microglia-mediated neuroinflammation and ameliorating neural injury and neurodegenerative diseases. PMID:28885615

Rare versus common variants in pharmacogenetics: SLCO1B1 variation and methotrexate disposition

PubMed Central

Ramsey, Laura B.; Bruun, Gitte H.; Yang, Wenjian; Treviño, Lisa R.; Vattathil, Selina; Scheet, Paul; Cheng, Cheng; Rosner, Gary L.; Giacomini, Kathleen M.; Fan, Yiping; Sparreboom, Alex; Mikkelsen, Torben S.; Corydon, Thomas J.; Pui, Ching-Hon; Evans, William E.; Relling, Mary V.

2012-01-01

Methotrexate is used to treat autoimmune diseases and malignancies, including acute lymphoblastic leukemia (ALL). Inter-individual variation in clearance of methotrexate results in heterogeneous systemic exposure, clinical efficacy, and toxicity. In a genome-wide association study of children with ALL, we identified SLCO1B1 as harboring multiple common polymorphisms associated with methotrexate clearance. The extent of influence of rare versus common variants on pharmacogenomic phenotypes remains largely unexplored. We tested the hypothesis that rare variants in SLCO1B1 could affect methotrexate clearance and compared the influence of common versus rare variants in addition to clinical covariates on clearance. From deep resequencing of SLCO1B1 exons in 699 children, we identified 93 SNPs, 15 of which were non-synonymous (NS). Three of these NS SNPs were common, with a minor allele frequency (MAF) >5%, one had low frequency (MAF 1%–5%), and 11 were rare (MAF <1%). NS SNPs (common or rare) predicted to be functionally damaging were more likely to be found among patients with the lowest methotrexate clearance than patients with high clearance. We verified lower function in vitro of four SLCO1B1 haplotypes that were associated with reduced methotrexate clearance. In a multivariate stepwise regression analysis adjusting for other genetic and non-genetic covariates, SLCO1B1 variants accounted for 10.7% of the population variability in clearance. Of that variability, common NS variants accounted for the majority, but rare damaging NS variants constituted 17.8% of SLCO1B1's effects (1.9% of total variation) and had larger effect sizes than common NS variants. Our results show that rare variants are likely to have an important effect on pharmacogenetic phenotypes. PMID:22147369

Postnatal TLR2 activation impairs learning and memory in adulthood.

PubMed

Madar, Ravit; Rotter, Aviva; Waldman Ben-Asher, Hiba; Mughal, Mohamed R; Arumugam, Thiruma V; Wood, W H; Becker, K G; Mattson, Mark P; Okun, Eitan

2015-08-01

Neuroinflammation in the central nervous system is detrimental for learning and memory, as evident form epidemiological studies linking developmental defects and maternal exposure to harmful pathogens. Postnatal infections can also induce neuroinflammatory responses with long-term consequences. These inflammatory responses can lead to motor deficits and/or behavioral disabilities. Toll like receptors (TLRs) are a family of innate immune receptors best known as sensors of microbial-associated molecular patterns, and are the first responders to infection. TLR2 forms heterodimers with either TLR1 or TLR6, is activated in response to gram-positive bacterial infections, and is expressed in the brain during embryonic development. We hypothesized that early postnatal TLR2-mediated neuroinflammation would adversely affect cognitive behavior in the adult. Our data indicate that postnatal TLR2 activation affects learning and memory in adult mice in a heterodimer-dependent manner. TLR2/6 activation improved motor function and fear learning, while TLR2/1 activation impaired spatial learning and enhanced fear learning. Moreover, developmental TLR2 deficiency significantly impairs spatial learning and enhances fear learning, stressing the involvement of the TLR2 pathway in learning and memory. Analysis of the transcriptional effects of TLR2 activation reveals both common and unique transcriptional programs following heterodimer-specific TLR2 activation. These results imply that adult cognitive behavior could be influenced in part, by activation or alterations in the TLR2 pathway at birth. Copyright © 2015 Elsevier Inc. All rights reserved.

MicroRNA-146a suppresses rheumatoid arthritis fibroblast-like synoviocytes proliferation and inflammatory responses by inhibiting the TLR4/NF-kB signaling

PubMed Central

Liu, Wei; Wu, Yuan-Hao; Zhang, Lei; Xue, Bin; Wang, Yi; Liu, Bin; Liu, Xiao-Ya; Zuo, Fang; Yang, Xiao-Yan; Chen, Fu-Yu; Duan, Ran; Cai, Yue; Zhang, Bo; Ji, Yang

