Effects of as-cast and wrought Cobalt-Chrome-Molybdenum and Titanium-Aluminium-Vanadium alloys on cytokine gene expression and protein secretion in J774A.1 macrophages.

PubMed

Jakobsen, Stig S; Larsen, A; Stoltenberg, M; Bruun, J M; Soballe, K

2007-09-11

Insertion of metal implants is associated with a possible change in the delicate balance between pro- and anti-inflammatory proteins, probably leading to an unfavourable predominantly pro-inflammatory milieu. The most likely cause is an inappropriate activation of macrophages in close relation to the metal implant and wear-products. The aim of the present study was to compare surfaces of as-cast and wrought Cobalt-Chrome-Molybdenum (CoCrMo) alloys and Titanium-Aluminium-Vanadium (TiAlV) alloy when incubated with mouse macrophage J774A.1 cell cultures. Changes in pro- and anti-inflammatory cytokines (TNF-alpha, IL-6, IL-alpha, IL-1beta, IL-10) and proteins known to induce proliferation (M-CSF), chemotaxis (MCP-1) and osteogenesis (TGF-beta, OPG) were determined by ELISA and Real Time reverse transcriptase - PCR (Real Time rt-PCR). Lactate dehydrogenase (LDH) was measured in the medium to asses the cell viability. Surface properties of the discs were characterised with a profilometer and with energy dispersive X-ray spectroscopy. We here report, for the first time, that the prosthetic material surface (non-phagocytable) of as-cast high carbon CoCrMo reduces the pro-inflammatory cytokine IL-6 transcription, the chemokine MCP-1 secretion, and M-CSF secretion by 77%, 36%, and 62%, respectively. Furthermore, we found that reducing surface roughness did not affect this reduction. The results suggest that as-cast CoCrMo alloy is more inert than wrought CoCrMo and wrought TiAlV alloys and could prove to be a superior implant material generating less inflammation which might result in less osteolysis.

Activation of macrophages stimulated by the bengkoang fiber extract through toll-like receptor 4.

PubMed

Kumalasari, Ika Dyah; Nishi, Kosuke; Putra, Agus Budiawan Naro; Sugahara, Takuya

2014-07-25

Bengkoang (Pachyrhizus erosus (L.) Urban) is an edible root tuber containing fairly large amounts of carbohydrates and crude fibers. Our previous studies showed that the bengkoang fiber extract (BFE) stimulates activation of macrophages, leading to induction of phagocytotic activity and cytokine production. In the present study we investigated the mechanism underlying activation of murine macrophages by BFE. BFE increased production of TNF-α, IL-6, and nitric oxide by J774.1 cells. In addition BFE also facilitated the gene expression levels of inducible nitric oxide synthase. We examined the effect of a TLR4 inhibitor on cytokine production to investigate the membrane receptor of macrophage activation by BFE. Treatment of J774.1 cells with the TLR4 inhibitor significantly inhibited production of IL-6 and TNF-α, suggesting that TLR4 is the target membrane receptor for BFE. The main signal molecules located downstream of TLR4 such as JNK, p38, ERK, and NF-κB were activated by BFE treatment. The immunostimulatory effect of BFE was cancelled by the pectinase treatment, suggesting that the active ingredient in BFE is pectin-like molecules. Overall results suggested that BFE activates J774.1 cells via the MAPK and NF-κB signaling pathways.

Fc-receptor induced cell spreading during frustrated phagocytosis in J774A.1 macrophages

NASA Astrophysics Data System (ADS)

Kovari, Daniel; Curtis, Jennifer; Wei, Wenbin

2014-03-01

Phagocytosis is the process where by cells engulf foreign particles. It is the primary mechanism through which macrophages and neutrophils (white blood cells) eliminate pathogens and debris from the body. The behavior is the result of a cascade of chemical and mechanical cues, which result in the actin-driven expansion of the cell's membrane around its target. For macrophages undergoing Fc-mediated phagocytosis, we show that above a minimum threshold the spreading rate and maximum cell-target contact area are independent of the target's opsonin density. Qualitatively, macrophage phagocytic spreading is similar to the spreading of other cell types (e.g. fibroblasts, lymphocytes, and Dict.d.). Early spreading is most likely the result of ``passive'' alignment of the cell to the target surface. This is followed by an active expansion period driven by actin. Finally upon reaching a maximum contact area, typically 2-3 times the size of ``non-activated'' cells, macrophages often undergo a period of rapid contraction not reported in other cell types. We hypothesize that this, as yet unexplained, transition may be specific to the chemical and mechanical machinery associated with phagocytosis. This work was funded by NSF grant PHYS 0848797 and NSF grant DMR 0820382.

A study to evaluate the effect of nootropic drug-piracetam on DNA damage in leukocytes and macrophages.

PubMed

Singh, Sarika; Goswami, Poonam; Swarnkar, Supriya; Singh, Sheelendra Pratap; Wahajuddin; Nath, Chandishwar; Sharma, Sharad

2011-11-27

Piracetam is a nootropic drug that protects neurons in neuropathological and age-related diseases and the activation and modulation of peripheral blood cells in patients with neuropathological conditions is well known. Therefore, in the present study, in vivo, ex vivo, and in vitro tests were conducted to investigate the effect of piracetam on leukocytes and macrophages. Lipopolysaccharide (LPS) causes oxidative DNA damage; thus, in the present study, LPS was used as a tool to induce DNA damage. In vivo experiments were conducted on Sprague Dawley rats, and piracetam (600mg/kg, oral) was provided for five consecutive days. On the fifth day, a single injection of LPS (10mg/kg, i.p.) was administered. Three hours after LPS injection, blood leukocytes and peritoneal macrophages were collected and processed, and a variety of different assays were conducted. Ex vivo treatments were performed on isolated rat blood leukocytes, and in vitro experiments were conducted on rat macrophage cell line J774A.1. Cell viability and the level of reactive oxygen species (ROS), mitochondrial membrane potential (MMP) and DNA damage were estimated in untreated (control) and piracetam-, LPS- and LPS+piracetam-treated leukocytes and macrophages. In vivo experiments revealed that rats pretreated with piracetam were significantly protected against LPS-induced increases in ROS levels and DNA damage. Ex vivo isolated leukocytes and J774A.1 cells treated with LPS exhibited augmented ROS levels and DNA damage, which were attenuated with piracetam treatment. Thus, the present study revealed the salutary effect of piracetam against LPS-induced oxidative stress and DNA damage in leukocytes and macrophages. Copyright © 2011 Elsevier B.V. All rights reserved.

Pomegranate Juice Polyphenols Induce Macrophage Death via Apoptosis as Opposed to Necrosis Induced by Free Radical Generation: A Central Role for Oxidative Stress.

PubMed

Rom, Oren; Volkova, Nina; Nandi, Sukhendu; Jelinek, Raz; Aviram, Michael

2016-08-01

At high concentrations, polyphenols induce cell death, and the polyphenols-rich pomegranate juice (PJ), known for its antioxidative/antiatherogenic properties, can possibly affect cell death, including macrophage death involved in atherogenesis. In the present study, apoptotic/necrotic macrophage death was analyzed in J774A.1 macrophages and in peritoneal macrophages isolated from atherosclerotic apoE-/- mice treated with PJ. The effects of PJ were compared with those of the free radical generator 2, 2'-azobis (2-amidinopropane) dihydrochloride (AAPH). Both PJ and AAPH significantly increased J774A.1 macrophage death; however, flow cytometric and microscopic analyses using annexin V/propidium iodide revealed that PJ increased the early apoptosis of the macrophage dose dependently (up to 2.5-fold, P < 0.01), whereas AAPH caused dose-dependent increases in late apoptosis/necrosis (up to 12-fold, P < 0.001). Unlike PJ, AAPH-induced macrophage death was associated with increased intracellular oxidative stress (up to 7-fold, P < 0.001) and with lipid stress demonstrated by triglyceride accumulation (up to 3-fold, P < 0.01) and greater chromatic vesicle response to culture medium (up to 5-fold, P < 0.001). Accordingly, recombinant paraoxonase 1, which hydrolyzes oxidized lipids, attenuated macrophage death induced by AAPH, but not by PJ. Similar apoptotic and oxidative effects were found in macrophages from apoE-/- mice treated with PJ or AAPH. As macrophage apoptotic/necrotic death has considerable impact on atherosclerosis progression, these findings may provide novel mechanisms for the antiatherogenicity of PJ.

Leucine supplementation attenuates macrophage foam-cell formation: Studies in humans, mice, and cultured macrophages.

PubMed

Grajeda-Iglesias, Claudia; Rom, Oren; Hamoud, Shadi; Volkova, Nina; Hayek, Tony; Abu-Saleh, Niroz; Aviram, Michael

2018-02-05

Whereas atherogenicity of dietary lipids has been largely studied, relatively little is known about the possible contribution of dietary amino acids to macrophage foam-cell formation, a hallmark of early atherogenesis. Recently, we showed that leucine has antiatherogenic properties in the macrophage model system. In this study, an in-depth investigation of the role of leucine in macrophage lipid metabolism was conducted by supplementing humans, mice, or cultured macrophages with leucine. Macrophage incubation with serum obtained from healthy adults supplemented with leucine (5 g/d, 3 weeks) significantly decreased cellular cholesterol mass by inhibiting the rate of cholesterol biosynthesis and increasing cholesterol efflux from macrophages. Similarly, leucine supplementation to C57BL/6 mice (8 weeks) resulted in decreased cholesterol content in their harvested peritoneal macrophages (MPM) in relation with reduced cholesterol biosynthesis rate. Studies in J774A.1 murine macrophages revealed that leucine dose-dependently decreased cellular cholesterol and triglyceride mass. Macrophages treated with leucine (0.2 mM) showed attenuated uptake of very low-density lipoproteins and triglyceride biosynthesis rate, with a concurrent down-regulation of diacylglycerol acyltransferase-1, a key enzyme catalyzing triglyceride biosynthesis in macrophages. Similar effects were observed when macrophages were treated with α-ketoisocaproate, a key leucine metabolite. Finally, both in vivo and in vitro leucine supplementation significantly improved macrophage mitochondrial respiration and ATP production. The above studies, conducted in human, mice, and cultured macrophages, highlight a protective role for leucine attenuating macrophage foam-cell formation by mechanisms related to the metabolism of cholesterol, triglycerides, and energy production. © 2018 BioFactors, 2018. © 2018 International Union of Biochemistry and Molecular Biology.

Characterization of lipopolysaccharide-stimulated cytokine expression in macrophages and monocytes

USDA-ARS?s Scientific Manuscript database

Inflammation plays a pivotal role in several chronic human conditions and diseases including atherosclerosis, ischemic heart disease, cancer, obesity, diabetes, and autoimmune diseases. In vitro cell culture models such as exposure of mouse macrophage J774A.1 and human monocyte THP-1 cells to bacter...

Monocyte-macrophage membrane possesses free radicals scavenging activity: stimulation by polyphenols or by paraoxonase 1 (PON1).

PubMed

Rosenblat, M; Elias, A; Volkova, N; Aviram, M

2013-04-01

In the current study, we analysed free radicals scavenging activity of monocytes-macrophages in the absence or presence of antioxidants such as polyphenols or paraoxonase 1 (PON1). THP-1 human monocytic cell line, murine J774A.1 macrophages, as well as human primary monocytes have the capability to scavenge free radicals, as measured by the 1-diphenyl-2-picryl-hydrazyl (DPPH) assay. This effect (which could be attributed to the cell's membrane) was cell number and incubation time dependent. Upon incubation of J774A.1 macrophages with acetylated LDL (Ac-LDL), with VLDL, or with the radical generator, AAPH, the cells' lipid peroxides content, and paraoxonase 2 (PON2) activity were significantly increased. While non-treated cells decreased DPPH absorbance by 65%, the Ac-LDL-, VLDL- or AAPH-treated cells, decreased it by only 33%, 30%, or 45%, respectively. We next analysed the effect of J774A.1 macrophage enrichment with antioxidants, such as polyphenols or PON1 on the cells' free radicals scavenging activity. Non-treated cells decreased DPPH absorbance by 50%, whereas vitamin E-, punicalagin- or PJ-treated cells significantly further decreased it, by 75%. Similarly, in PON1-treated cells DPPH absorbance was further decreased by 63%, in association with 23% increment in PON1 catalytic activity. In cells under oxidative stress [treated with AAPH-, or with oxidized LDL], PON1 activity was decreased by 31% or 40%, as compared to the activity observed in PON1 incubated with non-treated cells. We conclude that monocytes-macrophages possess free radicals scavenging activity, which is decreased under atherogenic conditions, and increased upon cell enrichment with potent antioxidants such as nutritional polyphenols, or PON1.

Macrophages clear refrigerator storage-damaged RBCs and subsequently secrete cytokines in vivo, but not in vitro, in a murine model

PubMed Central

Wojczyk, Boguslaw S.; Kim, Nina; Bandyopadhyay, Sheila; Francis, Richard O.; Zimring, James C.; Hod, Eldad A.; Spitalnik, Steven L.

2014-01-01

BACKGROUND In mice, refrigerator-stored red blood cells (RBCs) are cleared by extravascular hemolysis and induce cytokine production. To enhance understanding of this phenomenon, we sought to model it in vitro. STUDY DESIGN AND METHODS Ingestion of refrigerator-stored murine RBCs and subsequent cytokine production were studied using J774A.1 mouse macrophage cells and primary murine splenic macrophages. Wild-type and Ccl2-GFP-reporter mice were used for RBC clearance in vivo. RESULTS Although J774A.1 cells and primary macrophages preferentially ingested refrigerator-stored RBCs in vitro, as compared to freshly-isolated RBCs, neither produced increased cytokines following erythrophagocytosis. In contrast, phagocytosis of refrigerator-stored RBCs in vivo induced increases in circulating monocyte chemoattractant protein-1 (MCP-1) and keratinocyte chemoattractant (KC), and correspondingly increased mRNA levels in mouse spleen and liver. In the spleen, these were predominantly expressed by CD11b+ cells. Using Ccl2-GFP-reporter mice, the predominant splenic population responsible for MCP-1 mRNA production were tissue-resident macrophages (i.e., CD45+, CD11b+, F4/80+, Ly6c+, CD11clow cells). CONCLUSION J774A.1 cells and primary macrophages selectively ingested refrigerator-stored RBCs by phagocytosis. Although cytokine expression was not enhanced, this approach could be used to identify the relevant receptor-ligand combination(s). In contrast, cytokine levels increased following phagocytosis of refrigerator-stored RBCs in vivo. These were primarily cleared in the liver and spleen, which demonstrated increased MCP-1 and KC mRNA expression. Finally, in mouse spleen, tissue-resident macrophages were predominantly involved in MCP-1 mRNA production. The differences between cytokine production in vitro and in vivo are not yet well understood. PMID:25041478

Relaxation of ferromagnetic nanoparticles in macrophages: In vitro and in vivo studies

NASA Astrophysics Data System (ADS)

Möller, Winfried; Takenaka, Shinji; Buske, Norbert; Felten, Kathrin; Heyder, Joachim

2005-05-01

The relaxation characteristics of magnetic nanoparticles (CoFe 2O 4) were investigated in J774A.1 macrophages and after voluntary inhalation. In dry form 25% of the particles showed Néel relaxation. Relaxation in macrophages occurred within minutes and could be inhibited by fixation, showing Brownian relaxation and intracellular transport processes. Relaxation in the lung happened similarly, but was dependent on the time after deposition. The particles were cleared from the lung within 2 weeks.

Homocysteine elicits an M1 phenotype in murine macrophages through an EMMPRIN-mediated pathway.

PubMed

Winchester, Lee J; Veeranki, Sudhakar; Givvimani, Srikanth; Tyagi, Suresh C

2015-07-01

Hyperhomocysteinemia (HHcy) is associated with inflammatory diseases and is known to increase the production of reactive oxygen species (ROS), matrix metalloproteinase (MMP)-9, and inducible nitric oxide synthase, and to decrease endothelial nitric oxide production. However, the impact of HHcy on macrophage phenotype differentiation is not well-established. It has been documented that macrophages have 2 distinct phenotypes: the "classically activated/destructive" (M1), and the "alternatively activated/constructive" (M2) subtypes. We hypothesize that HHcy increases M1 macrophage differentiation through extracellular matrix metalloproteinase inducer (EMMPRIN), a known inducer of matrix metalloproteinases. murine J774A.1 and Raw 264.7 macrophages were treated with 100 and 500 μmol/L Hcy, respectively, for 24 h. Samples were analyzed using Western blotting and immunocytochemistry. Homocysteine treatment increased cluster of differentiation 40 (CD40; M1 marker) in J774A.1 and Raw 264.7 macrophages. MMP-9 was induced in both cell lines. EMMPRIN protein expression was also increased in both cell lines. Blocking EMMPRIN function by pre-treating cells with anti-EMMPRIN antibody, with or without Hcy, resulted in significantly lower expression of CD40 in both cell lines by comparison with the controls. A DCFDA assay demonstrated increased ROS production in both cell lines with Hcy treatment when compared with the controls. Our results suggest that HHcy results in an increase of the M1 macrophage phenotype. This effect seems to be at least partially mediated by EMMPRIN induction.

Macrophages clear refrigerator storage-damaged red blood cells and subsequently secrete cytokines in vivo, but not in vitro, in a murine model.

PubMed

Wojczyk, Boguslaw S; Kim, Nina; Bandyopadhyay, Sheila; Francis, Richard O; Zimring, James C; Hod, Eldad A; Spitalnik, Steven L

2014-12-01

In mice, refrigerator-stored red blood cells (RBCs) are cleared by extravascular hemolysis and induce cytokine production. To enhance understanding of this phenomenon, we sought to model it in vitro. Ingestion of refrigerator-stored murine RBCs and subsequent cytokine production were studied using J774A.1 mouse macrophage cells and primary murine splenic macrophages. Wild-type and Ccl2-GFP reporter mice were used for RBC clearance in vivo. Although J774A.1 cells and primary macrophages preferentially ingested refrigerator-stored RBCs in vitro, compared to freshly isolated RBCs, neither produced increased cytokines after erythrophagocytosis. In contrast, phagocytosis of refrigerator-stored RBCs in vivo induced increases in circulating monocyte chemoattractant protein-1 (MCP-1) and keratinocyte chemoattractant (KC) and correspondingly increased mRNA levels in mouse spleen and liver. In the spleen, these were predominantly expressed by CD11b+ cells. Using Ccl2-GFP reporter mice, the predominant splenic population responsible for MCP-1 mRNA production was tissue-resident macrophages (i.e., CD45+, CD11b+, F4/80+, Ly6c+, and CD11c(low) cells). J774A.1 cells and primary macrophages selectively ingested refrigerator-stored RBCs by phagocytosis. Although cytokine expression was not enhanced, this approach could be used to identify the relevant receptor-ligand combination(s). In contrast, cytokine levels increased after phagocytosis of refrigerator-stored RBCs in vivo. These were primarily cleared in the liver and spleen, which demonstrated increased MCP-1 and KC mRNA expression. Finally, in mouse spleen, tissue-resident macrophages were predominantly involved in MCP-1 mRNA production. The differences between cytokine production in vitro and in vivo are not yet well understood. © 2014 AABB.

Hyphal growth of phagocytosed Fusarium oxysporum causes cell lysis and death of murine macrophages.

PubMed

Schäfer, Katja; Bain, Judith M; Di Pietro, Antonio; Gow, Neil A R; Erwig, Lars P

2014-01-01

Fusarium oxysporum is an important plant pathogen and an opportunistic pathogen of humans. Here we investigated phagocytosis of F. oxysporum by J774.1 murine cell line macrophages using live cell video microscopy. Macrophages avidly migrated towards F. oxysporum germlings and were rapidly engulfed after cell-cell contact was established. F. oxysporum germlings continued hyphal growth after engulfment by macrophages, leading to associated macrophage lysis and escape. Macrophage killing depended on the multiplicity of infection. After engulfment, F. oxysporum inhibited macrophages from completing mitosis, resulting in large daughter cells fused together by means of a F. oxysporum hypha. These results shed new light on the initial stages of Fusarium infection and the innate immune response of the mammalian host.

Mouse IgA inhibits cell growth by stimulating tumor necrosis factor-alpha production and apoptosis of macrophage cell lines.

PubMed

Reljic, Rajko; Crawford, Carol; Challacombe, Stephen; Ivanyi, Juraj

2004-04-01

Potent Fcalpha-mediated actions of IgA have previously been shown for myeloid cells from man, but much less is known in relation to murine cells. Here, we report that mouse monoclonal IgA, irrespective of their antigenic specificity, inhibit the proliferation of mouse macrophage cell lines. The anti-proliferative activity was manifested by both monomeric and polymeric mouse IgA, but not by mouse monoclonal IgG and IgM. Growth of J774 cells was significantly inhibited during the 4-8 days of logarithmic growth, followed by a subsequent recovery of cell numbers prior to the stationary phase. We demonstrated that IgA binds to J774 cells, stimulates tumor necrosis factor (TNF)-alpha production and induces apoptosis which is not dependent on NO or FAS/CD95. We also demonstrated that IgA, in synergy with IFN-gamma, induced TNF-alpha production and apoptosis of thioglycollate-elicited mouse peritoneal macrophages. Thus, the in vitro actions of IgA described may also play a regulatory role for mouse macrophages in vivo.

The Impairment of Macrophage-to-Feces Reverse Cholesterol Transport during Inflammation Does Not Depend on Serum Amyloid A

PubMed Central

de Beer, Maria C.; Wroblewski, Joanne M.; Noffsinger, Victoria P.; Meyer, Jason M.; van der Westhuyzen, Deneys R.

2013-01-01

Studies suggest that inflammation impairs reverse cholesterol transport (RCT). We investigated whether serum amyloid A (SAA) contributes to this impairment using an established macrophage-to-feces RCT model. Wild-type (WT) mice and mice deficient in SAA1.1 and SAA2.1 (SAAKO) were injected intraperitoneally with 3H-cholesterol-labeled J774 macrophages 4 hr after administration of LPS or buffered saline. 3H-cholesterol in plasma 4 hr after macrophage injection was significantly reduced in both WT and SAAKO mice injected with LPS, but this was not associated with a reduced capacity of serum from LPS-injected mice to promote macrophage cholesterol efflux in vitro. Hepatic accumulation of 3H-cholesterol was unaltered in either WT or SAAKO mice by LPS treatment. Radioactivity present in bile and feces of LPS-injected WT mice 24 hr after macrophage injection was reduced by 36% (P < 0.05) and 80% (P < 0.001), respectively. In contrast, in SAAKO mice, LPS did not significantly reduce macrophage-derived 3H-cholesterol in bile, and fecal excretion was reduced by only 45% (P < 0.05). Injection of cholesterol-loaded allogeneic J774 cells, but not syngeneic bone-marrow-derived macrophages, transiently induced SAA in C57BL/6 mice. Our study confirms reports that acute inflammation impairs steps in the RCT pathway and establishes that SAA plays only a minor role in this impairment. PMID:23431457

The Impairment of Macrophage-to-Feces Reverse Cholesterol Transport during Inflammation Does Not Depend on Serum Amyloid A.

PubMed

de Beer, Maria C; Wroblewski, Joanne M; Noffsinger, Victoria P; Ji, Ailing; Meyer, Jason M; van der Westhuyzen, Deneys R; de Beer, Frederick C; Webb, Nancy R

2013-01-01

Studies suggest that inflammation impairs reverse cholesterol transport (RCT). We investigated whether serum amyloid A (SAA) contributes to this impairment using an established macrophage-to-feces RCT model. Wild-type (WT) mice and mice deficient in SAA1.1 and SAA2.1 (SAAKO) were injected intraperitoneally with (3)H-cholesterol-labeled J774 macrophages 4 hr after administration of LPS or buffered saline. (3)H-cholesterol in plasma 4 hr after macrophage injection was significantly reduced in both WT and SAAKO mice injected with LPS, but this was not associated with a reduced capacity of serum from LPS-injected mice to promote macrophage cholesterol efflux in vitro. Hepatic accumulation of (3)H-cholesterol was unaltered in either WT or SAAKO mice by LPS treatment. Radioactivity present in bile and feces of LPS-injected WT mice 24 hr after macrophage injection was reduced by 36% (P < 0.05) and 80% (P < 0.001), respectively. In contrast, in SAAKO mice, LPS did not significantly reduce macrophage-derived (3)H-cholesterol in bile, and fecal excretion was reduced by only 45% (P < 0.05). Injection of cholesterol-loaded allogeneic J774 cells, but not syngeneic bone-marrow-derived macrophages, transiently induced SAA in C57BL/6 mice. Our study confirms reports that acute inflammation impairs steps in the RCT pathway and establishes that SAA plays only a minor role in this impairment.

Hyphal Growth of Phagocytosed Fusarium oxysporum Causes Cell Lysis and Death of Murine Macrophages

PubMed Central

Schäfer, Katja; Bain, Judith M.

2014-01-01

Fusarium oxysporum is an important plant pathogen and an opportunistic pathogen of humans. Here we investigated phagocytosis of F. oxysporum by J774.1 murine cell line macrophages using live cell video microscopy. Macrophages avidly migrated towards F. oxysporum germlings and were rapidly engulfed after cell-cell contact was established. F. oxysporum germlings continued hyphal growth after engulfment by macrophages, leading to associated macrophage lysis and escape. Macrophage killing depended on the multiplicity of infection. After engulfment, F. oxysporum inhibited macrophages from completing mitosis, resulting in large daughter cells fused together by means of a F. oxysporum hypha. These results shed new light on the initial stages of Fusarium infection and the innate immune response of the mammalian host. PMID:25025395

Effect of low extracellular pH on NF-κB activation in macrophages.

PubMed

Gerry, A B; Leake, D S

2014-04-01

Many diseases, including atherosclerosis, involve chronic inflammation. The master transcription factor for inflammation is NF-κB. Inflammatory sites have a low extracellular pH. Our objective was to demonstrate the effect of pH on NF-κB activation and cytokine secretion. Mouse J774 macrophages or human THP-1 or monocyte-derived macrophages were incubated at pH 7.0-7.4 and inflammatory cytokine secretion and NF-κB activity were measured. A pH of 7.0 greatly decreased pro-inflammatory cytokine secretion (TNF or IL-6) by J774 macrophages, but not THP-1 or human monocyte-derived macrophages. Upon stimulation of mouse macrophages, the levels of IκBα, which inhibits NF-κB, fell but low pH prevented its later increase, which normally restores the baseline activity of NF-κB, even though the levels of mRNA for IκBα were increased. pH 7.0 greatly increased and prolonged NF-κB binding to its consensus promoter sequence, especially the anti-inflammatory p50:p50 homodimers. Human p50 was overexpressed using adenovirus in THP-1 macrophages and monocyte-derived macrophages to see if it would confer pH sensitivity to NF-κB activity in human cells. Overexpression of p50 increased p50:p50 DNA-binding and in THP-1 macrophages inhibited considerably TNF and IL-6 secretion, but there was still no effect of pH on p50:p50 DNA binding or cytokine secretion. A modest decrease in pH can sometimes have marked effects on NF-κB activation and cytokine secretion and might be one reason to explain why mice normally develop less atherosclerosis than do humans. Copyright © 2014 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

Macrophage Biocompatibility of CoCr Wear Particles Produced under Polarization in Hyaluronic Acid Aqueous Solution

PubMed Central

Perez-Maceda, Blanca Teresa; López-Fernández, María Encarnación; Díaz, Iván; Kavanaugh, Aaron; Billi, Fabrizio; Escudero, María Lorenza; García-Alonso, María Cristina; Lozano, Rosa María

2018-01-01

Macrophages are the main cells involved in inflammatory processes and in the primary response to debris derived from wear of implanted CoCr alloys. The biocompatibility of wear particles from a high carbon CoCr alloy produced under polarization in hyaluronic acid (HA) aqueous solution was evaluated in J774A.1 mouse macrophages cultures. Polarization was applied to mimic the electrical interactions observed in living tissues. Wear tests were performed in a pin-on-disk tribometer integrating an electrochemical cell in phosphate buffer solution (PBS) and in PBS supplemented with 3 g/L HA, an average concentration that is generally found in synovial fluid, used as lubricant solution. Wear particles produced in 3 g/L HA solution showed a higher biocompatibility in J774A.1 macrophages in comparison to those elicited by particles obtained in PBS. A considerable enhancement in macrophages biocompatibility in the presence of 3 g/L of HA was further observed by the application of polarization at potentials having current densities typical of injured tissues suggesting that polarization produces an effect on the surface of the metallic material that leads to the production of wear particles that seem to be macrophage-biocompatible and less cytotoxic. The results showed the convenience of considering the influence of the electric interactions in the chemical composition of debris detached from metallic surfaces under wear corrosion to get a better understanding of the biological effects caused by the wear products. PMID:29738506

Artemisia asiatica Nakai Attenuates the Expression of Proinflammatory Mediators in Stimulated Macrophages Through Modulation of Nuclear Factor-κB and Mitogen-Activated Protein Kinase Pathways

PubMed Central

Kim, Eun-Kyung; Tang, Yujiao; Cha, Kwang-Suk; Choi, Heeri; Lee, Chun Bok; Yoon, Jin-Hwan; Kim, Sang Bae; Kim, Jong-Shik; Kim, Jong Moon; Han, Weon Cheol; Choi, Suck-Jun; Lee, Sangmin; Choi, Eun-Ju; Kim, Sang-Hyun

2015-01-01

Abstract The present study aimed to examine the anti-inflammatory effects and potential mechanism of action of Artemisia asiatica Nakai (A. asiatica Nakai) extract in activated murine macrophages. A. asiatica Nakai extract showed dose-dependent suppression of lipopolysaccharide (LPS)-induced nitric oxide, inducible nitric oxide synthase, and cyclooxygenase-2 activity. It also showed dose-dependent inhibition of nuclear factor-κB (NF-κB) translocation from the cytosol to the nucleus and as an inhibitor of NF-κB-alpha phosphorylation. The extract's inhibitory effects were found to be mediated through NF-κB inhibition and phosphorylation of extracellular signal-regulated kinase 1/2 and p38 in LPS-stimulated J774A.1 murine macrophages, suggesting a potential mechanism for the anti-inflammatory activity of A. asiatica Nakai. To our knowledge, this is the first report of the anti-inflammatory effects of A. asiatica Nakai on J774A.1 murine macrophages; these results may help develop functional foods possessing an anti-inflammatory activity. PMID:26061361

SSN 774 Virginia Class Submarine (SSN 774)

DTIC Science & Technology

2015-12-01

Selected Acquisition Report ( SAR ) RCS: DD-A&T(Q&A)823-516 SSN 774 Virginia Class Submarine (SSN 774) As of FY 2017 President’s Budget Defense...Acquisition Management Information Retrieval (DAMIR) March 8, 2016 11:22:44 UNCLASSIFIED SSN 774 December 2015 SAR March 8, 2016 11:22:44 UNCLASSIFIED 2...Document OSD - Office of the Secretary of Defense O&S - Operating and Support PAUC - Program Acquisition Unit Cost SSN 774 December 2015 SAR March 8

Garlic compounds modulate macrophage and T-lymphocyte functions.

PubMed

Lau, B H; Yamasaki, T; Gridley, D S

1991-06-01

Organosulfur compounds of garlic have been shown to inhibit growth of animal tumors and to modulate the activity of diverse chemical carcinogens. There is also evidence that garlic may modulate antitumor immunity. In this study, we determined the effects of an aqueous garlic extract and a protein fraction isolated from the extract on the chemiluminescent oxidative burst of the murine J774 macrophage cell line and thioglycollate-elicited peritoneal macrophages obtained from BALB/c mice. T-lymphocyte activity was determined using mouse splenocytes incubated with phytohemagglutinin, labeled with [3H]-thymidine and assayed for lymphoproliferation. Significant dose-related augmentation of oxidative burst was observed with garlic extract and the protein fraction. The protein fraction also enhanced the T-lymphocyte blastogenesis. The data suggest that garlic compounds may serve as biological response modifiers by augmenting macrophage and T-lymphocyte functions.

Mouse macrophages primed with alendronate down-regulate monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1alpha (MIP-1alpha) production in response to Toll-like receptor (TLR) 2 and TLR4 agonist via Smad3 activation.

PubMed

Masuda, Takahiro; Deng, Xue; Tamai, Riyoko

2009-08-01

Alendronate is one of the nitrogen-containing bisphosphonates (NBPs) used as anti-bone resorptive drugs. However, NBPs have inflammatory side effects including osteomyelitis and osteonecrosis of the jaw. In the present study, we examined the effects of alendronate on chemokine production by the macrophage-like cell line, J774.1, when incubated with Pam(3)CSK(4) (a Toll-like receptor (TLR) 2 agonist) and Lipid A (a TLR4 agonist). Pretreatment of J774.1 cells with alendronate decreased the production of TLR ligand-induced monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1alpha (MIP-1alpha) but did not influence nuclear factor-kappaB (NF-kappaB) activation. While this agent induced caspase-8 activation, a caspase-8 inhibitor did not affect the decrease in MCP-1 production by alendronate and TLR ligands. Thus, the alendronate-mediated decrease in chemokine production was independent of NF-kappaB and caspase-8 activation. Although transforming growth factor-beta1 (TGF-beta1) is known to inhibit chemokine production by various cell types via Smad3 activation, pretreatment with alendronate did not increase TGF-beta1 production by J774.1 cells incubated in the presence or absence of TLR ligands. However, alendronate directly activated Smad3. These results suggest that by down-regulating MCP-1 and MIP-1alpha production via Smad3, long-term use of alendronate might inhibit normal activation and migration of osteoclasts and cause osteonecrosis.

Homocysteine enhances MMP-9 production in murine macrophages via ERK and Akt signaling pathways

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

Lee, Seung Jin; Lee, Yi Sle; Seo, Kyo Won

2012-04-01

Homocysteine (Hcy) at elevated levels is an independent risk factor of cardiovascular diseases, including atherosclerosis. In the present study, we investigated the effect of Hcy on the production of matrix metalloproteinases (MMP) in murine macrophages. Among the MMP known to regulate the activities of collagenase and gelatinase, Hcy exclusively increased the gelatinolytic activity of MMP-9 in J774A.1 cells as well as in mouse peritoneal macrophages. Furthermore, this activity was found to be correlated with Western blot findings in J774A.1 cells, which showed that MMP-9 expression was concentration- and time-dependently increased by Hcy. Inhibition of the ERK and Akt pathways ledmore » to a significant decrease in Hcy-induced MMP-9 expression, and combined treatment with inhibitors of the ERK and Akt pathways showed an additive effects. Activity assays for ERK and Akt showed that Hcy increased the phosphorylation of both, but these phosphorylation were not affected by inhibitors of the Akt and ERK pathways. In line with these findings, the molecular inhibition of ERK and Akt using siRNA did not affect the Hcy-induced phosphorylation of Akt and ERK, respectively. Taken together, these findings suggest that Hcy enhances MMP-9 production in murine macrophages by separately activating the ERK and Akt signaling pathways. -- Highlights: ► Homocysteine (Hcy) induced MMP-9 production in murine macrophages. ► Hcy induced MMP-9 production through ERK and Akt signaling pathways. ► ERK and Akt signaling pathways were activated by Hcy in murine macrophages. ► ERK and Akt pathways were additively act on Hcy-induced MMP-9 production. ► Hcy enhances MMP-9 production in macrophages via activation of ERK and Akt signaling pathways in an independent manner.« less

Plasminogen promotes macrophage phagocytosis in mice

PubMed Central

Ganapathy, Swetha; Settle, Megan; Plow, Edward F.

2014-01-01

The phagocytic function of macrophages plays a pivotal role in eliminating apoptotic cells and invading pathogens. Evidence implicating plasminogen (Plg), the zymogen of plasmin, in phagocytosis is extremely limited with the most recent in vitro study showing that plasmin acts on prey cells rather than on macrophages. Here, we use apoptotic thymocytes and immunoglobulin opsonized bodies to show that Plg exerts a profound effect on macrophage-mediated phagocytosis in vitro and in vivo. Plg enhanced the uptake of these prey by J774A.1 macrophage-like cells by 3.5- to fivefold Plg receptors and plasmin proteolytic activity were required for phagocytosis of both preys. Compared with Plg+/+ mice, Plg−/− mice exhibited a 60% delay in clearance of apoptotic thymocytes by spleen and an 85% reduction in uptake by peritoneal macrophages. Phagocytosis of antibody-mediated erythrocyte clearance by liver Kupffer cells was reduced by 90% in Plg−/− mice compared with Plg+/+ mice. A gene array of splenic and hepatic tissues from Plg−/− and Plg+/+ mice showed downregulation of numerous genes in Plg−/− mice involved in phagocytosis and regulation of phagocytic gene expression was confirmed in macrophage-like cells. Thus, Plg may play an important role in innate immunity by changing expression of genes that contribute to phagocytosis. PMID:24876560

Photodynamic therapy mediates innate immune responses via fibroblast-macrophage interactions.

PubMed

Zulaziz, N; Azhim, A; Himeno, N; Tanaka, M; Satoh, Y; Kinoshita, M; Miyazaki, H; Saitoh, D; Shinomiya, N; Morimoto, Y

2015-10-01

Antibacterial photodynamic therapy (PDT) has come to attract attention as an alternative therapy for drug-resistant bacteria. Recent reports revealed that antibacterial PDT induces innate immune response and stimulates abundant cytokine secretion as a part of inflammatory responses. However, the underlying mechanism how antibacterial PDT interacts with immune cells responsible for cytokine secretion has not been well outlined. In this study, we aimed to clarify the difference in gene expression and cytokine secretion between combined culture of fibroblasts and macrophages and their independent cultures. SCRC-1008, mouse fibroblast cell line and J774, mouse macrophage-like cell line were co-cultured and PDT treatments with different parameters were carried out. After various incubation periods (1-24 h), cells and culture medium were collected, and mRNA and protein levels for cytokines were measured using real-time PCR and ELISA, respectively. Our results showed that fibroblasts and macrophages interact with each other to mediate the immune response. We propose that fibroblasts initially respond to PDT by expressing Hspa1b, which regulates the NF-κB pathway via Tlr2 and Tlr4. Activation of the NF-κB pathway then results in an enhanced secretion of pro-inflammatory cytokines (TNF-α, IL-6 and IL-1β) and neutrophil chemoattractant MIP-2 and KC from macrophages.

Truncated EphA2 likely potentiates cell adhesion via integrins as well as infiltration and/or lodgment of a monocyte/macrophage cell line in the red pulp and marginal zone of the mouse spleen, where ephrin-A1 is prominently expressed in the vasculature.

PubMed

Konda, Naoko; Saeki, Noritaka; Nishino, Shingo; Ogawa, Kazushige

2017-03-01

We previously established a J774.1 monocyte/macrophage subline expressing a truncated EphA2 construct lacking the kinase domain. We demonstrated that following ephrin-A1 stimulation, endogenous EphA2 promotes cell adhesion through interaction with integrins and integrin ligands such as ICAM1 and that truncated EphA2 potentiates the adhesion and becomes associated with the integrin/integrin ligand complex. Based on these findings, we hypothesized that the EphA/ephrin-A system, particularly EphA2/ephrin-A1, regulates transendothelial migration/tissue infiltration of monocytes/macrophages, because ephrin-A1 is widely recognized to be upregulated in inflammatory vasculatures. To evaluate whether this hypothesis is applicable in the spleen, we screened for EphA2/ephrin-A1 expression and reexamined the cellular properties of the J774.1 subline. We found that ephrin-A1 was expressed in the vasculature of the marginal zone and the red pulp and that its expression was upregulated in response to phagocyte depletion; further, CD115, F4/80, and CXCR4 were expressed in J774.1 cells, which serve as a usable substitute for monocytes/macrophages. Moreover, following ephrin-A1 stimulation, truncated EphA2 did not detectably interfere with the phosphorylation of endogenous EphA2, and it potentiated cell adhesion possibly through modulation of integrin avidity. Accordingly, by intravenously injecting mice with equal numbers of J774.1 and the subline cells labeled with distinct fluorochromes, we determined that truncated EphA2 markedly potentiated preferential cell infiltration into the red pulp and the marginal zone. Thus, modulation of EphA2 signaling might contribute to effective transplantation of tissue-specific resident macrophages and/or monocytes.

Staphylococcus haemolyticus strains target mitochondria and induce caspase-dependent apoptosis of macrophages.

PubMed

Krzymińska, Sylwia; Szczuka, Ewa; Kaznowski, Adam

2012-11-01

The aim of this study was to investigate the interaction of Staphylococcus haemolyticus strains with a macrophage cell line. Infection with the strains resulted in macrophage injury. All strains exhibited cytotoxic effects towards J774 cells. Moreover, the bacteria triggered apoptosis of the cells. The lowest apoptotic index did not exceed 21 %, whereas the highest reached 70 % at 24 h and 85 % at 48 h after infection. Incubation with the bacteria caused loss of mitochondrial membrane potential (ΔΨm) in macrophages. The pro-apoptotic activity of the strains was blocked by a pan-caspase inhibitor z-VAD-fmk, indicating the involvement of caspases in the bacteria-mediated cell death. We observed that the induction of macrophage apoptosis could constitute an important mechanism of pathogenesis by which S. haemolyticus strains evade host immune defences and cause disease.

YopJ-induced caspase-1 activation in Yersinia-infected macrophages: independent of apoptosis, linked to necrosis, dispensable for innate host defense.

PubMed

Zheng, Ying; Lilo, Sarit; Mena, Patricio; Bliska, James B

2012-01-01

Yersinia outer protein J (YopJ) is a type III secretion system (T3SS) effector of pathogenic Yersinia (Yersinia pestis, Yersinia enterocolitica and Yersinia pseudotuberculosis) that is secreted into host cells. YopJ inhibits survival response pathways in macrophages, causing cell death. Allelic variation of YopJ is responsible for differential cytotoxicity in Yersinia strains. YopJ isoforms in Y. enterocolitica O:8 (YopP) and Y. pestis KIM (YopJ(KIM)) strains have high cytotoxic activity. In addition, YopJ(KIM)-induced macrophage death is associated with caspase-1 activation and interleukin-1β (IL-1β secretion. Here, the mechanism of YopJ(KIM)-induced cell death, caspase-1 activation, and IL-1β secretion in primary murine macrophages was examined. Caspase-3/7 activity was low and the caspase-3 substrate poly (ADP-ribose) polymerase (PARP) was not cleaved in Y. pestis KIM5-infected macrophages. In addition, cytotoxicity and IL-1β secretion were not reduced in the presence of a caspase-8 inhibitor, or in B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax)/Bcl-2 homologous antagonist/killer (Bak) knockout macrophages, showing that YopJ(KIM)-mediated cell death and caspase-1 activation occur independent of mitochondrial-directed apoptosis. KIM5-infected macrophages released high mobility group protein B1 (HMGB1), a marker of necrosis, and microscopic analysis revealed that necrotic cells contained active caspase-1, indicating that caspase-1 activation is associated with necrosis. Inhibitor studies showed that receptor interacting protein 1 (RIP1) kinase and reactive oxygen species (ROS) were not required for cytotoxicity or IL-β release in KIM5-infected macrophages. IL-1β secretion was reduced in the presence of cathepsin B inhibitors, suggesting that activation of caspase-1 requires cathepsin B activity. Ectopically-expressed YopP caused higher cytotoxicity and secretion of IL-1β in Y. pseudotuberculosis-infected macrophages than YopJ(KIM). Wild-type and

Effect of low-level laser therapy on the modulation of the mitochondrial activity of macrophages

PubMed Central

Souza, Nadhia H. C.; Ferrari, Raquel A. M.; Silva, Daniela F. T.; Nunes, Fabio D.; Bussadori, Sandra K.; Fernandes, Kristianne P. S.

2014-01-01

BACKGROUND: Macrophages play a major role among the inflammatory cells that invade muscle tissue following an injury. Low-level laser therapy (LLLT) has long been used in clinical practice to accelerate the muscle repair process. However, little is known regarding its effect on macrophages. OBJECTIVE: This study evaluated the effect of LLLT on the mitochondrial activity (MA) of macrophages. METHOD: J774 macrophages were treated with lipopolysaccharide (LPS) and interferon - gamma (IFN-γ) (activation) for 24 h to simulate an inflammatory process, then irradiated with LLLT using two sets of parameters (780 nm; 70 mW; 3 J/cm2 and 660 nm; 15 mW; 7.5 J/cm2). Non-activated/non-irradiated cells composed the control group. MA was evaluated by the cell mitochondrial activity (MTT) assay (after 1, 3 and 5 days) in three independent experiments. The data were analyzed statistically. RESULTS: After 1 day of culture, activated and 780 nm irradiated macrophages showed lower MA than activated macrophages, but activated and 660 nm irradiated macrophages showed MA similar to activated cells. After 3 days, activated and irradiated (660 nm and 780 nm) macrophages showed greater MA than activated macrophages, and after 5 days, the activated and irradiated (660 nm and 780 nm) macrophages showed similar MA to the activated macrophages. CONCLUSIONS: These results show that 660 nm and 780 nm LLLT can modulate the cellular activation status of macrophages in inflammation, highlighting the importance of this resource and of the correct determination of its parameters in the repair process of skeletal muscle. PMID:25076002

Lysozyme activates Enterococcus faecium to induce necrotic cell death in macrophages.

PubMed

Gröbner, Sabine; Fritz, Evelyn; Schoch, Friederike; Schaller, Martin; Berger, Alexander C; Bitzer, Michael; Autenrieth, Ingo B

2010-10-01

Enterococci are commensal organisms in the alimentary tract. However, they can cause a variety of life-threatening infections, especially in nosocomial settings. We hypothesized that induction of cell death might enable these facultative pathogenic bacteria to evade the innate immune response and to cause infections of their host. We demonstrate that E. faecium when exposed to lysozyme induces cell death in macrophages in vitro and in vivo. Flow cytometric analyses of J774A.1 macrophages infected with E. faecium revealed loss of cell membrane integrity indicated by uptake of propidium iodide and decrease of the inner mitochondrial transmembrane potential DeltaPsi(m). Inhibition of caspases, treatment of macrophages with cytochalasin D, or rifampicin did not prevent cells from dying, suggesting cell death mechanisms that are independent of caspase activation, bacterial uptake, and intracellular bacterial replication. Characteristics of necrotic cell death were demonstrated by both lack of procaspase 3 activation and cell shrinkage, electron microscopy, and release of lactate dehydrogenase. Pretreatment of E. faecium with lysozyme and subsequently with broad spectrum protease considerably reduced cell death, suggesting that a bacterial surface protein is causative for cell death induction. Moreover, in a mouse peritonitis model we demonstrated that E. faecium induces cell death of peritoneal macrophages in vivo. Altogether, our results show that enterococci, under specific conditions such as exposure to lysozyme, induce necrotic cell death in macrophages, which might contribute to disseminated infections by these facultative pathogenic bacteria.