2018-01-01

This study investigated whether microRNA-146a (miR-146a) mediating TLR4/NF-κB pathway affected proliferation and inflammatory responses of rheumatoid arthritis fibroblast-like synoviocytes from 12 RA patients (RA-FLSs). FLSs in the logarithmic growth phase were assigned into the control, miR-146a mimic miR-146a inhibitor, Tak-242 (treated with TLR4/NF-κB pathway inhibitor) and mimic + lipopolysaccharide (LPS) groups. Cell proliferation and apoptosis were detected using CCK-8 assay and flow cytometry. The expression of miR-146a, TLR4/NF-κB pathway-related proteins and cytokines were determined by RT-qPCR, western blotting and ELISA, and the release of NO by Greiss reaction. RA rat models were constructed and the primary cells were classified into the control, negative control (NC), miR-146a mimic, miR-146a inhibitor, Tak-242, mimic + LPS, and TLR4 groups. Immunohistochemistry was used to detect the expression of proliferating cell nuclear antigen (PCNA) and intercellular adhesion molecular-1 (ICAM-1). The results showed that miR-146a levels were lower in RA-FLSs than control fibroblasts. miR-146a mimic and Tak-242 decreased RA-FLS proliferation and increased RA-FLS apoptosis, while miR-146a inhibitor had an opposite trend. miR-146a mimic and Tak-242 also decreased expression of TLR4, NF-κB, IL-1β, IL-6, IL-8, IL-17, COX-2, MMP-3, Seprase, and iNOS, as well as reduced NO level in RA-FLSs while miR-146a inhibitor and TLR4 increased them. TLR4 and NF-κB levels and the positive rates of PCNA and ICAM-1 expressions were lower in RA-FLSs from RA rats given miR-146a mimic from control or miR-146a inhibitor-treated rats. These results suggest that miR-146a inhibits the proliferation and inflammatory response of RA-FLSs by down-regulating TLR4/NF-κB pathway.

TLR3 mediates release of IL-1β and cell death in keratinocytes in a caspase-4 dependent manner.

PubMed

Grimstad, Øystein; Husebye, Harald; Espevik, Terje

2013-10-01

Inflammation and timely cell death are important elements in host defence and healing processes. Keratinocytes express high levels of Toll-like receptor 3 (TLR3), and stimulation of the receptor with its ligand polyinosinic-polycytidylic acid (polyI:C) is a powerful signal for release of a variety of proinflammatory cytokines. Caspase-4 is required for maturation of pro-IL-1β through activation of caspase-1 in keratinocytes. TLR3 in keratinocytes was stimulated with polyI:C. Induction of messenger RNA of pro-IL-1β and inflammasomal components was measured using quantitative polymerase chain reaction methodology. Protein expression of IL-1β was analysed with ELISA and Western blot techniques. Activation of apoptotic caspases was measured with flow cytometry, and cytotoxicity was determined. TLR3 induced release of substantial amounts of pro-IL-1β in keratinocytes. NLRP3 or ASC dependent processing of IL-1β into its cleaved bioactive form was found to be minimal. The release of IL-1β was due to polyI:C induced cell death that occurred through a caspase-4 dependent manner. Caspase-1 did not seem to be involved in the polyI:C induced cytotoxicity despite that TLR3 stimulation induced activation of caspase-1. In addition, the apoptotic caspases -8, -9 and -3/7 were activated by polyI:C. TLR3 stimulation in keratinocytes induces a caspase-4 dependent release of pro-IL-1β, but further processing to active IL-1β is limited. Furthermore, TLR3 stimulation results in pyroptotic- and apoptotic cell death. Copyright © 2013 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

TAK-242 treatment ameliorates liver ischemia/reperfusion injury by inhibiting TLR4 signaling pathway in a swine model of Maastricht-category-III cardiac death.

PubMed

Shao, Zigong; Jiao, Baoping; Liu, Tingting; Cheng, Ying; Liu, Hao; Liu, Yongfeng

2016-12-01

This study aims to test the effects of TAK-242 on liver transplant viability in a model of swine Maastricht-category-III cardiac death. A swine DCD Maastricht-III model of cardiac death was established, and TAK-242 was administered prior to the induction of cardiac death. The protein and mRNA level of TLR4 signaling pathway molecules and cytokines that are important in mediating immune and inflammatory responses were assessed at different time points following the induction of cardiac death. After induction of cardiac death, both the mRNA and protein levels of key molecules (TLR4, TRAF6, NF-ϰB, ICAM-1, MCP-1 and MPO), TNF-α and IL-6 increased significantly. Infusion of TAK-242 1h before induction of cardiac death blocked the increase of immune and inflammatory response molecules. However, the increase of TLR4 level was not affected by infusion of TAK-242. Histology study showed that infusion of TAK-242 protect liver tissue from damage during cardiac death. These results indicates that TLR4 signaling pathway may contribute to ischemia/reperfusion injury in the liver grafts, and blocking TLR4 pathway with TAk-242 may reduce TLR4-mediated tissue damage. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Transcriptional profiling of TLR-4/7/8-stimulated guinea pig splenocytes and whole blood by bDNA assay

PubMed Central

Ching, Lance K.; Mompoint, Farah; Guderian, Jeffrey A.; Picone, Alex; Orme, Ian M.; Coler, Rhea N.; Reed, Steven G.; Baldwin, Susan L.