MIF-driven activation of macrophages induces killing of intracellular Trypanosoma cruzi dependent on endogenous production of tumor necrosis factor, nitric oxide and reactive oxygen species.

PubMed

Cutrullis, Romina A; Petray, Patricia B; Corral, Ricardo S

2017-02-01

The proinflammatory cytokine macrophage migration inhibitory factor (MIF) is a key player in innate immunity. MIF has been considered critical for controlling acute infection by the protozoan Trypanosoma cruzi, but the underlying mechanisms are poorly understood. Our study aimed to analyze whether MIF could favor microbicidal activity of the macrophage, a site where T. cruzi grows and the initial effector cell against this parasite. Using murine macrophages infected in vitro, we examined the effect of MIF on their parasiticidal ability and attempted to identify inflammatory agents involved in MIF-induced protection. Our findings show that MIF is readily secreted from peritoneal macrophages upon T. cruzi infection. MIF activates both primary and J774 phagocytes boosting the endogenous production of tumor necrosis factor-alpha via mitogen-activated protein kinase p38 signaling, as well as the release of nitric oxide and reactive oxygen species, leading to enhanced pathogen elimination. MIF can also potentiate the effect of interferon-gamma on T. cruzi killing by J774 and mouse peritoneal macrophages, rendering these cells more competent in reducing intracellular parasite burden. The present results unveil a novel innate immune pathway that contributes to host defense and broaden our understanding of the regulation of inflammatory mediators implicated in early parasite containment that is decisive for resistance to T. cruzi infection. Copyright © 2016 Elsevier GmbH. All rights reserved.

Dragon (repulsive guidance molecule b) inhibits IL-6 expression in macrophages.

PubMed

Xia, Yin; Cortez-Retamozo, Virna; Niederkofler, Vera; Salie, Rishard; Chen, Shanzhuo; Samad, Tarek A; Hong, Charles C; Arber, Silvia; Vyas, Jatin M; Weissleder, Ralph; Pittet, Mikael J; Lin, Herbert Y

2011-02-01

Repulsive guidance molecule (RGM) family members RGMa, RGMb/Dragon, and RGMc/hemojuvelin were found recently to act as bone morphogenetic protein (BMP) coreceptors that enhance BMP signaling activity. Although our previous studies have shown that hemojuvelin regulates hepcidin expression and iron metabolism through the BMP pathway, the role of the BMP signaling mediated by Dragon remains largely unknown. We have shown previously that Dragon is expressed in neural cells, germ cells, and renal epithelial cells. In this study, we demonstrate that Dragon is highly expressed in macrophages. Studies with RAW264.7 and J774 macrophage cell lines reveal that Dragon negatively regulates IL-6 expression in a BMP ligand-dependent manner via the p38 MAPK and Erk1/2 pathways but not the Smad1/5/8 pathway. We also generated Dragon knockout mice and found that IL-6 is upregulated in macrophages and dendritic cells derived from whole lung tissue of these mice compared with that in respective cells derived from wild-type littermates. These results indicate that Dragon is an important negative regulator of IL-6 expression in immune cells and that Dragon-deficient mice may be a useful model for studying immune and inflammatory disorders.

Alterations of zinc homeostasis in response to Cryptococcus neoformans in a murine macrophage cell line.

PubMed

Dos Santos, Francine Melise; Piffer, Alícia Corbellini; Schneider, Rafael de Oliveira; Ribeiro, Nicole Sartori; Garcia, Ane Wichine Acosta; Schrank, Augusto; Kmetzsch, Lívia; Vainstein, Marilene Henning; Staats, Charley Christian

2017-05-01

To evaluate alterations of zinc homeostasis in macrophages exposed to Cryptococcus neoformans. Materials & methods: Using a fluorescent zinc probe-based flow cytometry and atomic absorption spectrometry, zinc levels were evaluated in J774.A1 cell lines exposed to C. neoformans H99 cells. The transcription profile of macrophage zinc related homeostasis genes - metallothioneins and zinc transporters (ZnTs) of the SLC30 and SLC39 (Zrt-Irt-protein) families - was analyzed by quantitative PCR. Macrophage intracellular labile zinc levels decreased following exposure to C. neoformans. A significant decrease in transcription levels was detected in specific ZnTs from both the Zrt-Irt-protein and ZnT families, especially 24 h after infection. These findings suggest that macrophages may exhibit zinc depletion in response to C. neoformans infection.

Activation of macrophages and interference with CD4+ T-cell stimulation by Mycobacterium avium subspecies paratuberculosis and Mycobacterium avium subspecies avium

PubMed Central

Lage, Susanne Zur; Goethe, Ralph; Darji, Ayub; Valentin-Weigand, Peter; Weiss, Siegfried

2003-01-01

Mycobacterium avium subspecies paratuberculosis (M. ptb) and M. avium subspecies avium (M. avium) are closely related but exhibit significant differences in their interaction with the host immune system. The macrophage line, J774, was infected with M. ptb and M. avium and analysed for cytokine production and stimulatory capacity towards antigen-specific CD4+ T cells. Under all conditions J774 cells were activated to produce proinflammatory cytokines. No influence on the expression of major histocompatibility complex (MHC) class II, intracellular adhesion molecule-1 (ICAM-1), B7.1, B7.2 or CD40 was found. However, the antigen-specific stimulatory capacity of J774 cells for a CD4+ T-cell line was significantly inhibited after infection with M. ptb, but not with M. avium. When a T-cell hybridoma expressing a T-cell receptor identical to that of the T-cell line was used, this inhibition was not observed, suggesting that costimulation which is essential for the CD4+ T-cell line is influenced by the pathogenic bacterium M. ptb. PMID:12519304

Contribution of macrophages in the contrast loss in iron oxide-based MRI cancer cell tracking studies

PubMed Central

Danhier, Pierre; Deumer, Gladys; Joudiou, Nicolas; Bouzin, Caroline; Levêque, Philippe; Haufroid, Vincent; Jordan, Bénédicte F.; Feron, Olivier; Sonveaux, Pierre; Gallez, Bernard

2017-01-01

Magnetic resonance imaging (MRI) cell tracking of cancer cells labeled with superparamagnetic iron oxides (SPIO) allows visualizing metastatic cells in preclinical models. However, previous works showed that the signal void induced by SPIO on T2(*)-weighted images decreased over time. Here, we aim at characterizing the fate of iron oxide nanoparticles used in cell tracking studies and the role of macrophages in SPIO metabolism. In vivo MRI cell tracking of SPIO positive 4T1 breast cancer cells revealed a quick loss of T2* contrast after injection. We next took advantage of electron paramagnetic resonance (EPR) spectroscopy and inductively coupled plasma mass spectroscopy (ICP-MS) for characterizing the evolution of superparamagnetic and non-superparamagnetic iron pools in 4T1 breast cancer cells and J774 macrophages after SPIO labeling. These in vitro experiments and histology studies performed on 4T1 tumors highlighted the quick degradation of iron oxides by macrophages in SPIO-based cell tracking experiments. In conclusion, the release of SPIO by dying cancer cells and the subsequent uptake of iron oxides by tumor macrophages are limiting factors in MRI cell tracking experiments that plead for the use of (MR) reporter-gene based imaging methods for the long-term tracking of metastatic cells. PMID:28467814

Anti-inflammatory effect of a selective 11β-hydroxysteroid dehydrogenase type 1 inhibitor via the stimulation of heme oxygenase-1 in LPS-activated mice and J774.1 murine macrophages.

PubMed

Park, Sung Bum; Park, Ji Seon; Jung, Won Hoon; Kim, Hee Youn; Kwak, Hyun Jung; Ahn, Jin Hee; Choi, Kyoung-Jin; Na, Yoon-Ju; Choi, Sunhwa; Dal Rhee, Sang; Kim, Ki Young

2016-08-01

11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) converts inactive cortisone to the active cortisol. 11β-HSD1 may be involved in the resolution of inflammation. In the present study, we investigate the anti-inflammatory effects of 2-(3-benzoyl)-4-hydroxy-1,1-dioxo-2H-1,2-benzothiazine-2-yl-1-phenylethanone (KR-66344), a selective 11β-HSD1 inhibitor, in lipopolysaccharide (LPS)-activated C57BL/6J mice and macrophages. LPS increased 11β-HSD1 activity and expression in macrophages, which was inhibited by KR-66344. In addition, KR-66344 increased survival rate in LPS treated C57BL/6J mice. HO-1 mRNA expression level was increased by KR-66344, and this effect was reversed by the HO competitive inhibitor, ZnPP, in macrophages. Moreover, ZnPP reversed the suppression of ROS formation and cell death induced by KR-66344. ZnPP also suppressed animal survival rate in LPS plus KR-66344 treated C57BL/6J mice. In the spleen of LPS-treated mice, KR-66344 prevented cell death via suppression of inflammation, followed by inhibition of ROS, iNOS and COX-2 expression. Furthermore, LPS increased NFκB-p65 and MAPK phosphorylation, and these effects were abolished by pretreatment with KR-66344. Taken together, KR-66344 protects against LPS-induced animal death and spleen injury by inhibition of inflammation via induction of HO-1 and inhibition of 11β-HSD1 activity. Thus, we concluded that the selective 11β-HSD1 inhibitor may provide a novel strategy in the prevention/treatment of inflammatory disorders in patients. Copyright © 2016 The Authors. Production and hosting by Elsevier B.V. All rights reserved.

Low-dose Norfloxacin-treated leptospires induce less IL-1β release in J774A.1 cells following discrepant leptospiral gene expression.

PubMed

Cao, Yongguo; Xie, Xufeng; Zhang, Wenlong; Wu, Dianjun; Tu, Changchun

2018-06-01

Currently, accumulating evidence is challenging subtherapeutic therapy. Low-dose Norfloxacin (Nor) has been reported to suppress the immune response and worsen leptospirosis. In this study, we investigated the influence of low-dose Nor (0.03 μg/ml, 0.06 μg/ml, 0.125 μg/ml) on leptospiral gene expression and analyzed the immunomodulatory effects of low-dose Nor-treated leptospires in J774A.1 cells. To study the expression profiles of low-dose Nor-treated leptospires, we chose LipL71/LipL21 as reference genes determined by the geNorm applet in this experiment. The results showed that low-dose Nor up-regulated the expression of FlaB and inhibited the expression of 16S rRNA, LipL32, LipL41, Loa22, KdpA, and KdpB compared with the untreated leptospires. These results indicated that low-dose Nor could regulate leptospiral gene expression. Using RT-PCR, the gene expression of IL-1β and TNF-α in J774A.1 cells was detected. Nor-treated leptospires induced higher expression levels of both IL-1β and TNF-α. However, when analyzed by ELISA, the release of mature IL-1β was reduced compared with that observed in cells induced with no Nor-treated leptospires, although the TNF-α protein level showed no significant change. Our study indicated that the gene expression of leptospires could be modulated by low-dose Nor, which induced less IL-1β release in J774A.1 cells. Copyright © 2018 Elsevier Ltd. All rights reserved.

Regulation of adhesion behavior of murine macrophage using supported lipid membranes displaying tunable mannose domains

NASA Astrophysics Data System (ADS)

Kaindl, T.; Oelke, J.; Pasc, A.; Kaufmann, S.; Konovalov, O. V.; Funari, S. S.; Engel, U.; Wixforth, A.; Tanaka, M.

2010-07-01

Highly uniform, strongly correlated domains of synthetically designed lipids can be incorporated into supported lipid membranes. The systematic characterization of membranes displaying a variety of domains revealed that the equilibrium size of domains significantly depends on the length of fluorocarbon chains, which can be quantitatively interpreted within the framework of an equivalent dipole model. A mono-dispersive, narrow size distribution of the domains enables us to treat the inter-domain correlations as two-dimensional colloidal crystallization and calculate the potentials of mean force. The obtained results demonstrated that both size and inter-domain correlation can precisely be controlled by the molecular structures. By coupling α-D-mannose to lipid head groups, we studied the adhesion behavior of the murine macrophage (J774A.1) on supported membranes. Specific adhesion and spreading of macrophages showed a clear dependence on the density of functional lipids. The obtained results suggest that such synthetic lipid domains can be used as a defined platform to study how cells sense the size and distribution of functional molecules during adhesion and spreading.

YopJ-Induced Caspase-1 Activation in Yersinia-Infected Macrophages: Independent of Apoptosis, Linked to Necrosis, Dispensable for Innate Host Defense

PubMed Central

Zheng, Ying; Lilo, Sarit; Mena, Patricio; Bliska, James B.

2012-01-01

Yersinia outer protein J (YopJ) is a type III secretion system (T3SS) effector of pathogenic Yersinia (Yersinia pestis, Yersinia enterocolitica and Yersinia pseudotuberculosis) that is secreted into host cells. YopJ inhibits survival response pathways in macrophages, causing cell death. Allelic variation of YopJ is responsible for differential cytotoxicity in Yersinia strains. YopJ isoforms in Y. enterocolitica O:8 (YopP) and Y. pestis KIM (YopJKIM) strains have high cytotoxic activity. In addition, YopJKIM-induced macrophage death is associated with caspase-1 activation and interleukin-1β (IL-1β secretion. Here, the mechanism of YopJKIM-induced cell death, caspase-1 activation, and IL-1β secretion in primary murine macrophages was examined. Caspase-3/7 activity was low and the caspase-3 substrate poly (ADP-ribose) polymerase (PARP) was not cleaved in Y. pestis KIM5-infected macrophages. In addition, cytotoxicity and IL-1β secretion were not reduced in the presence of a caspase-8 inhibitor, or in B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax)/Bcl-2 homologous antagonist/killer (Bak) knockout macrophages, showing that YopJKIM-mediated cell death and caspase-1 activation occur independent of mitochondrial-directed apoptosis. KIM5-infected macrophages released high mobility group protein B1 (HMGB1), a marker of necrosis, and microscopic analysis revealed that necrotic cells contained active caspase-1, indicating that caspase-1 activation is associated with necrosis. Inhibitor studies showed that receptor interacting protein 1 (RIP1) kinase and reactive oxygen species (ROS) were not required for cytotoxicity or IL-β release in KIM5-infected macrophages. IL-1β secretion was reduced in the presence of cathepsin B inhibitors, suggesting that activation of caspase-1 requires cathepsin B activity. Ectopically-expressed YopP caused higher cytotoxicity and secretion of IL-1β in Y. pseudotuberculosis-infected macrophages than YopJKIM. Wild-type and congenic

Nanomaterial induction of oxidative stress in lung epithelial cells and macrophages

NASA Astrophysics Data System (ADS)

Wang, Lin; Pal, Anoop K.; Isaacs, Jacqueline A.; Bello, Dhimiter; Carrier, Rebecca L.

2014-09-01

Oxidative stress in the lung epithelial A549 cells and macrophages J774A.1 due to contact with commercially important nanomaterials [i.e., nano-silver (nAg), nano-alumina (nAl2O3), single-wall carbon nanotubes (CNT), and nano-titanium oxide anatase (nTiO2)] was evaluated. Nanomaterial-induced intracellular oxidative stress was analyzed by both H2DCFDA fluorescein probe and GSH depletion, extracellular oxidative stress was assessed by H2HFF fluorescein probes, and the secretion of chemokine IL-8 by A549 cells due to elevation of cellular oxidative stress was also monitored, in order to provide a comprehensive in vitro study on nanomaterial-induced oxidative stress in lung. In addition, results from this study were also compared with an acellular "ferric reducing ability of serum" (FRAS) assay and a prokaryotic cell-based assay in evaluating oxidative damage caused by the same set of nanomaterials, for comparison purposes. In general, it was found that nanomaterial-induced oxidative stress is highly cell-type dependent. In A549 lung epithelial cells, nAg appeared to induce highest level of oxidative stress and cell death followed by CNT, nTiO2, and nAl2O3. Different biological oxidative damage (BOD) assays' (i.e., H2DCFA, GSH, and IL-8 release) results generally agreed with each other, and the same trends of nanomaterial-induced BOD were also observed in acellular FRAS and prokaryotic E. coli K12-based assay. In macrophage J774A.1 cells, nAl2O3 and nTiO2 appeared to induce highest levels of oxidative stress. These results suggest that epithelial and macrophage cell models may provide complimentary information when conducting cell-based assays to evaluate nanomaterial-induced oxidative damage in lung.

Macrophage polarisation: an immunohistochemical approach for identifying M1 and M2 macrophages.

PubMed

Barros, Mário Henrique M; Hauck, Franziska; Dreyer, Johannes H; Kempkes, Bettina; Niedobitek, Gerald

2013-01-01

Macrophage polarization is increasingly recognised as an important pathogenetic factor in inflammatory and neoplastic diseases. Proinflammatory M1 macrophages promote T helper (Th) 1 responses and show tumoricidal activity. M2 macrophages contribute to tissue repair and promote Th2 responses. CD68 and CD163 are used to identify macrophages in tissue sections. However, characterisation of polarised macrophages in situ has remained difficult. Macrophage polarisation is regulated by transcription factors, pSTAT1 and RBP-J for M1, and CMAF for M2. We reasoned that double-labelling immunohistochemistry for the detection of macrophage markers together with transcription factors may be suitable to characterise macrophage polarisation in situ. To test this hypothesis, we have studied conditions associated with Th1- and Th2-predominant immune responses: infectious mononucleosis and Crohn's disease for Th1 and allergic nasal polyps, oxyuriasis, wound healing and foreign body granulomas for predominant Th2 response. In all situations, CD163+ cells usually outnumbered CD68+ cells. Moreover, CD163+ cells, usually considered as M2 macrophages, co-expressing pSTAT1 and RBP-J were found in all conditions examined. The numbers of putative M1 macrophages were higher in Th1- than in Th2-associated diseases, while more M2 macrophages were seen in Th2- than in Th1 related disorders. In most Th1-related diseases, the balance of M1 over M2 cells was shifted towards M1 cells, while the reverse was observed for Th2-related conditions. Hierarchical cluster analysis revealed two distinct clusters: cluster I included Th1 diseases together with cases with high numbers of CD163+pSTAT1+, CD68+pSTAT1+, CD163+RBP-J+ and CD68+RBP-J+ macrophages; cluster II comprised Th2 conditions together with cases displaying high numbers of CD163+CMAF+ and CD68+CMAF+ macrophages. These results suggest that the detection of pSTAT1, RBP-J, and CMAF in the context of CD68 or CD163 expression is a suitable tool for

The receptor tyrosine kinase MerTK activates phospholipase C γ2 during recognition of apoptotic thymocytes by murine macrophages

PubMed Central

Todt, Jill C.; Hu, Bin; Curtis, Jeffrey L.

2008-01-01

Apoptotic leukocytes must be cleared efficiently by macrophages (Mø). Apoptotic cell phagocytosis by Mø requires the receptor tyrosine kinase (RTK) MerTK (also known as c-Mer and Tyro12), the phosphatidylserine receptor (PS-R), and the classical protein kinase C (PKC) isoform βII, which translocates to Mø membrane and cytoskeletal fractions in a PS-R-dependent fashion. How these molecules cooperate to induce phagocytosis is unknown. Because the phosphatidylinositol-specific phospholipase (PI-PLC) PLC γ2 is downstream of RTKs in some cell types and can activate classical PKCs, we hypothesized that MerTK signals via PLC γ2. To test this hypothesis, we examined the interaction of MerTK and PLC γ2 in resident murine PMø and in the murine Mø cell line J774A.1 (J774) following exposure to apoptotic thymocytes. We found that, as with PMø, J774 phagocytosis of apoptotic thymocytes was inhibited by antibody against MerTK. Western blotting and immunoprecipitation showed that exposure to apoptotic cells produced three time-dependent changes in PMø and J774: (1) tyrosine phosphorylation of MerTK; (2) association of PLC γ2 with MerTK; and (3) tyrosine phosphorylation of PLC γ2. Phosphorylation of PLC γ2 and its association with MerTK was also induced by cross-linking MerTK using antibody. A PI-PLC appears to be required for phagocytosis of apoptotic cells because the PI-PLC inhibitor Et-18-OCH3 and the PLC inhibitor U73122, but not the inactive control U73343, blocked phagocytosis without impairing adhesion. On apoptotic cell adhesion to Mø, MerTK signals at least in part via PLC γ2. PMID:14704368

Cytotoxic activity and composition of petroleum ether extract from Magydaris tomentosa (Desf.) W. D. J. Koch (Apiaceae).

PubMed

Autore, Giuseppina; Marzocco, Stefania; Formisano, Carmen; Bruno, Maurizio; Rosselli, Sergio; Jemia, Mariem Ben; Senatore, Felice

2015-01-16

The petroleum ether extract of Magydaris tomentosa flowers (Desf.) W. D. J. Koch has been analyzed by GC-MS. It is mainly constituted by furanocoumarins such as xanthotoxin, xanthotoxol, isopimpinellin, and bergaptene. Other coumarins such as 7-methoxy-8-(2-formyl-2-methylpropyl) coumarin and osthole also occurred. The antiproliferative activity of Magydaris tomentosa flower extract has been evaluated in vitro on murine monocye/macrophages (J774A.1), human melanoma (A375) and human breast cancer (MCF-7) tumor cell lines, showing a major activity against the latter.

Evaluation of the leishmanicidal and cytotoxic potential of essential oils derived from ten colombian plants.

PubMed

Sanchez-Suarez, Jf; Riveros, I; Delgado, G

2013-01-01

The leishmanicidal and cytotoxic activity of ten essential oils obtained from ten plant specimens were evaluated. Essential oils were obtained by the steam distillation of plant leaves without any prior processing. Cytotoxicity was tested on J774 macrophages and leishmanicidal activity was assessed against four species of Leishmania associated with cutaneous leishmaniasis. Seven essential oils exhibited activity against Leishmania parasites, five of which were toxic against J774 macrophages. Selectivity indices of >6 and 13 were calculated for the essential oils of Ocimum basilicum and Origanum vulgare, respectively. The essential oil of Ocimum basilicum was active against promastigotes of Leishmania and innocuous to J774 macrophages at concentrations up to 1600 µg/mL and should be further investigated for leishmanicidal activity in others in vitro and in vivo experimental models.

Wallerian degeneration in C57BL/6J and A/J mice: differences in time course of neurofilament and myelin breakdown, macrophage recruitment and iNOS expression

PubMed Central

de la Hoz, Cristiane L R; Oliveira, Alexandre L R; de S Queiroz, Luciano; Langone, Francesco

2003-01-01

The lower regeneration potential reported for C57BL/6J mice strain after peripheral nerve lesion may result from alterations in crucial events during Wallerian degeneration. We analysed neurofilament and myelin breakdown, macrophage recruitment, NADPH-diaphorase reaction and inducible nitric oxide synthase (iNOS) expression in transected sciatic nerves of C57BL/6J and A/J mice. The neurofilament volume density was lower in C57BL/6J strain mice at 1 and 3 days after lesion, and later equalled the density observed in A/J. C57BL/6J mice presented a high number of cells containing myelin debris, 3 and 5 days after the lesion. In both strains iNOS immunoreactivity was intense in macrophages and less evident in Schwann cells. However, a delay in macrophage recruitment and a lower percentage of iNOS-expressing macrophages on the third day were observed in C57BL/6J mice. NADPH-diaphorase reaction disclosed a similar pattern for both strains until the seventh day. However, at 5 days, cells with slender processes involving ellipsoid segments showed a well-defined cytoplasmic labelling in C57BL/6J whereas in A/J most of these cells exhibited a more granular and disperse labelling. We propose that these differences between A/J and C57BL/6J strains during Wallerian degeneration may be implicated in the lower regeneration potential observed in the latter. PMID:14686692

Macrophage sphingolipids are essential for the entry of mycobacteria.

PubMed

Viswanathan, Gopinath; Jafurulla, Md; Kumar, G Aditya; Raghunand, Tirumalai R; Chattopadhyay, Amitabha

2018-07-01

Mycobacteria are intracellular pathogens that can invade and survive within host macrophages. Mycobacterial infections remain a major cause of mortality and morbidity worldwide, with serious concerns of emergence of multi and extensively drug-resistant tuberculosis. While significant advances have been made in identifying mycobacterial virulence determinants, the detailed molecular mechanism of internalization of mycobacteria into host cells remains poorly understood. Although several studies have highlighted the crucial role of sphingolipids in mycobacterial growth, persistence and establishment of infection, the role of sphingolipids in the entry of mycobacteria into host cells is not known. In this work, we explored the role of host membrane sphingolipids in the entry of Mycobacterium smegmatis into J774A.1 macrophages. Our results show that metabolic depletion of sphingolipids in host macrophages results in a significant reduction in the entry of M. smegmatis. Importantly, the entry of Escherichia coli into host macrophages under similar conditions remained invariant, implying the specificity of the requirement of sphingolipids in mycobacterial entry. To the best of our knowledge, our results constitute the first report demonstrating the role of host macrophage sphingolipids in the entry of mycobacteria. Our results could help in the development of novel therapeutic strategies targeting sphingolipid-mediated entry of mycobacteria into host cells. Copyright © 2018 Elsevier B.V. All rights reserved.

Differential activation of Fyn kinase distinguishes saturated and unsaturated fats in mouse macrophages

PubMed Central

Tarabra, Elena; An Lee, Ting-Wen; Zammit, Victor A.; Vatish, Manu; Yamada, Eijiro; Pessin, Jeffrey E.; Bastie, Claire C.

2017-01-01

Diet-induced obesity is associated with increased adipose tissue activated macrophages. Yet, how macrophages integrate fatty acid (FA) signals remains unclear. We previously demonstrated that Fyn deficiency (fynKO) protects against high fat diet-induced adipose tissue macrophage accumulation. Herein, we show that inflammatory markers and reactive oxygen species are not induced in fynKO bone marrow-derived macrophages exposed to the saturated FA palmitate, suggesting that Fyn regulates macrophage function in response to FA signals. Palmitate activates Fyn and re-localizes Fyn into the nucleus of RAW264.7, J774 and wild-type bone marrow-derived macrophages. Similarly, Fyn activity is increased in cells of adipose tissue stromal vascular fraction of high fat-fed control mice, with Fyn protein being located in the nucleus of these cells. We demonstrate that Fyn modulates palmitate-dependent oxidative stress in macrophages. Moreover, Fyn catalytic activity is necessary for its nuclear re-localization and downstream effects, as Fyn pharmacological inhibition abolishes palmitate-induced Fyn nuclear redistribution and palmitate-dependent increase of oxidative stress markers. Importantly, mono-or polyunsaturated FAs do not activate Fyn, and fail to re-localize Fyn to the nucleus. Together these data demonstrate that macrophages integrate nutritional FA signals via a differential activation of Fyn that distinguishes, at least partly, the effects of saturated versus unsaturated fats. PMID:29156823

Differential activation of Fyn kinase distinguishes saturated and unsaturated fats in mouse macrophages.

PubMed

Tarabra, Elena; An Lee, Ting-Wen; Zammit, Victor A; Vatish, Manu; Yamada, Eijiro; Pessin, Jeffrey E; Bastie, Claire C

2017-10-17

Diet-induced obesity is associated with increased adipose tissue activated macrophages. Yet, how macrophages integrate fatty acid (FA) signals remains unclear. We previously demonstrated that Fyn deficiency ( fynKO ) protects against high fat diet-induced adipose tissue macrophage accumulation. Herein, we show that inflammatory markers and reactive oxygen species are not induced in fynKO bone marrow-derived macrophages exposed to the saturated FA palmitate, suggesting that Fyn regulates macrophage function in response to FA signals. Palmitate activates Fyn and re-localizes Fyn into the nucleus of RAW264.7, J774 and wild-type bone marrow-derived macrophages. Similarly, Fyn activity is increased in cells of adipose tissue stromal vascular fraction of high fat-fed control mice, with Fyn protein being located in the nucleus of these cells. We demonstrate that Fyn modulates palmitate-dependent oxidative stress in macrophages. Moreover, Fyn catalytic activity is necessary for its nuclear re-localization and downstream effects, as Fyn pharmacological inhibition abolishes palmitate-induced Fyn nuclear redistribution and palmitate-dependent increase of oxidative stress markers. Importantly, mono-or polyunsaturated FAs do not activate Fyn, and fail to re-localize Fyn to the nucleus. Together these data demonstrate that macrophages integrate nutritional FA signals via a differential activation of Fyn that distinguishes, at least partly, the effects of saturated versus unsaturated fats.

Evaluation of the Leishmanicidal and Cytotoxic Potential of Essential Oils Derived From Ten Colombian Plants

PubMed Central

Sanchez-Suarez, JF; Riveros, I; Delgado, G

2013-01-01

Background The leishmanicidal and cytotoxic activity of ten essential oils obtained from ten plant specimens were evaluated. Methods Essential oils were obtained by the steam distillation of plant leaves without any prior processing. Cytotoxicity was tested on J774 macrophages and leishmanicidal activity was assessed against four species of Leishmania associated with cutaneous leishmaniasis. Results Seven essential oils exhibited activity against Leishmania parasites, five of which were toxic against J774 macrophages. Selectivity indices of >6 and 13 were calculated for the essential oils of Ocimum basilicum and Origanum vulgare, respectively. Conclusion The essential oil of Ocimum basilicum was active against promastigotes of Leishmania and innocuous to J774 macrophages at concentrations up to 1600 µg/mL and should be further investigated for leishmanicidal activity in others in vitro and in vivo experimental models. PMID:23682270

Anti-inflammatory activity of horseradish (Armoracia rusticana) root extracts in LPS-stimulated macrophages.

PubMed

Marzocco, Stefania; Calabrone, Luana; Adesso, Simona; Larocca, Marilena; Franceschelli, Silvia; Autore, Giuseppina; Martelli, Giuseppe; Rossano, Rocco

2015-12-01

Horseradish (Armoracia rusticana) is a perennial crop belonging to the Brassicaceae family. Horseradish root is used as a condiment due to its extremely pungent flavour, deriving from the high content of glucosinolates and their breakdown products such as isothiocyanates and other sulfur compounds. Horseradish also has a long history in ethnomedicine. In this study the anti-inflammatory potential of three accessions of Armoracia rusticana on lipopolysaccharide from E. coli treated J774A.1 murine macrophages was evaluated. Our results demonstrate that Armoracia rusticana reduced nitric oxide, tumor necrosis factor-α and interleukin-6 release and nitric oxide synthase and cyclooxygenase-2 expression in macrophages, acting on nuclear transcription factor NF-κB p65 activation. Moreover Armoracia rusticana reduced reactive oxygen species release and increased heme-oxygenase-1 expression, thus contributing to the cytoprotective cellular effect during inflammation.

The scavenger receptor SR-A I/II (CD204) signals via the receptor tyrosine kinase Mertk during apoptotic cell uptake by murine macrophages

PubMed Central

Todt, Jill C.; Hu, Bin; Curtis, Jeffrey L.

2008-01-01

Apoptotic cells (AC) must be cleared by macrophages (Mø) to resolve inflammation effectively. Mertk and scavenger receptor A (SR-A) are two of many receptors involved in AC clearance. As SR-A lacks enzymatic activity or evident intracellular signaling motifs, yet seems to signal in some cell types, we hypothesized that SR-A signals via Mer receptor tyrosine kinase (Mertk), which contains a multisubstrate docking site. We induced apoptosis in murine thymocytes by dexamethasone and used Western blotting and immunoprecipitation to analyze the interaction of Mertk and SR-A in the J774A.1 (J774) murine Mø cell line and in peritoneal Mø of wild-type mice and SR-A−/− mice. Phagocytosis (but not adhesion) of AC by J774 was inhibited by anti-SR-A or function-blocking SR-A ligands. In resting J774, SR-A was associated minimally with unphosphorylated (monomeric) Mertk; exposure to AC induced a time-dependent increase in association of SR-A with Mertk in a direct or indirect manner. Anti-SR-A inhibited AC-induced phosphorylation of Mertk and of phospholipase Cγ2, essential steps in AC ingestion. Relative to tissue Mø of wild-type mice, AC-induced Mertk phosphorylation was reduced and delayed in tissue Mø of SR-A−/− mice, as was in vitro AC ingestion at early time-points. Thus, during AC uptake by murine Mø, SR-A is essential for optimal phosphorylation of Mertk and subsequent signaling required for AC ingestion. These data support the Mertk/SR-A complex as a potential target to manipulate AC clearance and hence, resolution of inflammation and infections. PMID:18511575

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

Rodriguez-Acebes, Sara; CIBER de Fisiopatologia de la Obesidad y Nutricion; Cueva, Paloma de la

We addressed the ability of native, oxidized and acetylated low-density lipoproteins (nLDL, oxLDL and acLDL, respectively) and desmosterol to act as sources of sterol for the proliferation of J774A.1 macrophages. Treatment with 0.5 {mu}M lovastatin and lipoprotein-deficient serum suppressed cell proliferation. This inhibition was effectively prevented by nLDL, but only to a lesser extent by oxLDL. AcLDL, despite its ability to deliver a higher amount of cholesterol to J774 macrophages than the other LDLs, was dependent on mevalonate supply to sustain cell proliferation. Similarly, exogenous desmosterol, which is not converted into cholesterol in J774 cells, required the simultaneous addition ofmore » mevalonate to support optimal cell growth. Expression of hydroxymethyl glutaryl coenzyme A reductase mRNA was potently down-regulated by acLDL and exogenous desmosterol, but the effect was weaker with other sterol sources. We conclude that nLDL is more efficient than modified LDL in sustaining macrophage proliferation. Despite the requirement of cholesterol or desmosterol for J774 cell proliferation, excessive provision of either sterol limits mevalonate availability, thus suppressing cell proliferation.« less

30 CFR 774.9 - Information collection.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 30 Mineral Resources 3 2011-07-01 2011-07-01 false Information collection. 774.9 Section 774.9... OTHER ACTIONS BASED ON OWNERSHIP, CONTROL, AND VIOLATION INFORMATION § 774.9 Information collection. (a) The collections of information contained in part 774 have been approved by the Office of Management...

30 CFR 774.9 - Information collection.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 30 Mineral Resources 3 2012-07-01 2012-07-01 false Information collection. 774.9 Section 774.9... OTHER ACTIONS BASED ON OWNERSHIP, CONTROL, AND VIOLATION INFORMATION § 774.9 Information collection. (a) The collections of information contained in part 774 have been approved by the Office of Management...

Entrance and Survival of Brucella pinnipedialis Hooded Seal Strain in Human Macrophages and Epithelial Cells

PubMed Central

Briquemont, Benjamin; Sørensen, Karen K.; Godfroid, Jacques

2013-01-01

Marine mammal Brucella spp. have been isolated from pinnipeds (B. pinnipedialis) and cetaceans (B. ceti) from around the world. Although the zoonotic potential of marine mammal brucellae is largely unknown, reports of human disease exist. There are few studies of the mechanisms of bacterial intracellular invasion and multiplication involving the marine mammal Brucella spp. We examined the infective capacity of two genetically different B. pinnipedialis strains (reference strain; NTCT 12890 and a hooded seal isolate; B17) by measuring the ability of the bacteria to enter and replicate in cultured phagocytes and epithelial cells. Human macrophage-like cells (THP-1), two murine macrophage cell lines (RAW264.7 and J774A.1), and a human malignant epithelial cell line (HeLa S3) were challenged with bacteria in a gentamicin protection assay. Our results show that B. pinnipedialis is internalized, but is then gradually eliminated during the next 72 – 96 hours. Confocal microscopy revealed that intracellular B. pinnipedialis hooded seal strain colocalized with lysosomal compartments at 1.5 and 24 hours after infection. Intracellular presence of B. pinnipedialis hooded seal strain was verified by transmission electron microscopy. By using a cholesterol-scavenging lipid inhibitor, entrance of B. pinnipedialis hooded seal strain in human macrophages was significantly reduced by 65.8 % (± 17.3), suggesting involvement of lipid-rafts in intracellular entry. Murine macrophages invaded by B. pinnipedialis do not release nitric oxide (NO) and intracellular bacterial presence does not induce cell death. In summary, B. pinnipedialis hooded seal strain can enter human and murine macrophages, as well as human epithelial cells. Intracellular entry of B. pinnipedialis hooded seal strain involves, but seems not to be limited to, lipid-rafts in human macrophages. Brucella pinnipedialis does not multiply or survive for prolonged periods intracellulary. PMID:24376851

Nanospheres Encapsulating Anti-Leishmanial Drugs for Their Specific Macrophage Targeting, Reduced Toxicity, and Deliberate Intracellular Release

PubMed Central

Shukla, Anil Kumar; Patra, Sanjukta

2012-01-01

Abstract The current work focuses on the study of polymeric, biodegradable nanoparticles (NPs) for the encapsulation of doxorubicin and mitomycin C (anti-leishmanial drugs), and their efficient delivery to macrophages, the parasite's home. The biodegradable polymer methoxypoly-(ethylene glycol)-b-poly (lactic acid) (MPEG-PLA) was used to prepare polymeric NPs encapsulating doxorubicin and mitomycin C. The morphology, mean diameter, and surface area of spherical NPs were determined by transmission electron microscopy (TEM), field emission scanning electron microscopy (FESEM), and BET surface area analysis. X-ray diffraction was performed to validate drug encapsulation. An in vitro release profile of the drugs suggested a fairly slow release. These polymeric NPs were efficiently capable of releasing drug inside macrophages at a slower pace than the free drug, which was monitored by epi-fluorescence microscopy. Encapsulation of doxorubicin and mitomycin C into NPs also decreases cellular toxicity in mouse macrophages (J774.1A). PMID:22925019

Antibacterial Activity of Ciprofloxacin-Encapsulated Cockle Shells Calcium Carbonate (Aragonite) Nanoparticles and Its Biocompatability in Macrophage J774A.1

PubMed Central

Isa, Tijani; Zakaria, Zuki Abu Bakar; Rukayadi, Yaya; Mohd Hezmee, Mohd Noor; Jaji, Alhaji Zubair; Imam, Mustapha Umar; Hammadi, Nahidah Ibrahim; Mahmood, Saffanah Khuder

2016-01-01

The use of nanoparticle delivery systems to enhance intracellular penetration of antibiotics and their retention time is becoming popular. The challenge, however, is that the interaction of nanoparticles with biological systems at the cellular level must be established prior to biomedical applications. Ciprofloxacin–cockle shells-derived calcium carbonate (aragonite) nanoparticles (C-CSCCAN) were developed and characterized. Antibacterial activity was determined using a modified disc diffusion protocol on Salmonella Typhimurium (S. Typhimurium). Biocompatibilittes with macrophage were evaluated using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 5-Bromo-2′-deoxyuridine (BrdU) assays. Transcriptional regulation of interleukin 1 beta (IL-1β) was determined using reverse transcriptase-polymerase chain reaction (RT-PCR). C-CSCCAN were spherical in shape, with particle sizes ranging from 11.93 to 22.12 nm. Encapsulation efficiency (EE) and loading content (LC) were 99.5% and 5.9%, respectively, with negative ζ potential. X-ray diffraction patterns revealed strong crystallizations and purity in the formulations. The mean diameter of inhibition zone was 18.6 ± 0.5 mm, which was better than ciprofloxacin alone (11.7 ± 0.9 mm). Study of biocompatability established the cytocompatability of the delivery system without upregulation of IL-1β. The results indicated that ciprofloxacin–nanoparticles enhanced the antibacterial efficacy of the antibiotic, and could act as a suitable delivery system against intracellular infections. PMID:27213349

Antibacterial Activity of Ciprofloxacin-Encapsulated Cockle Shells Calcium Carbonate (Aragonite) Nanoparticles and Its Biocompatability in Macrophage J774A.1.

PubMed

Isa, Tijani; Zakaria, Zuki Abu Bakar; Rukayadi, Yaya; Mohd Hezmee, Mohd Noor; Jaji, Alhaji Zubair; Imam, Mustapha Umar; Hammadi, Nahidah Ibrahim; Mahmood, Saffanah Khuder

2016-05-19

The use of nanoparticle delivery systems to enhance intracellular penetration of antibiotics and their retention time is becoming popular. The challenge, however, is that the interaction of nanoparticles with biological systems at the cellular level must be established prior to biomedical applications. Ciprofloxacin-cockle shells-derived calcium carbonate (aragonite) nanoparticles (C-CSCCAN) were developed and characterized. Antibacterial activity was determined using a modified disc diffusion protocol on Salmonella Typhimurium (S. Typhimurium). Biocompatibilittes with macrophage were evaluated using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 5-Bromo-2'-deoxyuridine (BrdU) assays. Transcriptional regulation of interleukin 1 beta (IL-1β) was determined using reverse transcriptase-polymerase chain reaction (RT-PCR). C-CSCCAN were spherical in shape, with particle sizes ranging from 11.93 to 22.12 nm. Encapsulation efficiency (EE) and loading content (LC) were 99.5% and 5.9%, respectively, with negative ζ potential. X-ray diffraction patterns revealed strong crystallizations and purity in the formulations. The mean diameter of inhibition zone was 18.6 ± 0.5 mm, which was better than ciprofloxacin alone (11.7 ± 0.9 mm). Study of biocompatability established the cytocompatability of the delivery system without upregulation of IL-1β. The results indicated that ciprofloxacin-nanoparticles enhanced the antibacterial efficacy of the antibiotic, and could act as a suitable delivery system against intracellular infections.

Anti-leishmanial and toxicity activities of some selected Iranian medicinal plants.

PubMed

Kheiri Manjili, Hamidreza; Jafari, Hamidreza; Ramazani, Ali; Davoudi, Noushin

2012-11-01

Leishmaniasis is caused by protozoan parasites belonging to the genus Leishmania. Cutaneous leishmaniasis is the most common form of leishmaniasis in Iran. As there is not any vaccine for leishmaniasis, treatment is important to prevent the spreading of parasites. There is, therefore, a need to develop newer drugs from different sources. The aim of this study was to assess anti-leishmanial activity of the ethanolic extracts of 17 different medicinal plants against Leishmania major promastigotes and macrophage cell line J774. The selection of the hereby studied 17 plants was based on the existing information on their local ethnobotanic history. Plants were dried, powdered, and macerated in a hydroalcoholic solution. Resulting extracts have been assessed for in vitro anti-leishmanial and brine shrimp toxicity activities. Four plants, Caesalpinia gilliesii, Satureia hortensis, Carum copticum heirm, and Thymus migricus, displayed high anti-leishmanial activity (IC50, 9.76 ± 1.27, 15.625 ± 3.76, 15.625 ± 5.46, and 31.25 ± 15.44 μM, respectively) and were toxic against the J774 macrophage cell line at higher concentrations than those needed to inhibit the parasite cell growth (IC50, 45.13 ± 3.17, 100.44 ± 17.48, 43.76 ± 0.78, and 39.67 ± 3.29 μM, respectively). Glucantime as positive control inhibited the growth of L. major promastigotes with IC50 = 254 μg/ml on promastigotes (1 × 10(6)/100 μ/well) of a log phase culture, without affecting the growth of J774 macrophages. These data revealed that C. gilliesii, S. hortensis, C. copticum heirm, and T. migricus extracts contain active compounds, which could serve as alternative agents in the control of cutaneous leishmaniasis. The activity of these herbs against L. major promastigotes and macrophage cell line J774 was reported for the first time in our study.

ALV-J strain SCAU-HN06 induces innate immune responses in chicken primary monocyte-derived macrophages.

PubMed

Feng, Min; Dai, Manman; Cao, Weisheng; Tan, Yan; Li, Zhenhui; Shi, Meiqing; Zhang, Xiquan

2017-01-01

Avian leucosis virus subgroup J (ALV-J) can cause lifelong infection and can escape from the host immune defenses in chickens. Since macrophages act as the important defense line against invading pathogens in host innate immunity, we investigated the function and innate immune responses of chicken primary monocyte-derived macrophages (MDM) after ALV-J infection in this study. Our results indicated that ALV-J was stably maintained in MDM cells but that the viral growth rate was significantly lower than that in DF-1 cells. We also found that ALV-J infection significantly increased nitric oxide (NO) production, but had no effect on MDM phagocytic capacity. Interestingly, infection with ALV-J rapidly promoted the expression levels of Myxovirus resistance 1 (Mx) (3 h, 6 h), ISG12 (6 h), and interleukin-1β (IL-1β) (3 h, 12 h) at an early infection stage, whereas it sharply decreased the expression of Mx (24 h, 36 h), ISG12 (36 h), and made little change on IL-1β (24 h, 36 h) production at a late infection stage in MDM cells. Moreover, the protein levels of interferon-β (IFN-β) and interleukin-6 (IL-6) had sharply increased in infected MDM cells from 3 to 36 h post infection (hpi) of ALV-J. And, the protein level of interleukin-10 (IL-10) was dramatically decreased at 36 hpi in MDM cells infected with ALV-J. These results demonstrate that ALV-J can induce host innate immune responses and we hypothesize that macrophages play an important role in host innate immune attack and ALV-J immune escape. © The Author 2016. Published by Oxford University Press on behalf of Poultry Science Association.

Toxicological Profiling of Metal Oxide Nanoparticles in Liver Context Reveals Pyroptosis in Kupffer Cells and Macrophages versus Apoptosis in Hepatocytes.

PubMed

Mirshafiee, Vahid; Sun, Bingbing; Chang, Chong Hyun; Liao, Yu-Pei; Jiang, Wen; Jiang, Jinhong; Liu, Xiangsheng; Wang, Xiang; Xia, Tian; Nel, André E

2018-04-24

The liver and the mononuclear phagocyte system are a frequent target for engineered nanomaterials, either as a result of particle uptake and spread from primary exposure sites or systemic administration of therapeutic and imaging nanoparticles. In this study, we performed a comparative analysis of the toxicological impact of 29 metal oxide nanoparticles (NPs), some commonly used in consumer products, in transformed or primary Kupffer cells (KCs) and hepatocytes. We not only observed differences between KCs and hepatocytes, but also differences in the toxicological profiles of transition-metal oxides (TMOs, e. g., Co 3 O 4 ) versus rare-earth oxide (REO) NPs ( e. g., Gd 2 O 3 ). While pro-oxidative TMOs induced the activation of caspases 3 and 7, resulting in apoptotic cell death in both cell types, REOs induced lysosomal damage, NLRP3 inflammasome activation, caspase 1 activation, and pyroptosis in KCs. Pyroptosis was accompanied by cell swelling, membrane blebbing, IL-1β release, and increased membrane permeability, which could be reversed by knockdown of the pore forming protein, gasdermin D. Though similar features were not seen in hepatocytes, the investigation of the cytotoxic effects of REO NPs could also be seen to affect macrophage cell lines such as J774A.1 and RAW 264.7 cells as well as bone marrow-derived macrophages. These phagocytic cell types also demonstrated features of pyroptosis and increased IL-1β production. Collectively, these findings demonstrate important mechanistic considerations that can be used for safety evaluation of metal oxides, including commercial products that are developed from these materials.