2011-01-01

Toll-like receptor (TLR) agonists are currently being examined as adjuvants for vaccines, with several lead candidates now in licensed products or in late-stage clinical development. Guinea pigs are widely used for preclinical testing of drugs and vaccines; however, evaluation of TLR agonists in this model is hindered by the limited availability of immunological tools and reagents. In this study, we validated the use of a branched-chain DNA (bDNA) assay known as the QuantiGene Plex 2.0 Reagent System for measuring innate cytokine and chemokine mRNA levels following TLR stimulation of guinea pig cells. Gene expression for T-helper-1 (Th1) polarizing cytokines (TNF-α, IL-1β, IL-12) and chemokines (CXCL1, CCL2) was upregulated following ex vivo stimulation of guinea pig splenocytes and whole blood with TLR-4 or TLR-7/8 agonists. These data confirm the utility of the QuantiGene system both as an alternative to RT-PCR for measuring transcript levels and as a high-throughput screening tool for dissecting the immunological response to TLR stimulation in guinea pigs. Overall, the QuantiGene platform is reliable, reproducible, and sensitive. These agonists have the potential to be used as adjuvant components in vaccines against various pathogens. PMID:21839740

Vinpocetine alleviate cerebral ischemia/reperfusion injury by down-regulating TLR4/MyD88/NF-κB signaling.

PubMed

Wu, Li-Rong; Liu, Liang; Xiong, Xiao-Yi; Zhang, Qin; Wang, Fa-Xiang; Gong, Chang-Xiong; Zhong, Qi; Yang, Yuan-Rui; Meng, Zhao-You; Yang, Qing-Wu

2017-10-06

Inflammatory responses play crucial roles in cerebral ischemia/reperfusion injury. Toll-like receptor 4 (TLR4) is an important mediator of the neuroinflammatory response to cerebral ischemia/reperfusion injury. Vinpocetine is a derivative of the alkaloid vincamine and exerts an anti-inflammatory effect by inhibiting NF-κB activation. However, the effects of vinpocetine on pathways upstream of NF-κB signaling, such as TLR4, have not been fully elucidated. Here, we used mouse middle cerebral artery occlusion (MCAO) and cell-based oxygen-glucose deprivation (OGD) models to evaluate the therapeutic effects and mechanisms of vinpocetine treatment. The vinpocetine treatment significantly reduced mice cerebral infarct volumes and neurological scores. Moreover, the numbers of TUNEL+ and Fluoro-Jade B+ cells were significantly decreased in the ischemic brain tissues after vinpocetine treatment. In the OGD model, the vinpocetine treatment also increased the viability of cultured cortical neurons. Interestingly, vinpocetine exerted a neuroprotective effect on the mouse MCAO model and cell-based OGD model by inhibiting TLR4-mediated inflammatory responses and decreasing proinflammatory cytokine release through the MyD88-dependent signaling pathway, independent of TRIF signaling pathway. In conclusion, vinpocetine exerts anti-inflammatory effects to ameliorate cerebral ischemia/reperfusion injury in vitro and in vivo. Vinpocetine may inhibit inflammatory responses through the TLR4/MyD88/NF-κB signaling pathway, independent of TRIF-mediated inflammatory responses. Thus, vinpocetine may be an attractive therapeutic candidate for the treatment of ischemic cerebral injury or other inflammatory diseases.

Comprehensive genotyping in dyslipidemia: mendelian dyslipidemias caused by rare variants and Mendelian randomization studies using common variants.

PubMed

Tada, Hayato; Kawashiri, Masa-Aki; Yamagishi, Masakazu

2017-04-01

Dyslipidemias, especially hyper-low-density lipoprotein cholesterolemia and hypertriglyceridemia, are important causal risk factors for coronary artery disease. Comprehensive genotyping using the 'next-generation sequencing' technique has facilitated the investigation of Mendelian dyslipidemias, in addition to Mendelian randomization studies using common genetic variants associated with plasma lipids and coronary artery disease. The beneficial effects of low-density lipoprotein cholesterol-lowering therapies on coronary artery disease have been verified by many randomized controlled trials over the years, and subsequent genetic studies have supported these findings. More recently, Mendelian randomization studies have preceded randomized controlled trials. When the on-target/off-target effects of rare variants and common variants exhibit the same direction, novel drugs targeting molecules identified by investigations of rare Mendelian lipid disorders could be promising. Such a strategy could aid in the search for drug discovery seeds other than those for dyslipidemias.

Saturated fatty acids activate TLR-mediated pro-inflammatory signaling pathways

USDA-ARS?s Scientific Manuscript database

Toll-like receptor 4 (TLR4) and TLR2 were shown to be activated by saturated fatty acids (SFAs) but inhibited by docosahexaenoic acid (DHA). However, one report (ATVB 11:1944, 2009) suggested that SFA-induced TLR activation in cell culture systems is due to contaminants in BSA used for conjugating f...

Association of TLR2 S450S and ICAM1 K469E polymorphisms with polycystic ovary syndrome (PCOS) and obesity.