7 CFR 774.9 - Environmental requirements.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 7 Agriculture 7 2013-01-01 2013-01-01 false Environmental requirements. 774.9 Section 774.9 Agriculture Regulations of the Department of Agriculture (Continued) FARM SERVICE AGENCY, DEPARTMENT OF AGRICULTURE SPECIAL PROGRAMS EMERGENCY LOAN FOR SEED PRODUCERS PROGRAM § 774.9 Environmental requirements. The...

7 CFR 774.9 - Environmental requirements.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 7 2011-01-01 2011-01-01 false Environmental requirements. 774.9 Section 774.9 Agriculture Regulations of the Department of Agriculture (Continued) FARM SERVICE AGENCY, DEPARTMENT OF AGRICULTURE SPECIAL PROGRAMS EMERGENCY LOAN FOR SEED PRODUCERS PROGRAM § 774.9 Environmental requirements. The...

7 CFR 774.9 - Environmental requirements.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 7 2010-01-01 2010-01-01 false Environmental requirements. 774.9 Section 774.9 Agriculture Regulations of the Department of Agriculture (Continued) FARM SERVICE AGENCY, DEPARTMENT OF AGRICULTURE SPECIAL PROGRAMS EMERGENCY LOAN FOR SEED PRODUCERS PROGRAM § 774.9 Environmental requirements. The...

7 CFR 774.9 - Environmental requirements.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 7 Agriculture 7 2014-01-01 2014-01-01 false Environmental requirements. 774.9 Section 774.9 Agriculture Regulations of the Department of Agriculture (Continued) FARM SERVICE AGENCY, DEPARTMENT OF AGRICULTURE SPECIAL PROGRAMS EMERGENCY LOAN FOR SEED PRODUCERS PROGRAM § 774.9 Environmental requirements. The...

7 CFR 774.9 - Environmental requirements.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 7 Agriculture 7 2012-01-01 2012-01-01 false Environmental requirements. 774.9 Section 774.9 Agriculture Regulations of the Department of Agriculture (Continued) FARM SERVICE AGENCY, DEPARTMENT OF AGRICULTURE SPECIAL PROGRAMS EMERGENCY LOAN FOR SEED PRODUCERS PROGRAM § 774.9 Environmental requirements. The...

7 CFR 774.19 - Processing applications.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 7 2011-01-01 2011-01-01 false Processing applications. 774.19 Section 774.19 Agriculture Regulations of the Department of Agriculture (Continued) FARM SERVICE AGENCY, DEPARTMENT OF AGRICULTURE SPECIAL PROGRAMS EMERGENCY LOAN FOR SEED PRODUCERS PROGRAM § 774.19 Processing applications...

7 CFR 774.19 - Processing applications.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 7 Agriculture 7 2012-01-01 2012-01-01 false Processing applications. 774.19 Section 774.19 Agriculture Regulations of the Department of Agriculture (Continued) FARM SERVICE AGENCY, DEPARTMENT OF AGRICULTURE SPECIAL PROGRAMS EMERGENCY LOAN FOR SEED PRODUCERS PROGRAM § 774.19 Processing applications...

7 CFR 774.19 - Processing applications.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 7 2010-01-01 2010-01-01 false Processing applications. 774.19 Section 774.19 Agriculture Regulations of the Department of Agriculture (Continued) FARM SERVICE AGENCY, DEPARTMENT OF AGRICULTURE SPECIAL PROGRAMS EMERGENCY LOAN FOR SEED PRODUCERS PROGRAM § 774.19 Processing applications...

7 CFR 774.19 - Processing applications.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 7 Agriculture 7 2014-01-01 2014-01-01 false Processing applications. 774.19 Section 774.19 Agriculture Regulations of the Department of Agriculture (Continued) FARM SERVICE AGENCY, DEPARTMENT OF AGRICULTURE SPECIAL PROGRAMS EMERGENCY LOAN FOR SEED PRODUCERS PROGRAM § 774.19 Processing applications...

7 CFR 774.19 - Processing applications.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 7 Agriculture 7 2013-01-01 2013-01-01 false Processing applications. 774.19 Section 774.19 Agriculture Regulations of the Department of Agriculture (Continued) FARM SERVICE AGENCY, DEPARTMENT OF AGRICULTURE SPECIAL PROGRAMS EMERGENCY LOAN FOR SEED PRODUCERS PROGRAM § 774.19 Processing applications...

7 CFR 774.20 - Funding applications.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 7 Agriculture 7 2013-01-01 2013-01-01 false Funding applications. 774.20 Section 774.20 Agriculture Regulations of the Department of Agriculture (Continued) FARM SERVICE AGENCY, DEPARTMENT OF AGRICULTURE SPECIAL PROGRAMS EMERGENCY LOAN FOR SEED PRODUCERS PROGRAM § 774.20 Funding applications. Loan...

7 CFR 774.20 - Funding applications.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 7 Agriculture 7 2012-01-01 2012-01-01 false Funding applications. 774.20 Section 774.20 Agriculture Regulations of the Department of Agriculture (Continued) FARM SERVICE AGENCY, DEPARTMENT OF AGRICULTURE SPECIAL PROGRAMS EMERGENCY LOAN FOR SEED PRODUCERS PROGRAM § 774.20 Funding applications. Loan...

7 CFR 774.20 - Funding applications.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 7 Agriculture 7 2014-01-01 2014-01-01 false Funding applications. 774.20 Section 774.20 Agriculture Regulations of the Department of Agriculture (Continued) FARM SERVICE AGENCY, DEPARTMENT OF AGRICULTURE SPECIAL PROGRAMS EMERGENCY LOAN FOR SEED PRODUCERS PROGRAM § 774.20 Funding applications. Loan...

7 CFR 774.20 - Funding applications.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 7 2011-01-01 2011-01-01 false Funding applications. 774.20 Section 774.20 Agriculture Regulations of the Department of Agriculture (Continued) FARM SERVICE AGENCY, DEPARTMENT OF AGRICULTURE SPECIAL PROGRAMS EMERGENCY LOAN FOR SEED PRODUCERS PROGRAM § 774.20 Funding applications. Loan...

7 CFR 774.20 - Funding applications.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 7 2010-01-01 2010-01-01 false Funding applications. 774.20 Section 774.20 Agriculture Regulations of the Department of Agriculture (Continued) FARM SERVICE AGENCY, DEPARTMENT OF AGRICULTURE SPECIAL PROGRAMS EMERGENCY LOAN FOR SEED PRODUCERS PROGRAM § 774.20 Funding applications. Loan...

Fibronectin induces macrophage migration through a SFK-FAK/CSF-1R pathway.

PubMed

Digiacomo, Graziana; Tusa, Ignazia; Bacci, Marina; Cipolleschi, Maria Grazia; Dello Sbarba, Persio; Rovida, Elisabetta

2017-07-04

Integrins, following binding to proteins of the extracellular matrix (ECM) including collagen, laminin and fibronectin (FN), are able to transduce molecular signals inside the cells and to regulate several biological functions such as migration, proliferation and differentiation. Besides activation of adaptor molecules and kinases, integrins transactivate Receptor Tyrosine Kinases (RTK). In particular, adhesion to the ECM may promote RTK activation in the absence of growth factors. The Colony-Stimulating Factor-1 Receptor (CSF-1R) is a RTK that supports the survival, proliferation, and motility of monocytes/macrophages, which are essential components of innate immunity and cancer development. Macrophage interaction with FN is recognized as an important aspect of host defense and wound repair. The aim of the present study was to investigate on a possible cross-talk between FN-elicited signals and CSF-1R in macrophages. FN induced migration in BAC1.2F5 and J774 murine macrophage cell lines and in human primary macrophages. Adhesion to FN determined phosphorylation of the Focal Adhesion Kinase (FAK) and Src Family Kinases (SFK) and activation of the SFK/FAK complex, as witnessed by paxillin phosphorylation. SFK activity was necessary for FAK activation and macrophage migration. Moreover, FN-induced migration was dependent on FAK in either murine macrophage cell lines or human primary macrophages. FN also induced FAK-dependent/ligand-independent CSF-1R phosphorylation, as well as the interaction between CSF-1R and β1. CSF-1R activity was necessary for FN-induced macrophage migration. Indeed, genetic or pharmacological inhibition of CSF-1R prevented FN-induced macrophage migration. Our results identified a new SFK-FAK/CSF-1R signaling pathway that mediates FN-induced migration of macrophages.

7 CFR 774.17 - Loan application.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 7 2010-01-01 2010-01-01 false Loan application. 774.17 Section 774.17 Agriculture Regulations of the Department of Agriculture (Continued) FARM SERVICE AGENCY, DEPARTMENT OF AGRICULTURE SPECIAL PROGRAMS EMERGENCY LOAN FOR SEED PRODUCERS PROGRAM § 774.17 Loan application. A complete...

Evidence for apoptosis of murine macrophages by Actinobacillus actinomycetemcomitans infection.

PubMed

Kato, S; Muro, M; Akifusa, S; Hanada, N; Semba, I; Fujii, T; Kowashi, Y; Nishihara, T

1995-10-01

The gram-negative bacterium Actinobacillus actinomycetemcomitans is considered an important etiological agent in periodontal diseases. In this study, we show that A. actinomycetemcomitans strains are cytotoxic for the murine macrophage cell line J774.1. On the other hand, Porphyromonas gingivalis strains, other gram-negative oral species implicated in adult periodontitis, showed weak cytotoxic effects. For this to occur, A. actinomycetemcomitans had to gain entry into the macrophages, since cytotoxicity was prevented by cytochalasin D. We demonstrate that cell death induced by A. actinomycetemcomitans Y4 occurs through apoptosis, as shown by changes in nuclear morphology, an increase in the proportion of fragmented DNA, and the typical ladder pattern of DNA fragmentation indicative of apoptosis. We further sought to determine whether the cytotoxicity induced by A. actinomycetemcomitans Y4 could be modulated by the protein kinase inhibitors H7 and HA1004. Apoptotic cell death induced by A. actinomycetemcomitans Y4 was suppressed by H7 but was relatively unaffected by HA1004. These findings suggest that the signals of protein kinases may regulate apoptosis induced by A. actinomycetemcomitans Y4. The ability of A. actinomycetemcomitans to promote the apoptosis of macrophages may be important for the initiation of infection and the development of periodontal diseases.

Evidence for apoptosis of murine macrophages by Actinobacillus actinomycetemcomitans infection.

PubMed Central

Kato, S; Muro, M; Akifusa, S; Hanada, N; Semba, I; Fujii, T; Kowashi, Y; Nishihara, T

1995-01-01

The gram-negative bacterium Actinobacillus actinomycetemcomitans is considered an important etiological agent in periodontal diseases. In this study, we show that A. actinomycetemcomitans strains are cytotoxic for the murine macrophage cell line J774.1. On the other hand, Porphyromonas gingivalis strains, other gram-negative oral species implicated in adult periodontitis, showed weak cytotoxic effects. For this to occur, A. actinomycetemcomitans had to gain entry into the macrophages, since cytotoxicity was prevented by cytochalasin D. We demonstrate that cell death induced by A. actinomycetemcomitans Y4 occurs through apoptosis, as shown by changes in nuclear morphology, an increase in the proportion of fragmented DNA, and the typical ladder pattern of DNA fragmentation indicative of apoptosis. We further sought to determine whether the cytotoxicity induced by A. actinomycetemcomitans Y4 could be modulated by the protein kinase inhibitors H7 and HA1004. Apoptotic cell death induced by A. actinomycetemcomitans Y4 was suppressed by H7 but was relatively unaffected by HA1004. These findings suggest that the signals of protein kinases may regulate apoptosis induced by A. actinomycetemcomitans Y4. The ability of A. actinomycetemcomitans to promote the apoptosis of macrophages may be important for the initiation of infection and the development of periodontal diseases. PMID:7558299

Modulating macrophage polarization with divalent cations in nanostructured titanium implant surfaces

NASA Astrophysics Data System (ADS)

Lee, Chung-Ho; Kim, Youn-Jeong; Jang, Je-Hee; Park, Jin-Woo

2016-02-01

Nanoscale topographical modification and surface chemistry alteration using bioactive ions are centrally important processes in the current design of the surface of titanium (Ti) bone implants with enhanced bone healing capacity. Macrophages play a central role in the early tissue healing stage and their activity in response to the implant surface is known to affect the subsequent healing outcome. Thus, the positive modulation of macrophage phenotype polarization (i.e. towards the regenerative M2 rather than the inflammatory M1 phenotype) with a modified surface is essential for the osteogenesis funtion of Ti bone implants. However, relatively few advances have been made in terms of modulating the macrophage-centered early healing capacity in the surface design of Ti bone implants for the two important surface properties of nanotopography and and bioactive ion chemistry. We investigated whether surface bioactive ion modification exerts a definite beneficial effect on inducing regenerative M2 macrophage polarization when combined with the surface nanotopography of Ti. Our results indicate that nanoscale topographical modification and surface bioactive ion chemistry can positively modulate the macrophage phenotype in a Ti implant surface. To the best of our knowledge, this is the first demonstration that chemical surface modification using divalent cations (Ca and Sr) dramatically induces the regenerative M2 macrophage phenotype of J774.A1 cells in nanostructured Ti surfaces. In this study, divalent cation chemistry regulated the cell shape of adherent macrophages and markedly up-regulated M2 macrophage phenotype expression when combined with the nanostructured Ti surface. These results provide insight into the surface engineering of future Ti bone implants that are harmonized between the macrophage-governed early wound healing process and subsequent mesenchymal stem cell-centered osteogenesis function.

45 CFR 77.4 - Remedial actions.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 45 Public Welfare 1 2011-10-01 2011-10-01 false Remedial actions. 77.4 Section 77.4 Public Welfare DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL ADMINISTRATION REMEDIAL ACTIONS APPLICABLE TO LETTER OF CREDIT ADMINISTRATION § 77.4 Remedial actions. If, after the conclusion of the procedures set forth in...

45 CFR 77.4 - Remedial actions.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 45 Public Welfare 1 2010-10-01 2010-10-01 false Remedial actions. 77.4 Section 77.4 Public Welfare DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL ADMINISTRATION REMEDIAL ACTIONS APPLICABLE TO LETTER OF CREDIT ADMINISTRATION § 77.4 Remedial actions. If, after the conclusion of the procedures set forth in...

7 CFR 774.22 - Loan closing.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 7 2010-01-01 2010-01-01 false Loan closing. 774.22 Section 774.22 Agriculture... SPECIAL PROGRAMS EMERGENCY LOAN FOR SEED PRODUCERS PROGRAM § 774.22 Loan closing. (a) Conditions. The... approval prior to closing. (b) Loan instruments and legal documents. The applicant will execute all loan...

Macrosialin, a macrophage-restricted membrane sialoprotein differentially glycosylated in response to inflammatory stimuli [published erratum appears in J Exp Med 1992 Jan 1;175(1):309

PubMed Central

1991-01-01

Rat monoclonal antibody FA/11 has been used to identify macrosialin, a sialoglycoprotein confined to murine mononuclear phagocytes and related cells. Originally identified as a macrophage-associated glycoprotein predominantly localized in intracellular membranes (Smith, M.J., and G.L.E. Koch. 1987. J. Cell Sci. 87:113), the antigen is widely expressed on tissue macrophages, including those in lymphoid areas, and is expressed at low levels on isolated dendritic cells. Immuno- adsorption experiments reported here show that macrosialin is identical to the major 87-115-kD sialoglycoprotein previously identified by lectin blotting in exudate but not resident peritoneal macrophages (Rabinowitz, S., and S. Gordon. 1989. J. Cell Sci. 93:623). Resident peritoneal macrophages express low levels of macrosialin antigen in a glycoform that does not bind 125I wheat germ agglutinin or 125I peanut agglutinin; inflammatory stimuli upregulate expression of this antigen (up to 17-fold), in an alternative glycoform that is detected by these lectins. Pulse-chase experiments reveal a 44-kD core peptide that initially bears high-mannose chains (giving Mr 66 kD) and is subsequently processed to a mature protein of Mr 87-104 kD. Each glycoform contains N-linked glycan, as well as O-linked sugar structures that show alternative processing. Poly-N-acetyllactosamine structures are detected in the exudate cell glycoform only. This new marker for mononuclear phagocytes illustrates two strategies by which macrophages remodel their membranes in response to inflammatory stimuli. Its predominantly intracellular location and restricted cell distribution suggest a possible role in membrane fusion or antigen processing. PMID:1919437

Induction of different activated phenotypes of mouse peritoneal macrophages grown in different tissue culture media.

PubMed

Kawakami, Tomoya; Koike, Atsushi; Amano, Fumio

2017-08-01

The role of activated macrophages in the host defense against pathogens or tumor cells has been investigated extensively. Many researchers have been using various culture media in in vitro experiments using macrophages. We previously reported that J774.1/JA-4 macrophage-like cells showed great differences in their activated macrophage phenotypes, such as production of reactive oxygen, nitric oxide (NO) or cytokines depending on the culture medium used, either F-12 (Ham's F-12 nutrient mixture) or Dulbecco modified Eagle's medium (DMEM). To examine whether a difference in the culture medium would influence the functions of primary macrophages, we used BALB/c mouse peritoneal macrophages in this study. Among the activated macrophage phenotypes, the expression of inducible NO synthase in LPS- and/or IFN-γ-treated peritoneal macrophages showed the most remarkable differences between F-12 and DMEM; i.e., NO production by LPS- and/or IFN-γ-treated cells was far lower in DMEM than in F-12. Similar results were obtained with C57BL mouse peritoneal macrophages. Besides, dilution of F-12 medium with saline resulted in a slight decrease in NO production, whereas that of DMEM with saline resulted in a significant increase, suggesting the possibility that DMEM contained some inhibitory factor(s) for NO production. However, such a difference in NO production was not observed when macrophage-like cell lines were examined. These results suggest that phenotypes of primary macrophages could be changed significantly with respect to host inflammatory responses by the surrounding environment including nutritional factors and that these altered macrophage phenotypes might influence the biological host defense.

Use of Microsphere Technology for Targeted Delivery of Rifampin to Mycobacterium tuberculosis-Infected Macrophages

PubMed Central

Barrow, Esther L. W.; Winchester, Gary A.; Staas, Jay K.; Quenelle, Debra C.; Barrow, William W.

1998-01-01

Microsphere technology was used to develop formulations of rifampin for targeted delivery to host macrophages. These formulations were prepared by using biocompatible polymeric excipients of lactide and glycolide copolymers. Release characteristics were examined in vitro and also in two monocytic cell lines, the murine J774 and the human Mono Mac 6 cell lines. Bioassay assessment of cell culture supernatants from monocyte cell lines showed release of bioactive rifampin during a 7-day experimental period. Treatment of Mycobacterium tuberculosis H37Rv-infected monocyte cell lines with rifampin-loaded microspheres resulted in a significant decrease in numbers of CFU at 7 days following initial infection, even though only 8% of the microsphere-loaded rifampin was released. The levels of rifampin released from microsphere formulations within monocytes were more effective at reducing M. tuberculosis intracellular growth than equivalent doses of rifampin given as a free drug. These results demonstrate that rifampin-loaded microspheres can be formulated for effective sustained and targeted delivery to host macrophages. PMID:9756777

7 CFR 774.23 - Loan servicing.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 7 2010-01-01 2010-01-01 false Loan servicing. 774.23 Section 774.23 Agriculture... SPECIAL PROGRAMS EMERGENCY LOAN FOR SEED PRODUCERS PROGRAM § 774.23 Loan servicing. Loans will be serviced as a Non-program loan in accordance with 7 CFR part 766. If the loan is not repaid as agreed and...

Digital holographic microscopy as a technique to monitor macrophages infected by leishmania

NASA Astrophysics Data System (ADS)

Mendoza-Rodríguez, E.; Organista-Castelblanco, C.; Camacho, M.; Monroy-Ramírez, F.

2017-06-01

The Digital Holographic Microscopy in Transmission technique (DHM) is considered a useful tool in the noninvasive quantifying of transparent biological objects like living cells. In this work, we propose this technique to study and to monitor control macrophages infected by Leishmania (mouse lineJ774.A1). When the promastigotes enter in contact with healthy macrophages, they got phagocytosed and latterly confined in the formed parasitophorous vacuole. These processes change the morphology and density of the host macrophage. Both parameters can be measured in a label-free analysis of cells with the aid of the DHM technique. Our technique begins with the optical record of the holograms using a modified Mach-Zehnder interferometer and the reconstruction of the complex optical field transmitted by macrophages. In the latter point, we employ the angular spectrum algorithm. With the complex optical field reconstruction, we compute the field amplitude and the phase difference maps, which leads to describe one morphological characterization for the samples. Using phase difference maps is possible to measure internal variations for the integral refractive index, estimating the infection level of macrophages. Through the changes in the integral refractive index, it is also possible to describe and quantify in two different states the evolution of the infection. With these results some parameters of cells have been quantified, making the DHM technique a viable tool for diagnosis of biological samples under the presence of some pathogen.

Role of urease in megasome formation and Helicobacter pylori survival in macrophages

PubMed Central

Schwartz, Justin T.; Allen, Lee-Ann H.

2007-01-01

Previous studies have demonstrated that Helicobacter pylori (Hp) delays its entry into macrophages and persists inside megasomes, which are poorly acidified and accumulate early endosome autoantigen 1. Herein, we explored the role of Hp urease in bacterial survival in murine peritoneal macrophages and J774 cells. Plasmid-free mutagenesis was used to replace ureA and ureB with cat in Hp Strains 11637 and 11916. ureAB null Hp lacked detectable urease activity and did not express UreA or UreB as judged by immunoblotting. Deletion of ureAB had no effect on Hp binding to macrophages or the rate or extent of phagocytosis. However, intracellular survival of mutant organisms was impaired significantly. Immunofluorescence microscopy demonstrated that (in contrast to parental organisms) mutant Hp resided in single phagosomes, which were acidic and accumulated the lysosome marker lysosome-associated membrane protein-1 but not early endosome autoantigen 1. A similar phenotype was observed for spontaneous urease mutants derived from Hp Strain 60190. Treatment of macrophages with bafilomycin A1, NH4Cl, or chloroquine prevented acidification of phagosomes containing mutant Hp. However, only ammonium chloride enhanced bacterial viability significantly. Rescue of ureAB null organisms was also achieved by surface adsorption of active urease. Altogether, our data indicate a role for urease and urease-derived ammonia in megasome formation and Hp survival. PMID:16543403

Reduced expression of IL-12 p35 by SJL/J macrophages responding to Theiler's virus infection is associated with constitutive activation of IRF-3

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

Dahlberg, Angela; Auble, Mark R.; Petro, Thomas M.

2006-09-30

Macrophages responding to viral infections may contribute to autoimmune demyelinating diseases (ADD). Macrophages from ADD-susceptible SJL/J mice responding to Theiler's Virus (TMEV) infection, the TLR7 agonist loxoribine, or the TLR4 agonist-LPS expressed less IL-12 p35 but more IL-12/23 p40 and IFN-{beta} than macrophages from ADD-resistant B10.S mice. While expression of IRF-1 and -7 was similar between B10.S and SJL/J TMEV-infected macrophages, SJL/J but not B10.S macrophages exhibited constitutively active IRF-3. In contrast to overexpressed IRF-1, IRF-5, and IRF-7, which stimulated p35 promoter reporter activity, overexpressed IRF-3 repressed p35 promoter activity in response to TMEV infection, loxoribine, IFN-{gamma}/LPS, but not IFN-{gamma}more » alone. IRF-3 lessened but did not eliminate IRF-1-stimulated p35 promoter activity. Repression by IRF-3 required bp -172 to -122 of the p35 promoter. The data suggest that pre-activated IRF-3 is a major factor in the differences in IL-12 production between B10.S and SJL/J macrophages responding to TMEV.« less

Rho is Required for the Initiation of Calcium Signaling and Phagocytosis by Fcγ Receptors in Macrophages

PubMed Central

Hackam, David J.; Rotstein, Ori D.; Schreiber, Alan; Zhang, Wei-jian; Grinstein, Sergio

1997-01-01

Phagocytosis of bacteria by macrophages and neutrophils is an essential component of host defense against infection. The mechanism whereby the interaction of opsonized particles with Fcγ receptors triggers the engulfment of opsonized particles remains incompletely understood, although activation of tyrosine kinases has been recognized as an early step. Recent studies in other systems have demonstrated that tyrosine kinases can in turn signal the activation of small GTPases of the ras superfamily. We therefore investigated the possible role of Rho in Fc receptor–mediated phagocytosis. To this end we microinjected J774 macrophages with C3 exotoxin from Clostridium botulinum, which ADP-ribosylates and inactivates Rho. C3 exotoxin induced the retraction of filopodia, the disappearance of focal complexes, and a global decrease in the F-actin content of J774 cells. In addition, these cells exhibited increased spreading and the formation of vacuolar structures. Importantly, inactivation of Rho resulted in the complete abrogation of phagocytosis. Inhibition of Fcγ receptor–mediated phagocytosis by C3 exotoxin was confirmed in COS cells, which become phagocytic upon transfection of the FcγRIIA receptor. Rho was found to be essential for the accumulation of phosphotyrosine and of F-actin around phagocytic cups and for Fcγ receptor–mediated Ca2+ signaling. The clustering of receptors in response to opsonin, an essential step in Fcγ-induced signaling, was the earliest event shown to be inhibited by C3 exotoxin. The effect of the toxin was specific, since clustering and internalization of transferrin receptors were unaffected by microinjection of C3. These data identify a role for small GTPases in Fcγ receptor–mediated phagocytosis by leukocytes. PMID:9294149

48 CFR 731.774 - Overseas recruitment incentive.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 48 Federal Acquisition Regulations System 5 2010-10-01 2010-10-01 false Overseas recruitment incentive. 731.774 Section 731.774 Federal Acquisition Regulations System AGENCY FOR INTERNATIONAL... Organizations 731.774 Overseas recruitment incentive. USAID's policies regarding overseas recruitment incentives...

Macrophage activation by heparanase is mediated by TLR-2 and TLR-4 and associates with plaque progression.

PubMed

Blich, Miry; Golan, Amnon; Arvatz, Gil; Sebbag, Anat; Shafat, Itay; Sabo, Edmond; Cohen-Kaplan, Victoria; Petcherski, Sirouch; Avniel-Polak, Shani; Eitan, Amnon; Hammerman, Haim; Aronson, Doron; Axelman, Elena; Ilan, Neta; Nussbaum, Gabriel; Vlodavsky, Israel

2013-02-01

Factors and mechanisms that activate macrophages in atherosclerotic plaques are incompletely understood. We examined the capacity of heparanase to activate macrophages. Highly purified heparanase was added to mouse peritoneal macrophages and macrophage-like J774 cells, and the levels of tumor necrosis factor-α, matrix metalloproteinase-9, interlukin-1, and monocyte chemotactic protein-1 were evaluated by ELISA. Gene expression was determined by RT-PCR. Cells collected from Toll-like receptor-2 and Toll-like receptor-4 knockout mice were evaluated similarly. Heparanase levels in the plasma of patients with acute myocardial infarction, stable angina, and healthy subjects were determined by ELISA. Immunohistochemistry was applied to detect the expression of heparanase in control specimens and specimens of patients with stable angina or acute myocardial infarction. Addition or overexpression of heparanase variants resulted in marked increase in tumor necrosis factor-α, matrix metalloproteinase-9, interlukin-1, and monocyte chemotactic protein-1 levels. Mouse peritoneal macrophages harvested from Toll-like receptor-2 or Toll-like receptor-4 knockout mice were not activated by heparanase. Plasma heparanase level was higher in patients with acute myocardial infarction, compared with patients with stable angina and healthy subjects. Pathologic coronary specimens obtained from vulnerable plaques showed increased heparanase staining compared with specimens of stable plaque and controls. Heparanase activates macrophages, resulting in marked induction of cytokine expression associated with plaque progression toward vulnerability.

Phagocytosis of Advanced Glycation End Products (AGEs) in Macrophages Induces Cell Apoptosis.

PubMed

Gao, Yuan; Wake, Hidenori; Morioka, Yuta; Liu, Keyue; Teshigawara, Kiyoshi; Shibuya, Megumi; Zhou, Jingxiu; Mori, Shuji; Takahashi, Hideo; Nishibori, Masahiro

2017-01-01

Advanced glycation end products (AGEs) are the products of a series of nonenzymatic modifications of proteins by reducing sugars. AGEs play a pivotal role in development of diabetic complications and atherosclerosis. Accumulation of AGEs in a vessel wall may contribute to the development of vascular lesions. Although AGEs have a diverse range of bioactivities, the clearance process of AGEs from the extracellular space, including the incorporation of AGEs into specific cells, subcellular localization, and the fate of AGEs, remains unclear. In the present study, we examined the kinetics of the uptake of AGEs by mouse macrophage J774.1 cells in vitro and characterized the process. We demonstrated that AGEs bound to the surface of the cells and were also incorporated into the cytoplasm. The temperature- and time-dependent uptake of AGEs was saturable with AGE concentration and was inhibited by cytochalasin D but not chlorpromazine. We also observed the granule-like appearance of AGE immunoreactivity in subcellular localizations in macrophages. Higher concentrations of AGEs induced intracellular ROS and 4-HNE, which were associated with activation of the NF- κ B pathway and caspase-3. These results suggest that incorporation of AGEs occurred actively by endocytosis in macrophages, leading to apoptosis of these cells through NF- κ B activation.

Phagocytosis of Advanced Glycation End Products (AGEs) in Macrophages Induces Cell Apoptosis

PubMed Central

Wake, Hidenori; Morioka, Yuta; Liu, Keyue; Shibuya, Megumi; Zhou, Jingxiu; Mori, Shuji; Takahashi, Hideo

2017-01-01

Advanced glycation end products (AGEs) are the products of a series of nonenzymatic modifications of proteins by reducing sugars. AGEs play a pivotal role in development of diabetic complications and atherosclerosis. Accumulation of AGEs in a vessel wall may contribute to the development of vascular lesions. Although AGEs have a diverse range of bioactivities, the clearance process of AGEs from the extracellular space, including the incorporation of AGEs into specific cells, subcellular localization, and the fate of AGEs, remains unclear. In the present study, we examined the kinetics of the uptake of AGEs by mouse macrophage J774.1 cells in vitro and characterized the process. We demonstrated that AGEs bound to the surface of the cells and were also incorporated into the cytoplasm. The temperature- and time-dependent uptake of AGEs was saturable with AGE concentration and was inhibited by cytochalasin D but not chlorpromazine. We also observed the granule-like appearance of AGE immunoreactivity in subcellular localizations in macrophages. Higher concentrations of AGEs induced intracellular ROS and 4-HNE, which were associated with activation of the NF-κB pathway and caspase-3. These results suggest that incorporation of AGEs occurred actively by endocytosis in macrophages, leading to apoptosis of these cells through NF-κB activation. PMID:29430285

Agglomeration, sedimentation, and cellular toxicity of alumina nanoparticles in cell culture medium

NASA Astrophysics Data System (ADS)

Yoon, Dokyung; Woo, Daekwang; Kim, Jung Heon; Kim, Moon Ki; Kim, Taesung; Hwang, Eung-Soo; Baik, Seunghyun

2011-06-01

The cytotoxicity of alumina nanoparticles (NPs) was investigated for a wide range of concentration (25-200 μg/mL) and incubation time (0-72 h) using floating cells (THP-1) and adherent cells (J774A.1, A549, and 293). Alumina NPs were gradually agglomerated over time although a significant portion of sedimentation occurred at the early stage within 6 h. A decrease of the viability was found in floating (THP-1) and adherent (J774A.1 and A549) cells in a dose-dependent manner. However, the time-dependent decrease in cell viability was observed only in adherent cells (J774A.1 and A549), which is predominantly related with the sedimentation of alumina NPs in cell culture medium. The uptake of alumina NPs in macrophages and an increased cell-to-cell adhesion in adherent cells were observed. There was no significant change in the viability of 293 cells. This in vitro test suggests that the agglomeration and sedimentation of alumina NPs affected cellular viability depending on cell types such as monocytes (THP-1), macrophages (J774A.1), lung carcinoma cells (A549), and embryonic kidney cells (293).

A Nitric Oxide Storage and Transport System That Protects Activated Macrophages from Endogenous Nitric Oxide Cytotoxicity*

PubMed Central

Lok, Hiu Chuen; Sahni, Sumit; Jansson, Patric J.; Kovacevic, Zaklina; Hawkins, Clare L.; Richardson, Des R.

2016-01-01

Nitric oxide (NO) is integral to macrophage cytotoxicity against tumors due to its ability to induce iron release from cancer cells. However, the mechanism for how activated macrophages protect themselves from endogenous NO remains unknown. We previously demonstrated by using tumor cells that glutathione S-transferase P1 (GSTP1) sequesters NO as dinitrosyl-dithiol iron complexes (DNICs) and inhibits NO-mediated iron release from cells via the transporter multidrug resistance protein 1 (MRP1/ABCC1). These prior studies also showed that MRP1 and GSTP1 protect tumor cells against NO cytotoxicity, which parallels their roles in defending cancer cells from cytotoxic drugs. Considering this, and because GSTP1 and MRP1 are up-regulated during macrophage activation, this investigation examined whether this NO storage/transport system protects macrophages against endogenous NO cytotoxicity in two well characterized macrophage cell types (J774 and RAW 264.7). MRP1 expression markedly increased upon macrophage activation, and the role of MRP1 in NO-induced 59Fe release was demonstrated by Mrp1 siRNA and the MRP1 inhibitor, MK571, which inhibited NO-mediated iron efflux. Furthermore, Mrp1 silencing increased DNIC accumulation in macrophages, indicating a role for MRP1 in transporting DNICs out of cells. In addition, macrophage 59Fe release was enhanced by silencing Gstp1, suggesting GSTP1 was responsible for DNIC binding/storage. Viability studies demonstrated that GSTP1 and MRP1 protect activated macrophages from NO cytotoxicity. This was confirmed by silencing nuclear factor-erythroid 2-related factor 2 (Nrf2), which decreased MRP1 and GSTP1 expression, concomitant with reduced 59Fe release and macrophage survival. Together, these results demonstrate a mechanism by which macrophages protect themselves against NO cytotoxicity. PMID:27866158

Testing Nucleoside Analogues as Inhibitors of Bacillus anthracis Spore Germination In Vitro and in Macrophage Cell Culture ▿

PubMed Central

Alvarez, Zadkiel; Lee, Kyungae; Abel-Santos, Ernesto

2010-01-01

Bacillus anthracis, the etiological agent of anthrax, has a dormant stage in its life cycle known as the endospore. When conditions become favorable, spores germinate and transform into vegetative bacteria. In inhalational anthrax, the most fatal manifestation of the disease, spores enter the organism through the respiratory tract and germinate in phagosomes of alveolar macrophages. Germinated cells can then produce toxins and establish infection. Thus, germination is a crucial step for the initiation of pathogenesis. B. anthracis spore germination is activated by a wide variety of amino acids and purine nucleosides. Inosine and l-alanine are the two most potent nutrient germinants in vitro. Recent studies have shown that germination can be hindered by isomers or structural analogues of germinants. 6-Thioguanosine (6-TG), a guanosine analogue, is able to inhibit germination and prevent B. anthracis toxin-mediated necrosis in murine macrophages. In this study, we screened 46 different nucleoside analogues as activators or inhibitors of B. anthracis spore germination in vitro. These compounds were also tested for their ability to protect the macrophage cell line J774a.1 from B. anthracis cytotoxicity. Structure-activity relationship analysis of activators and inhibitors clarified the binding mechanisms of nucleosides to B. anthracis spores. In contrast, no structure-activity relationships were apparent for compounds that protected macrophages from B. anthracis-mediated killing. However, multiple inhibitors additively protected macrophages from B. anthracis. PMID:20921305

49 CFR 236.774 - Movement, facing.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 49 Transportation 4 2010-10-01 2010-10-01 false Movement, facing. 236.774 Section 236.774 Transportation Other Regulations Relating to Transportation (Continued) FEDERAL RAILROAD ADMINISTRATION... Movement, facing. The movement of a train over the points of a switch which face in a direction opposite to...

36 CFR 7.74 - Virgin Islands National Park.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 36 Parks, Forests, and Public Property 1 2013-07-01 2013-07-01 false Virgin Islands National Park. 7.74 Section 7.74 Parks, Forests, and Public Property NATIONAL PARK SERVICE, DEPARTMENT OF THE INTERIOR SPECIAL REGULATIONS, AREAS OF THE NATIONAL PARK SYSTEM § 7.74 Virgin Islands National Park. (a...

36 CFR 7.74 - Virgin Islands National Park.

Code of Federal Regulations, 2012 CFR

2012-07-01

... 36 Parks, Forests, and Public Property 1 2012-07-01 2012-07-01 false Virgin Islands National Park. 7.74 Section 7.74 Parks, Forests, and Public Property NATIONAL PARK SERVICE, DEPARTMENT OF THE INTERIOR SPECIAL REGULATIONS, AREAS OF THE NATIONAL PARK SYSTEM § 7.74 Virgin Islands National Park. (a...

36 CFR 7.74 - Virgin Islands National Park.

Code of Federal Regulations, 2014 CFR

2014-07-01

... 36 Parks, Forests, and Public Property 1 2014-07-01 2014-07-01 false Virgin Islands National Park. 7.74 Section 7.74 Parks, Forests, and Public Property NATIONAL PARK SERVICE, DEPARTMENT OF THE INTERIOR SPECIAL REGULATIONS, AREAS OF THE NATIONAL PARK SYSTEM § 7.74 Virgin Islands National Park. (a...

Evidence for a gamma-interferon receptor that regulates macrophage tumoricidal activity

PubMed Central

1984-01-01

Gamma-interferon (IFN-gamma) is the macrophage-activating factor (MAF) produced by normal murine splenic cells and the murine T cell hybridoma 24/G1 that induces nonspecific tumoricidal activity in macrophages. Incubation of 24/G1 supernatants diluted to 8.3 IRU IFN-gamma/ml with 6 X 10(6) elicited peritoneal macrophages or bone marrow-derived macrophages for 4 h at 37 degrees C, resulted in removal of 80% of the MAF activity from the lymphokine preparation. Loss of activity appeared to result from absorption and not consumption because (a) 40% of the activity was removed after exposure to macrophage for 30 min at 4 degrees C, (b) no reduction of MAF activity was detected when the 24/G1 supernatant was incubated with macrophage culture supernatants, and (c) macrophage-treated supernatants showed a selective loss of MAF activity but not interleukin 2 (IL-2) activity. Absorption was dependent on the input of either IFN-gamma or macrophages and was time dependent at 37 degrees C but not at 4 degrees C. With four rodent species tested, absorption of murine IFN-gamma displayed species specificity. However, cultured human peripheral blood monocytes and the human histiocytic lymphoma cell line U937 were able to absorb the murine lymphokine. Although the majority of murine cell lines tested absorbed 24/G1 MAF activity, two murine macrophage cell lines, P388D1 and J774, were identified which absorbed significantly reduced amounts of natural IFN- gamma. Purified murine recombinant IFN-gamma was absorbed by elicited macrophages but not by P388D1. Normal macrophages but not P388D1 bound fluoresceinated microspheres coated with recombinant IFN-gamma and binding was inhibited by pretreatment of the normal cells with 24/G1 supernatants. Scatchard plot analysis showed that 12,000 molecules of soluble 125I-recombinant IFN-gamma bound per bone marrow macrophage with a Ka of 0.9 X 10(8) M-1. Binding was quantitatively inhibitable by natural IFN-gamma but not by murine IFN alpha. IFN

Multi-walled carbon nanotubes injure the plasma membrane of macrophages

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

Hirano, Seishiro; Kanno, Sanae; Furuyama, Akiko

2008-10-15

Carbon nanotubes (CNTs) are emerging nanotechnology materials which are likely to be mass-produced in the near future. However, prior to mass-production, certain health-related concerns should first be addressed. For example, when inhaled, the thin-fibrous shape and the biopersistent characteristics of CNTs may cause pulmonary diseases, in a manner similar to asbestos. In the present study, mouse macrophages (J774.1) were exposed to highly-purified multi-walled CNTs (MWCNTs, 67 nm) or to UICC crocidolite in order to evaluate the toxicity of these nano-size fibers. The cytotoxicity of MWCNTs was found to be higher than that of crocidolite. The toxic effect of MWCNTs wasmore » not affected by N-acetylcysteine, an antioxidant, or buthionine sulfoximine, a glutathione synthesis inhibitor. cDNA microarray analyses suggested that the cytotoxicity of MWCNTs could not be explained satisfactorily by either an increase or decrease of gene expression, although mRNA levels of some cytokines were slightly increased by MWCNTs. Moreover, MWCNTs did not significantly activate either MAP kinases such as ERK, JNK and p38, nor common apoptosis pathways such as caspase 3 and PARP. Electron microscopic studies indicated that MWCNTs associate with the plasma membrane of macrophages and disrupt the integrity of the membrane. Several proteins were found to adsorb onto MWCNTs when MWCNT-exposed macrophages were gently lysed. One of these proteins was macrophage receptor with collagenous structure (MARCO). MARCO-transfected CHO-K1 cells associated with MWCNTs more rapidly than mock-transfected cells. These results indicate that MWCNTs probably trigger cytotoxic effects in phagocytotic cells by reacting with MARCO on the plasma membrane and rupturing the plasma membrane.« less

Dual Transcriptome Profiling of Leishmania-Infected Human Macrophages Reveals Distinct Reprogramming Signatures

PubMed Central

Fernandes, Maria Cecilia; Dillon, Laura A. L.; Belew, Ashton Trey; Bravo, Hector Corrada; Mosser, David M.

2016-01-01

ABSTRACT Macrophages are mononuclear phagocytes that constitute a first line of defense against pathogens. While lethal to many microbes, they are the primary host cells of Leishmania spp. parasites, the obligate intracellular pathogens that cause leishmaniasis. We conducted transcriptomic profiling of two Leishmania species and the human macrophage over the course of intracellular infection by using high-throughput RNA sequencing to characterize the global gene expression changes and reprogramming events that underlie the interactions between the pathogen and its host. A systematic exclusion of the generic effects of large-particle phagocytosis revealed a vigorous, parasite-specific response of the human macrophage early in the infection that was greatly tempered at later time points. An analogous temporal expression pattern was observed with the parasite, suggesting that much of the reprogramming that occurs as parasites transform into intracellular forms generally stabilizes shortly after entry. Following that, the parasite establishes an intracellular niche within macrophages, with minimal communication between the parasite and the host cell later during the infection. No significant difference was observed between parasite species transcriptomes or in the transcriptional response of macrophages infected with each species. Our comparative analysis of gene expression changes that occur as mouse and human macrophages are infected by Leishmania spp. points toward a general signature of the Leishmania-macrophage infectome. PMID:27165796

48 CFR 731.774 - Overseas recruitment incentive.

Code of Federal Regulations, 2011 CFR

2011-10-01

... incentive. 731.774 Section 731.774 Federal Acquisition Regulations System AGENCY FOR INTERNATIONAL DEVELOPMENT GENERAL CONTRACTING REQUIREMENTS CONTRACT COST PRINCIPLES AND PROCEDURES Contracts With Nonprofit... are set forth in AIDAR 731.205-70. These policies are also applicable to contracts with a nonprofit...

Genetic and Phenotypic Characterization of a Salmonella enterica serovar Enteritidis Emerging Strain with Superior Intra-macrophage Replication Phenotype

PubMed Central

Shomer, Inna; Avisar, Alon; Desai, Prerak; Azriel, Shalhevet; Smollan, Gill; Belausov, Natasha; Keller, Nathan; Glikman, Daniel; Maor, Yasmin; Peretz, Avi; McClelland, Michael; Rahav, Galia; Gal-Mor, Ohad

2016-01-01

Salmonella enterica serovar Enteritidis (S. Enteritidis) is one of the ubiquitous Salmonella serovars worldwide and a major cause of food-born outbreaks, which are often associated with poultry and poultry derivatives. Here we report a nation-wide S. Enteritidis clonal outbreak that occurred in Israel during the last third of 2015. Pulsed field gel electrophoresis and whole genome sequencing identified genetically related strains that were circulating in Israel as early as 2008. Global comparison linked this outbreak strain to several clinical and marine environmental isolates that were previously isolated in California and Canada, indicating that similar strains are prevalent outside of Israel. Phenotypic comparison between the 2015 outbreak strain and other clinical and reference S. Enteritidis strains showed only limited intra-serovar phenotypic variation in growth in rich medium, invasion into Caco-2 cells, uptake by J774.1A macrophages, and host cell cytotoxicity. In contrast, significant phenotypic variation was shown among different S. Enteritidis isolates when biofilm-formation, motility, invasion into HeLa cells and uptake by THP-1 human macrophages were studied. Interestingly, the 2015 outbreak clone was found to possess superior intra-macrophage replication ability within both murine and human macrophages in comparison to the other S. Enteritidis strains studied. This phenotype is likely to play a role in the virulence and host-pathogen interactions of this emerging clone. PMID:27695450

Biotin deficiency up-regulates TNF-alpha production in murine macrophages.

PubMed

Kuroishi, Toshinobu; Endo, Yasuo; Muramoto, Koji; Sugawara, Shunji

2008-04-01

Biotin, a water-soluble vitamin of the B complex, functions as a cofactor of carboxylases that catalyze an indispensable cellular metabolism. Although significant decreases in serum biotin levels have been reported in patients with chronic inflammatory diseases, the biological roles of biotin in inflammatory responses are unclear. In this study, we investigated the effects of biotin deficiency on TNF-alpha production. Mice were fed a basal diet or a biotin-deficient diet for 8 weeks. Serum biotin levels were significantly lower in biotin-deficient mice than biotin-sufficient mice. After i.v. administration of LPS, serum TNF-alpha levels were significantly higher in biotin-deficient mice than biotin-sufficient mice. A murine macrophage-like cell line, J774.1, was cultured in a biotin-sufficient or -deficient medium for 4 weeks. Cell proliferation and biotinylation of intracellular proteins were decreased significantly in biotin-deficient cells compared with biotin-sufficient cells. Significantly higher production and mRNA expression of TNF-alpha were detected in biotin-deficient J774.1 cells than biotin-sufficient cells in response to LPS and even without LPS stimulation. Intracellular TNF-alpha expression was inhibited by actinomycin D, indicating that biotin deficiency up-regulates TNF-alpha production at the transcriptional level. However, the expression levels of TNF receptors, CD14, and TLR4/myeloid differentiation protein 2 complex were similar between biotin-sufficient and -deficient cells. No differences were detected in the activities of the NF-kappaB family or AP-1. The TNF-alpha induction by biotin deficiency was down-regulated by biotin supplementation in vitro and in vivo. These results indicate that biotin deficiency may up-regulate TNF-alpha production or that biotin excess down-regulates TNF-alpha production, suggesting that biotin status may influence inflammatory diseases.

Pancreatic Cancer Cell Exosome-Mediated Macrophage Reprogramming and the Role of MicroRNAs 155 and 125b2 Transfection using Nanoparticle Delivery Systems

PubMed Central

Su, Mei-Ju; Aldawsari, Hibah; Amiji, Mansoor

2016-01-01

Exosomes are nano-sized endosome-derived small intraluminal vesicles, which are important facilitators of intercellular communication by transporting contents, such as protein, mRNA, and microRNAs, between neighboring cells, such as in the tumor microenvironment. The purpose of this study was to understand the mechanisms of exosomes-mediated cellular communication between human pancreatic cancer (Panc-1) cells and macrophages (J771.A1) using a Transwell co-culture system. Following characterization of exosome-mediated cellular communication and pro-tumoral baseline M2 macrophage polarization, the Panc-1 cells were transfected with microRNA-155 (miR-155) and microRNA-125b-2 (miR-125b2) expressing plasmid DNA using hyaluronic acid-poly(ethylene imine)/hyaluronic acid-poly(ethylene glycol) (HA-PEI/HA-PEG) self-assembling nanoparticle-based non-viral vectors. Our results show that upon successful transfection of Panc-1 cells, the exosome content was altered leading to differential communication and reprogramming of the J774.A1 cells to an M1 phenotype. Based on these results, genetic therapies targeted towards selective manipulation of tumor cell-derived exosome content may be very promising for cancer therapy. PMID:27443190

Dual Transcriptome Profiling of Leishmania-Infected Human Macrophages Reveals Distinct Reprogramming Signatures.

PubMed

Fernandes, Maria Cecilia; Dillon, Laura A L; Belew, Ashton Trey; Bravo, Hector Corrada; Mosser, David M; El-Sayed, Najib M

2016-05-10

Macrophages are mononuclear phagocytes that constitute a first line of defense against pathogens. While lethal to many microbes, they are the primary host cells of Leishmania spp. parasites, the obligate intracellular pathogens that cause leishmaniasis. We conducted transcriptomic profiling of two Leishmania species and the human macrophage over the course of intracellular infection by using high-throughput RNA sequencing to characterize the global gene expression changes and reprogramming events that underlie the interactions between the pathogen and its host. A systematic exclusion of the generic effects of large-particle phagocytosis revealed a vigorous, parasite-specific response of the human macrophage early in the infection that was greatly tempered at later time points. An analogous temporal expression pattern was observed with the parasite, suggesting that much of the reprogramming that occurs as parasites transform into intracellular forms generally stabilizes shortly after entry. Following that, the parasite establishes an intracellular niche within macrophages, with minimal communication between the parasite and the host cell later during the infection. No significant difference was observed between parasite species transcriptomes or in the transcriptional response of macrophages infected with each species. Our comparative analysis of gene expression changes that occur as mouse and human macrophages are infected by Leishmania spp. points toward a general signature of the Leishmania-macrophage infectome. Little is known about the transcriptional changes that occur within mammalian cells harboring intracellular pathogens. This study characterizes the gene expression signatures of Leishmania spp. parasites and the coordinated response of infected human macrophages as the pathogen enters and persists within them. After accounting for the generic effects of large-particle phagocytosis, we observed a parasite-specific response of the human macrophages early in

Immunostimulatory activity of snake fruit (Salacca edulis Reinw.) cultivar Pondoh Hitam extract on the activation of macrophages in vitro

NASA Astrophysics Data System (ADS)

Wijanarti, Sri; Putra, Agus Budiawan Naro; Nishi, Kosuke; Harmayani, Eni; Sugahara, Takuya

2017-05-01

Snake fruit (Salacca edulis Reinw) cultivar Pondoh Hitam is a tropical fruit produced in Indonesia. It is consumed freshly or processed and believed as the most delicious snake fruit cultivar. Snake fruit flesh contains high polisaccharides such as pectin and dietary fiber. Therefore, snake fruit is a potential immunostimulator candidates but the immunological effect of snake fruit flesh has not been reported. In the present study, immunostimulatory activity of snake fruit flesh extract (SFFE) on macrophages activation was evaluated. SFFE was prepared by extracting from snake fruit flesh with water, methanol 70%, and ethanol 70% for 15 h at 4°C. Then obtained SFFE was used to stimulated cytokine production in vitro using J774.1 cell line. The extract giving strongest stimulation was sellected for in vivo assay to stimulate cytokines production and gene expression using peritoneal macrophage (P-mac) of BALB/c mice. The results showed that SFFE exhibited immunostimulatory activities. Immunostimulatory activity could be indicated by macrophages activation characteristics such as cytokines production. Water extract of SFFE gave strongest stimulation on cytokines production in vitro and sellected for in vivo assay. In vivo assay showed that SFFE stimulated cytokines production as well as their gene expression levels. The optimum stimulation was demonstrated by SFFE 16.7 mg/g. Overall findings suggest that SFFE has a potent beneficial effects to promote the body health through activating macrophages.

Eicosapentaenoic acid membrane incorporation impairs ABCA1-dependent cholesterol efflux via a protein kinase A signaling pathway in primary human macrophages.

PubMed

Fournier, Natalie; Tardivel, Sylviane; Benoist, Jean-François; Vedie, Benoît; Rousseau-Ralliard, Delphine; Nowak, Maxime; Allaoui, Fatima; Paul, Jean-Louis

2016-04-01

A diet rich in n-3/n-6 polyunsaturated fatty acids (PUFAs) is cardioprotective. Dietary PUFAs affect the cellular phospholipids composition, which may influence the function of membrane proteins. We investigated the impact of the membrane incorporation of several PUFAs on ABCA1-mediated cholesterol efflux, a key antiatherogenic pathway. Arachidonic acid (AA) (C20:4 n-6) and docosahexaenoic acid (DHA) (C22:6 n-3) decreased or increased cholesterol efflux from J774 mouse macrophages, respectively, whereas they had no effect on efflux from human monocyte-derived macrophages (HMDM). Importantly, eicosapentaenoic acid (EPA) (C20:5 n-3) induced a dose-dependent reduction of ABCA1 functionality in both cellular models (-28% for 70μM of EPA in HMDM), without any alterations in ABCA1 expression. These results show that PUFA membrane incorporation does not have the same consequences on cholesterol efflux from mouse and human macrophages. The EPA-treated HMDM exhibited strong phospholipid composition changes, with high levels of both EPA and its elongation product docosapentaenoic acid (DPA) (C22:5 n-3), which is associated with a decreased level of AA. In HMDM, EPA reduced the ATPase activity of the membrane transporter. Moreover, the activation of adenylate cyclase by forskolin and the inhibition of cAMP phosphodiesterase by isobutylmethylxanthine restored ABCA1 cholesterol efflux in EPA-treated human macrophages. In conclusion, EPA membrane incorporation reduces ABCA1 functionality in mouse macrophages as well as in primary human macrophages and this effect seems to be PKA-dependent in human macrophages. Copyright © 2016 Elsevier B.V. All rights reserved.

Flavonoid Apigenin Inhibits Lipopolysaccharide-Induced Inflammatory Response through Multiple Mechanisms in Macrophages

PubMed Central

Zhang, Xiaoxuan; Wang, Guangji; Gurley, Emily C.; Zhou, Huiping

2014-01-01

Background Apigenin is a non-toxic natural flavonoid that is abundantly present in common fruits and vegetables. It has been reported that apigenin has various beneficial health effects such as anti-inflammation and chemoprevention. Multiple studies have shown that inflammation is an important risk factor for atherosclerosis, diabetes, sepsis, various liver diseases, and other metabolic diseases. Although it has been long realized that apigenin has anti-inflammatory activities, the underlying functional mechanisms are still not fully understood. Methodology and Principal Findings In the present study, we examined the effect of apigenin on LPS-induced inflammatory response and further elucidated the potential underlying mechanisms in human THP-1-induced macrophages and mouse J774A.1 macrophages. By using the PrimePCR array, we were able to identify the major target genes regulated by apigenin in LPS-mediated immune response. The results indicated that apigenin significantly inhibited LPS-induced production of pro-inflammatory cytokines, such as IL-6, IL-1β, and TNF-α through modulating multiple intracellular signaling pathways in macrophages. Apigenin inhibited LPS-induced IL-1β production by inhibiting caspase-1 activation through the disruption of the NLRP3 inflammasome assembly. Apigenin also prevented LPS-induced IL-6 and IL-1β production by reducing the mRNA stability via inhibiting ERK1/2 activation. In addition, apigenin significantly inhibited TNF-α and IL-1β-induced activation of NF-κB. Conclusion and Significance Apigenin Inhibits LPS-induced Inflammatory Response through multiple mechanisms in macrophages. These results provided important scientific evidences for the potential application of apigenin as a therapeutic agent for inflammatory diseases. PMID:25192391

Direct recognition of superparamagnetic nanocrystals by macrophage scavenger receptor SR-AI.

PubMed

Chao, Ying; Karmali, Priya P; Mukthavaram, Rajesh; Kesari, Santosh; Kouznetsova, Valentina L; Tsigelny, Igor F; Simberg, Dmitri

2013-05-28

Scavenger receptors (SRs) are molecular pattern recognition receptors that have been shown to mediate opsonin-independent uptake of therapeutic and imaging nanoparticles, underlying the importance of SRs in nanomedicine. Unlike pathogens, engineered nanomaterials offer great flexibility in control of surface properties, allowing addressing specific questions regarding the molecular mechanisms of nanoparticle recognition. Recently, we showed that SR-type AI/II mediates opsonin-independent internalization of dextran superparamagnetic iron oxide (SPIO) nanoparticles via positively charged extracellular collagen-like domain. To understand the mechanism of opsonin-independent SPIO recognition, we tested the binding and uptake of nanoparticles with different surface coatings by SR-AI. SPIO coated with 10 kDa dextran was efficiently recognized and taken up by SR-AI transfected cells and J774 macrophages, while SPIO with 20 kDa dextran coating or cross-linked dextran hydrogel avoided the binding and uptake. Nanoparticle negative charge density and zeta-potential did not correlate with SR-AI binding/uptake efficiency. Additional experiments and computer modeling revealed that recognition of the iron oxide crystalline core by the positively charged collagen-like domain of SR-AI is sterically hindered by surface polymer coating. Importantly, the modeling revealed a strong complementarity between the surface Fe-OH groups of the magnetite crystal and the charged lysines of the collagen-like domain of SR-AI, suggesting a specific recognition of SPIO crystalline surface. These data provide an insight into the molecular recognition of nanocrystals by innate immunity receptors and the mechanisms whereby polymer coatings promote immune evasion.

Differential Kinetics of Aspergillus nidulans and Aspergillus fumigatus Phagocytosis.

PubMed

Gresnigt, Mark S; Becker, Katharina L; Leenders, Floris; Alonso, M Fernanda; Wang, Xiaowen; Meis, Jacques F; Bain, Judith M; Erwig, Lars P; van de Veerdonk, Frank L

2018-01-01

Invasive aspergillosis mainly occurs in immunocompromised patients and is commonly caused by Aspergillus fumigatus, while A.nidulans is rarely the causative agent. However, in chronic granulomatous disease (CGD) patients, A. nidulans is a frequent cause of invasive aspergillosis and is associated with higher mortality. Immune recognition of A. nidulans was compared to A. fumigatus to offer an insight into why A. nidulans infections are prevalent in CGD. Live cell imaging with J774A.1 macrophage-like cells and LC3-GFP-mCherry bone marrow-derived macrophages (BMDMs) revealed that phagocytosis of A. nidulans was slower compared to A. fumigatus. This difference could be attributed to slower migration of J774A.1 cells and a lower percentage of migrating BMDMs. In addition, delayed phagosome acidification and LC3-associated phagocytosis was observed with A. nidulans. Cytokine and oxidative burst measurements in human peripheral blood mononuclear cells revealed a lower oxidative burst upon challenge with A. nidulans. In contrast, A. nidulans induced significantly higher concentrations of cytokines. Collectively, our data demonstrate that A. nidulans is phagocytosed and processed at a slower rate compared to A. fumigatus, resulting in reduced fungal killing and increased germination of conidia. This slower rate of A. nidulans clearance may be permissive for overgrowth within certain immune settings. The Author(s). Published by S. Karger AG, Basel.

40 CFR 77.4 - Administrator's action on proposed offset plans.

Code of Federal Regulations, 2013 CFR

2013-07-01

... 40 Protection of Environment 17 2013-07-01 2013-07-01 false Administrator's action on proposed offset plans. 77.4 Section 77.4 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS (CONTINUED) EXCESS EMISSIONS § 77.4 Administrator's action on proposed offset plans. (a...

Proteomic analysis reveals different composition of extracellular vesicles released by two Trypanosoma cruzi strains associated with their distinct interaction with host cells.

PubMed

Ribeiro, Kleber Silva; Vasconcellos, Camilla Ioshida; Soares, Rodrigo Pedro; Mendes, Maria Tays; Ellis, Cameron C; Aguilera-Flores, Marcela; de Almeida, Igor Correia; Schenkman, Sergio; Iwai, Leo Kei; Torrecilhas, Ana Claudia

2018-01-01

Trypanosoma cruzi , the aetiologic agent of Chagas disease, releases vesicles containing a wide range of surface molecules known to affect the host immunological responses and the cellular infectivity. Here, we compared the secretome of two distinct strains (Y and YuYu) of T. cruzi , which were previously shown to differentially modulate host innate and acquired immune responses. Tissue culture-derived trypomastigotes of both strains secreted extracellular vesicles (EVs), as demonstrated by electron scanning microscopy. EVs were purified by exclusion chromatography or ultracentrifugation and quantitated using nanoparticle tracking analysis. Trypomastigotes from YuYu strain released higher number of EVs than those from Y strain, enriched with virulence factors trans -sialidase (TS) and cruzipain. Proteomic analysis confirmed the increased abundance of proteins coded by the TS gene family, mucin-like glycoproteins, and some typical exosomal proteins in the YuYu strain, which also showed considerable differences between purified EVs and vesicle-free fraction as compared to the Y strain. To evaluate whether such differences were related to parasite infectivity, J774 macrophages and LLC-MK2 kidney cells were preincubated with purified EVs from both strains and then infected with Y strain trypomastigotes. EVs released by YuYu strain caused a lower infection but higher intracellular proliferation in J774 macrophages than EVs from Y strain. In contrast, YuYu strain-derived EVs caused higher infection of LLC-MK2 cells than Y strain-derived EVs. In conclusion, quantitative and qualitative differences in EVs and secreted proteins from different T. cruzi strains may correlate with infectivity/virulence during the host-parasite interaction.

Proteomic analysis reveals different composition of extracellular vesicles released by two Trypanosoma cruzi strains associated with their distinct interaction with host cells

PubMed Central

Ribeiro, Kleber Silva; Vasconcellos, Camilla Ioshida; Soares, Rodrigo Pedro; Ellis, Cameron C.; Aguilera-Flores, Marcela; de Almeida, Igor Correia

2018-01-01

ABSTRACT Trypanosoma cruzi, the aetiologic agent of Chagas disease, releases vesicles containing a wide range of surface molecules known to affect the host immunological responses and the cellular infectivity. Here, we compared the secretome of two distinct strains (Y and YuYu) of T. cruzi, which were previously shown to differentially modulate host innate and acquired immune responses. Tissue culture-derived trypomastigotes of both strains secreted extracellular vesicles (EVs), as demonstrated by electron scanning microscopy. EVs were purified by exclusion chromatography or ultracentrifugation and quantitated using nanoparticle tracking analysis. Trypomastigotes from YuYu strain released higher number of EVs than those from Y strain, enriched with virulence factors trans-sialidase (TS) and cruzipain. Proteomic analysis confirmed the increased abundance of proteins coded by the TS gene family, mucin-like glycoproteins, and some typical exosomal proteins in the YuYu strain, which also showed considerable differences between purified EVs and vesicle-free fraction as compared to the Y strain. To evaluate whether such differences were related to parasite infectivity, J774 macrophages and LLC-MK2 kidney cells were preincubated with purified EVs from both strains and then infected with Y strain trypomastigotes. EVs released by YuYu strain caused a lower infection but higher intracellular proliferation in J774 macrophages than EVs from Y strain. In contrast, YuYu strain-derived EVs caused higher infection of LLC-MK2 cells than Y strain-derived EVs. In conclusion, quantitative and qualitative differences in EVs and secreted proteins from different T. cruzi strains may correlate with infectivity/virulence during the host–parasite interaction. PMID:29696081

7 CFR 774.10 - Other Federal, State, and local requirements.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 7 Agriculture 7 2013-01-01 2013-01-01 false Other Federal, State, and local requirements. 774.10 Section 774.10 Agriculture Regulations of the Department of Agriculture (Continued) FARM SERVICE AGENCY, DEPARTMENT OF AGRICULTURE SPECIAL PROGRAMS EMERGENCY LOAN FOR SEED PRODUCERS PROGRAM § 774.10 Other Federal...

7 CFR 774.10 - Other Federal, State, and local requirements.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 7 Agriculture 7 2012-01-01 2012-01-01 false Other Federal, State, and local requirements. 774.10 Section 774.10 Agriculture Regulations of the Department of Agriculture (Continued) FARM SERVICE AGENCY, DEPARTMENT OF AGRICULTURE SPECIAL PROGRAMS EMERGENCY LOAN FOR SEED PRODUCERS PROGRAM § 774.10 Other Federal...

7 CFR 774.10 - Other Federal, State, and local requirements.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 7 Agriculture 7 2011-01-01 2011-01-01 false Other Federal, State, and local requirements. 774.10 Section 774.10 Agriculture Regulations of the Department of Agriculture (Continued) FARM SERVICE AGENCY, DEPARTMENT OF AGRICULTURE SPECIAL PROGRAMS EMERGENCY LOAN FOR SEED PRODUCERS PROGRAM § 774.10 Other Federal...

7 CFR 774.10 - Other Federal, State, and local requirements.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 7 2010-01-01 2010-01-01 false Other Federal, State, and local requirements. 774.10 Section 774.10 Agriculture Regulations of the Department of Agriculture (Continued) FARM SERVICE AGENCY, DEPARTMENT OF AGRICULTURE SPECIAL PROGRAMS EMERGENCY LOAN FOR SEED PRODUCERS PROGRAM § 774.10 Other Federal...

7 CFR 774.10 - Other Federal, State, and local requirements.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 7 Agriculture 7 2014-01-01 2014-01-01 false Other Federal, State, and local requirements. 774.10 Section 774.10 Agriculture Regulations of the Department of Agriculture (Continued) FARM SERVICE AGENCY, DEPARTMENT OF AGRICULTURE SPECIAL PROGRAMS EMERGENCY LOAN FOR SEED PRODUCERS PROGRAM § 774.10 Other Federal...

CYP epoxygenase 2J2 prevents cardiac fibrosis by suppression of transmission of pro-inflammation from cardiomyocytes to macrophages

PubMed Central

Yang, Lei; Ni, Li; Duan, Quanlu; Wang, Xingxu; Chen, Chen; Chen, Song; Chaugai, Sandip; Zeldin, D.C.; Tang, Jia Rong; Wang, Dao Wen

2017-01-01

Cytochrome P450 epoxygenase (CYP450)-derived epoxyeicosatrienoic acids (EETs) are important regulators of cardiac remodeling; but the underlying mechanism remains unclear. The present study aimed to elucidate how EETs regulated cardiac fibrosis in response to isoprenaline (Iso) or angiotensin (Ang) II. Cardiac-specific human CYP2J2 transgenic mice (Tr) and wild-type (WT) C57BL/6 littermates were infused with Iso- or Ang II. Two weeks after infusion, Tr mice showed more alleviative cardiac fibrosis and inflammation compared with WT mice. In vitro, we found Iso or Ang II induced nuclear transfer of NF-κB p65 and inflammatory cytokines expression in cardiomyocytes. Furthermore, inflammation response emerged in macrophages cultured in cardiomyocytes-conditioned medium. When pretreatment with 14,15-EET in cardiomyocytes, the inflammatory response was markedly suppressed and the transmission of inflammation from cardiomyocytes to macrophages was reduced. In conclusion, CYP2J2 and EETs prevent cardiac fibrosis and cardiac dysfunction by suppressing transmission of pro-inflammation from cardiomyocytes to macrophages in heart, suggesting that elevation of EETs level could be a potential strategy to prevent cardiac fibrosis. PMID:25686540

CRISPR/Cas9-Mediated Gene Editing in Human iPSC-Derived Macrophage Reveals Lysosomal Acid Lipase Function in Human Macrophages-Brief Report.

PubMed

Zhang, Hanrui; Shi, Jianting; Hachet, Melanie A; Xue, Chenyi; Bauer, Robert C; Jiang, Hongfeng; Li, Wenjun; Tohyama, Junichiro; Millar, John; Billheimer, Jeffrey; Phillips, Michael C; Razani, Babak; Rader, Daniel J; Reilly, Muredach P

2017-11-01

To gain mechanistic insights into the role of LIPA (lipase A), the gene encoding LAL (lysosomal acid lipase) protein, in human macrophages. We used CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) technology to knock out LIPA in human induced pluripotent stem cells and then differentiate to macrophage (human-induced pluripotent stem cells-derived macrophage [IPSDM]) to explore the human macrophage LIPA loss-of-function phenotypes. LIPA was abundantly expressed in monocyte-derived macrophages and was markedly induced on IPSDM differentiation to comparable levels as in human monocyte-derived macrophage. IPSDM with knockout of LIPA ( LIPA -/- ) had barely detectable LAL enzymatic activity. Control and LIPA -/- IPSDM were loaded with [ 3 H]-cholesteryl oleate-labeled AcLDL (acetylated low-density lipoprotein) followed by efflux to apolipoprotein A-I. Efflux of liberated [ 3 H]-cholesterol to apolipoprotein A-I was abolished in LIPA -/- IPSDM, indicating deficiency in LAL-mediated lysosomal cholesteryl ester hydrolysis. In cells loaded with [ 3 H]-cholesterol-labeled AcLDL, [ 3 H]-cholesterol efflux was, however, not different between control and LIPA -/- IPSDM. ABCA1 (ATP-binding cassette, subfamily A, member 1) expression was upregulated by AcLDL loading but to a similar extent between control and LIPA -/- IPSDM. In nonlipid loaded state, LIPA -/- IPSDM had high levels of cholesteryl ester mass compared with minute amounts in control IPSDM. Yet, with AcLDL loading, overall cholesteryl ester mass was increased to similar levels in both control and LIPA -/- IPSDM. LIPA -/- did not impact lysosomal apolipoprotein-B degradation or expression of IL1B , IL6 , and CCL5. CONCLUSIONS: LIPA -/- IPSDM reveals macrophage-specific hallmarks of LIPA deficiency. CRISPR/Cas9 and IPSDM provide important tools to study human macrophage biology and more broadly for future studies of disease-associated LIPA genetic variation in human

Acrolein increases macrophage atherogenicity in association with gut microbiota remodeling in atherosclerotic mice: protective role for the polyphenol-rich pomegranate juice.

PubMed

Rom, Oren; Korach-Rechtman, Hila; Hayek, Tony; Danin-Poleg, Yael; Bar, Haim; Kashi, Yechezkel; Aviram, Michael

2017-04-01

The unsaturated aldehyde acrolein is pro-atherogenic, and the polyphenol-rich pomegranate juice (PJ), known for its anti-oxidative/anti-atherogenic properties, inhibits macrophage foam cell formation, the hallmark feature of early atherosclerosis. This study aimed to investigate two unexplored areas of acrolein atherogenicity: macrophage lipid metabolism and the gut microbiota composition. The protective effects of PJ against acrolein atherogenicity were also evaluated. Atherosclerotic apolipoprotein E-deficient (apoE -/- ) mice that were fed acrolein (3 mg/kg/day) for 1 month showed significant increases in serum and aortic cholesterol, triglycerides, and lipid peroxides. In peritoneal macrophages isolated from the mice and in J774A.1 cultured macrophages, acrolein exposure increased intracellular oxidative stress and stimulated cholesterol and triglyceride accumulation via enhanced rates of their biosynthesis and over-expression of key regulators of cellular lipid biosynthesis: sterol regulatory element-binding proteins (SREBPs), 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR), and diacylglycerol acyltransferase1 (DGAT1). Acrolein-fed mice demonstrated a major shift in the gut microbiota composition, including a significant phylum-level change in increased Firmicutes and decreased Bacteroidetes. At the family level, acrolein significantly increased the prevalence of Ruminococcaceae and Lachnospiraceae of which the Coprococcus genus was significantly and positively correlated with serum, aortic and macrophage lipid levels and peroxidation. The pro-atherogenic effects of acrolein on serum, aortas, macrophages, and the gut microbiota were substantially abolished by PJ. In conclusion, these findings provide novel mechanisms by which acrolein increases macrophage lipid accumulation and alters the gut microbiota composition in association with enhanced atherogenesis. Moreover, PJ was found as an effective strategy against acrolein atherogenicity.

Implication of matrix metalloproteinases 2 and 9 in ceramide 1-phosphate-stimulated macrophage migration.

PubMed

Ordoñez, Marta; Rivera, Io-Guané; Presa, Natalia; Gomez-Muñoz, Antonio

2016-08-01

Cell migration is a complex biological function involved in both physiologic and pathologic processes. Although this is a subject of intense investigation, the mechanisms by which cell migration is regulated are not completely understood. In this study we show that the bioactive sphingolipid ceramide 1-phosphate (C1P), which is involved in inflammatory responses, causes upregulation of metalloproteinases (MMP) -2 and -9 in J774A.1 macrophages. This effect was shown to be dependent on stimulation of phosphatidylinositol 3-kinase (PI3K) and extracellularly regulated kinases 1-2 (ERK1-2) as demonstrated by treating the cells with specific siRNA to knockdown the p85 regulatory subunit of PI3K, or ERK1-2. Inhibition of MMP-2 or MMP-9 pharmacologically or with specific siRNA to silence the genes encoding these MMPs abrogated C1P-stimulated macrophage migration. Also, C1P induced actin polymerization and potently increased phosphorylation of the focal adhesion protein paxillin, which are essential factors in the regulation of cell migration. As expected, blockade of paxillin activation with specific siRNA significantly reduced actin polymerization. In addition, inhibition of actin polymerization with cytochalasin D completely blocked C1P-induced MMP-2 and -9 expression as well as C1P-stimulated macrophage migration. It was also observed that pertussis toxin (Ptx) inhibited Akt, ERK1-2, and paxillin phosphorylation, and completely blocked cell migration. The latter findings support the notion that C1P-stimulated macrophage migration is a receptor mediated effect, and point to MMP-2 and -9 as possible therapeutic targets to control inflammation. Copyright © 2016 Elsevier Inc. All rights reserved.

Essential role of protein kinase C zeta in transducing a motility signal induced by superoxide and a chemotactic peptide, fMLP.

PubMed

Kuribayashi, Kageaki; Nakamura, Kiminori; Tanaka, Maki; Sato, Tsutomu; Kato, Junji; Sasaki, Katsunori; Takimoto, Rishu; Kogawa, Katsuhisa; Terui, Takeshi; Takayama, Tetsuji; Onuma, Takayuki; Matsunaga, Takuya; Niitsu, Yoshiro

2007-03-26

Under various pathological conditions, including infection, malignancy, and autoimmune diseases, tissues are incessantly exposed to reactive oxygen species produced by infiltrating inflammatory cells. We show augmentation of motility associated with morphological changes of human squamous carcinoma SASH1 cells, human peripheral monocytes (hPMs), and murine macrophage-like cell line J774.1 by superoxide stimulation. We also disclose that motility of hPMs and J774.1 induced by a chemotactic peptide (N-formyl-methionyl-leucyl-phenylalanine [fMLP]) was inhibited by superoxide dismutase or N-acetylcystein, indicating stimulation of motility by superoxide generated by fMLP stimulation. In these cells, protein kinase C (PKC) zeta was activated to phosphorylate RhoGDI-1, which liberated RhoGTPases, leading to their activation. These events were inhibited by dominant-negative PKCzeta in SASH1 cells, myristoylated PKCzeta peptides in hPMs and J774.1, or a specific inhibitor of RhoGTPase in SASH1, hPMs, and J774.1. These results suggest a new approach for manipulation of inflammation as well as tumor cell invasion by targeting this novel signaling pathway.

P2Y receptors and atherosclerosis in apolipoprotein E-deficient mice

PubMed Central

Guns, Pieter-Jan DF; Hendrickx, Jan; Van Assche, Tim; Fransen, Paul; Bult, Hidde

2010-01-01

Background and purpose: P2Y nucleotide receptors are involved in the regulation of vascular tone, smooth muscle cell (SMC) proliferation and inflammatory responses. The present study investigated whether they are involved in atherosclerosis. Experimental approach: mRNA of P2Y receptors was quantified (RT-PCR) in atherosclerotic and plaque-free aorta segments of apolipoprotein E-deficient (apoE–/–) mice. Macrophage activation was assessed in J774 macrophages, and effects of non-selective purinoceptor antagonists on atherosclerosis were evaluated in cholesterol-fed apoE–/– mice. Key results: P2Y6 receptor mRNA was consistently elevated in segments with atherosclerosis, whereas P2Y2 receptor expression remained unchanged. Expression of P2Y1 or P2Y4 receptor mRNA was low or undetectable, and not influenced by atherosclerosis. P2Y6 mRNA expression was higher in cultured J774 macrophages than in cultured aortic SMCs. Furthermore, immunohistochemical staining of plaques demonstrated P2Y6-positive macrophages, but few SMCs, suggesting that macrophage recruitment accounted for the increase in P2Y6 receptor mRNA during atherosclerosis. In contrast to ATP, the P2Y6-selective agonist UDP increased mRNA expression and activity of inducible nitric oxide synthase and interleukin-6 in J774 macrophages; this effect was blocked by suramin (100–300 µM) or pyridoxal-phosphate-6-azophenyl-2′-4′-disulphonic acid (PPADS, 10–30 µM). Finally, 4-week treatment of cholesterol-fed apoE–/– mice with suramin or PPADS (50 and 25 mg·kg−1·day−1 respectively) reduced plaque size, without changing plaque composition (relative SMC and macrophage content) or cell replication. Conclusions and implications: These results suggest involvement of nucleotide receptors, particularly P2Y6 receptors, during atherosclerosis, and warrant further research with selective purinoceptor antagonists or P2Y6 receptor-deficient mice. PMID:20050854

Sirtuin inhibition attenuates the production of inflammatory cytokines in lipopolysaccharide-stimulated macrophages

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

Fernandes, Claudia A.; Fievez, Laurence; Neyrinck, Audrey M.

Highlights: Black-Right-Pointing-Pointer Lipopolysaccharide-stimulated macrophages were treated with cambinol and sirtinol. Black-Right-Pointing-Pointer Cambinol and sirtinol decreased lipopolysaccharide-induced cytokines. Black-Right-Pointing-Pointer Cambinol decreased NF-{kappa}B activity but had no impact on p38 MAPK activation. Black-Right-Pointing-Pointer Sirtuins are an interesting target for the treatment of inflammatory diseases. -- Abstract: In several inflammatory conditions such as rheumatoid arthritis or sepsis, the regulatory mechanisms of inflammation are inefficient and the excessive inflammatory response leads to damage to the host. Sirtuins are class III histone deacetylases that modulate the activity of several transcription factors that are implicated in immune responses. In this study, we evaluated the impact ofmore » sirtuin inhibition on the activation of lipopolysaccharide (LPS)-stimulated J774 macrophages by assessing the production of inflammatory cytokines. The pharmacologic inhibition of sirtuins decreased the production of tumour necrosis factor-alpha (TNF-{alpha}) interleukin 6 (IL-6) and Rantes. The reduction of cytokine production was associated with decreased nuclear factor kappa B (NF-{kappa}B) activity and inhibitor kappa B alpha (I{kappa}B{alpha}) phosphorylation while no impact was observed on the phosphorylation status of p38 mitogen-activated kinase (p38 MAPK). This work shows that sirtuin pharmacologic inhibitors are a promising tool for the treatment of inflammatory conditions.« less

The Role of CD38 in Fcγ Receptor (FcγR)-mediated Phagocytosis in Murine Macrophages*

PubMed Central

Kang, John; Park, Kwang-Hyun; Kim, Jwa-Jin; Jo, Eun-Kyeong; Han, Myung-Kwan; Kim, Uh-Hyun

2012-01-01

Phagocytosis is a crucial event in the immune system that allows cells to engulf and eliminate pathogens. This is mediated through the action of immunoglobulin (IgG)-opsonized microbes acting on Fcγ receptors (FcγR) on macrophages, which results in sustained levels of intracellular Ca2+ through the mobilization of Ca2+ second messengers. It is known that the ADP-ribosyl cyclase is responsible for the rise in Ca2+ levels after FcγR activation. However, it is unclear whether and how CD38 is involved in FcγR-mediated phagocytosis. Here we show that CD38 is recruited to the forming phagosomes during phagocytosis of IgG-opsonized particles and produces cyclic-ADP-ribose, which acts on ER Ca2+ stores, thus allowing an increase in FcγR activation-mediated phagocytosis. Ca2+ data show that pretreatment of J774A.1 macrophages with 8-bromo-cADPR, ryanodine, blebbistatin, and various store-operated Ca2+ inhibitors prevented the long-lasting Ca2+ signal, which significantly reduced the number of ingested opsonized particles. Ex vivo data with macrophages extracted from CD38−/− mice also shows a reduced Ca2+ signaling and phagocytic index. Furthermore, a significantly reduced phagocytic index of Mycobacterium bovis BCG was shown in macrophages from CD38−/− mice in vivo. This study suggests a crucial role of CD38 in FcγR-mediated phagocytosis through its recruitment to the phagosome and mobilization of cADPR-induced intracellular Ca2+ and store-operated extracellular Ca2+ influx. PMID:22396532

42 CFR 423.774 - Eligibility determinations, redeterminations, and applications.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 42 Public Health 3 2010-10-01 2010-10-01 false Eligibility determinations, redeterminations, and applications. 423.774 Section 423.774 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICARE PROGRAM VOLUNTARY MEDICARE PRESCRIPTION DRUG BENEFIT...

The role of lipopolysaccharide in infectious bone resorption of periapical lesion.

PubMed

Hong, Chi-Yuan; Lin, Sze-Kwan; Kok, Sang-Heng; Cheng, Shih-Jung; Lee, Ming-Shu; Wang, Tong-Mei; Chen, Chuan-Shuo; Lin, Li-Deh; Wang, Juo-Song

2004-03-01

The role of lipopolysaccharide (LPS) in periapical lesion-induced bone resorption was investigated. Polymyxin B (PMB), a specific inhibitor of LPS, was evaluated to treat the apical lesion. Lipopolysaccharide isolated from two common endodontic pathogens, Fusobacterium nucleatum and Porphyromonas endodontalis, stimulated mouse macrophage (J774) to release interleukin-1alpha (IL-1 alpha) and tumor necrosis factor-alpha (TNF-alpha) in a time-dependent manner. Combination of LPS further enhanced the stimulation. PMB inhibited these effects significantly. LPS also stimulated matrix metalloproteinase-1 (MMP-1) gene expression in J774, whereas anti-IL-1 alpha and anti-TNF-alpha antibodies, as well as PMB, diminished this effect. A disease model of periapical lesion was established in Wistar rat. Administration of PMB reduced the extent of lesion-associated bone resorption by 76% to approximately 80%, and simultaneously reduced the numbers of MMP-1-producing macrophages. It is suggested that LPS released from the infected root canal triggers the synthesis of IL-1 alpha and TNF-alpha from macrophages. These pro-inflammatory cytokines up-regulate the production of MMP-1 by macrophages to promote periapical bone resorption.

Advanced glycation end products affect cholesterol homeostasis by impairing ABCA1 expression on macrophages.

PubMed

Kamtchueng Simo, Olivier; Ikhlef, Souade; Berrougui, Hicham; Khalil, Abdelouahed

2017-08-01

Reverse cholesterol transport (RCT), which is intimately linked to high-density lipoproteins (HDLs), plays a key role in cholesterol homeostasis and the prevention of atherosclerosis. The goal of the present study was to investigate the effect of aging and advanced glycation end products (AGEs) on RCT as well as on other factors that may affect the antiatherogenic property of HDLs. The transfer of macrophage-derived cholesterol to the plasma and liver and then to the feces for elimination was significantly lower in aged mice than in young mice. Chronic injection of d -galactose (D-gal) or AGEs also significantly reduced RCT (65.3% reduction in [ 3 H]cholesterol levels in the plasma of D-gal-treated mice after 48 h compared with control mice, P < 0.01). The injection of both D-gal and aminoguanidine hydrochloride increased [ 3 H]cholesterol levels in the plasma, although the levels were lower than those of control mice. The in vitro incubation of HDLs with dicarbonyl compounds increased the carbonyl and conjugated diene content of HDLs and significantly reduced PON1 paraoxonase activity (87.4% lower than control HDLs, P < 0.0001). Treating J774A.1 macrophages with glycated fetal bovine serum increased carbonyl formation (39.5% increase, P < 0.003) and reduced ABCA1 protein expression and the capacity of macrophages to liberate cholesterol (69.1% decrease, P < 0.0001). Our results showed, for the first time, that RCT is altered with aging and that AGEs contribute significantly to this alteration.

7 CFR 774.3 - Appeals.

Code of Federal Regulations, 2011 CFR

2011-01-01

... PROGRAMS EMERGENCY LOAN FOR SEED PRODUCERS PROGRAM § 774.3 Appeals. A loan applicant or borrower may request an appeal or review of an adverse decision made by the Agency in accordance with 7 CFR part 11. ...

7 CFR 774.3 - Appeals.

Code of Federal Regulations, 2010 CFR

2010-01-01

... PROGRAMS EMERGENCY LOAN FOR SEED PRODUCERS PROGRAM § 774.3 Appeals. A loan applicant or borrower may request an appeal or review of an adverse decision made by the Agency in accordance with 7 CFR part 11. ...

Inhibitory effect of trichodermanone C, a sorbicillinoid produced by Trichoderma citrinoviride associated to the green alga Cladophora sp., on nitrite production in LPS-stimulated macrophages.

PubMed

Marra, Roberta; Nicoletti, Rosario; Pagano, Ester; DellaGreca, Marina; Salvatore, Maria Michela; Borrelli, Francesca; Lombardi, Nadia; Vinale, Francesco; Woo, Sheridan L; Andolfi, Anna

2018-05-31

From the green alga Cladophora sp. collected in Italy, the marine fungal strain A12 of Trichoderma citrinoviride was isolated, identified and characterized. LC-MS qTOF analysis was applied to perform a metabolic profile of the fungal culture. Chromatographic techniques and spectroscopic methods were used to isolate and characterize the major secondary metabolites produced by this strain in liquid culture. In particular, four known sorbicillinoids (trichodermanone C, spirosorbicillinol A, vertinolide and sorbicillin) were purified and identified, together with 2-phenylethanol and tyrosol. Moreover, metabolomic analysis allowed to detect small amounts of trichodimerol, rezishanone A, 2',3'-dihydrosorbicillin and bisvertinol. For the first time a significant inhibitory effect on nitrite levels has been shown for trichodermanone C in lipopolysaccharide-stimulated J774A.1 macrophages.

Brucella spp. of amphibians comprise genomically diverse motile strains competent for replication in macrophages and survival in mammalian hosts

PubMed Central

Al Dahouk, Sascha; Köhler, Stephan; Occhialini, Alessandra; Jiménez de Bagüés, María Pilar; Hammerl, Jens Andre; Eisenberg, Tobias; Vergnaud, Gilles; Cloeckaert, Axel; Zygmunt, Michel S.; Whatmore, Adrian M.; Melzer, Falk; Drees, Kevin P.; Foster, Jeffrey T.; Wattam, Alice R.; Scholz, Holger C.

2017-01-01

Twenty-one small Gram-negative motile coccobacilli were isolated from 15 systemically diseased African bullfrogs (Pyxicephalus edulis), and were initially identified as Ochrobactrum anthropi by standard microbiological identification systems. Phylogenetic reconstructions using combined molecular analyses and comparative whole genome analysis of the most diverse of the bullfrog strains verified affiliation with the genus Brucella and placed the isolates in a cluster containing B. inopinata and the other non-classical Brucella species but also revealed significant genetic differences within the group. Four representative but molecularly and phenotypically diverse strains were used for in vitro and in vivo infection experiments. All readily multiplied in macrophage-like murine J774-cells, and their overall intramacrophagic growth rate was comparable to that of B. inopinata BO1 and slightly higher than that of B. microti CCM 4915. In the BALB/c murine model of infection these strains replicated in both spleen and liver, but were less efficient than B. suis 1330. Some strains survived in the mammalian host for up to 12 weeks. The heterogeneity of these novel strains hampers a single species description but their phenotypic and genetic features suggest that they represent an evolutionary link between a soil-associated ancestor and the mammalian host-adapted pathogenic Brucella species. PMID:28300153

Toxicity and Carcinogenicity Mechanisms of Fibrous Antigorite

PubMed Central

Cardile, Venera; Lombardo, Laura; Belluso, Elena; Panico, Annamaria; Capella, Silvana; Balazy, Michael

2007-01-01

We studied the effects of fibrous antigorite on mesothelial MeT-5A and monocyte-macrophage J774 cell lines to further understand cellular mechanisms induced by asbestos fibers leading to lung damage and cancer. Antigorite is a mineral with asbestiform properties, which tends to associate with chrysotile or tremolite, and frequently occurs as the predominant mineral in the veins of several serpentinite rocks found abundantly in the Western Alps. Particles containing antigorite are more abundant in the breathing air of this region than those typically found in urban ambient air. Exposure of MeT-5A and J774 cells to fibrous antigorite at concentrations of 5–100 μg/ml for 72 hr induced dose-dependent cytotoxicity. Antigorite also stimulated the ROS production, induced the generation of nitrite and PGE2. MeT-5A cells were more sensitive to antigorite than J774 cells. The results of this study revealed that the fibrous antigorite stimulates cyclooxygenase and formation of hydroxyl and nitric oxide radicals. These changes represent early cellular responses to antigorite fibers, which lead to a host of pathological and neoplastic conditions because free radicals and PGE2 play important roles as mediators of tumor pathogenesis. Understanding the mechanisms of the cellular responses to antigorite and other asbestos particles should be helpful in designing rational prevention and treatment approaches. PMID:17431308

Asian and Siberian ginseng as a potential modulator of immune function: an in vitro cytokine study using mouse macrophages.

PubMed

Wang, Huamin; Actor, Jeffrey K; Indrigo, Jessica; Olsen, Margaret; Dasgupta, Amitava

2003-01-01

Ginseng is a widely used herbal product in China, other Asian countries, and in the Unites States. There is a traditional belief that ginseng stimulates immune functions. In this study, the innate effects of Asian and Siberian ginsengs on cytokines and chemokines produced by cultured macrophages were examined. The effects of Asian and Siberian ginseng on cytokines and chemokines produced by cultured macrophages were examined. Mouse macrophages (J774A.1) were incubated with Asian or Siberian ginseng at varying concentrations (1, 10, 100, and 1000 microg/ml) for 24 h and then harvested for RNA isolation. The expression levels of IL-1beta, IL-12, TNF-alpha, MIP-1 alpha, and MIP-2 mRNA were measured by quantitative PCR. Our data showed that Asian ginseng induced a statistically significant increase in IL-12 expression at both mRNA and protein levels. However, the minor twofold increase is probably biologically insignificant. No significant increase of IL-12 by Siberian ginseng was observed at any dose level studied. No significant change in IL-1beta, IL-15, TNF-alpha, or MIP-1alpha mRNA was observed by either Asian or Siberian ginseng treatment. Our data showed statistically significant differential regulation of IL-12 by Asian ginseng. Siberian ginseng did not show a statistically significant increase. We conclude that both Asian ginseng and Siberian ginseng cannot significantly stimulate innate macrophage immune functions that influence cellular immune responses. Therefore, contrary to the popular belief, Asian and Siberian ginseng may not stimulate immune function.

30 CFR 937.774 - Revision; renewal; and transfer, assignment, or sale of permit rights.

Code of Federal Regulations, 2010 CFR

2010-07-01

... sale of permit rights. 937.774 Section 937.774 Mineral Resources OFFICE OF SURFACE MINING RECLAMATION... WITHIN EACH STATE OREGON § 937.774 Revision; renewal; and transfer, assignment, or sale of permit rights. (a) Part 774 of this chapter, Revision; Renewal; and Transfer, Assignment, or Sale of Permit Rights...

30 CFR 921.774 - Revision; renewal; and transfer, assignment, or sale of permit rights.

Code of Federal Regulations, 2010 CFR

2010-07-01

... sale of permit rights. 921.774 Section 921.774 Mineral Resources OFFICE OF SURFACE MINING RECLAMATION... WITHIN EACH STATE MASSACHUSETTS § 921.774 Revision; renewal; and transfer, assignment, or sale of permit rights. (a) Part 774 of this chapter, Revision; Renewal; and Transfer, Assignment, or Sale of Permit...

30 CFR 910.774 - Revision; renewal; and transfer, assignment, or sale of permit rights.

Code of Federal Regulations, 2010 CFR

2010-07-01

... sale of permit rights. 910.774 Section 910.774 Mineral Resources OFFICE OF SURFACE MINING RECLAMATION... WITHIN EACH STATE GEORGIA § 910.774 Revision; renewal; and transfer, assignment, or sale of permit rights. (a) Part 774 of this chapter, Revision; Renewal; and Transfer, Assignment, or Sale of Permit Rights...

30 CFR 939.774 - Revision; renewal; and transfer, assignment, or sale of permit rights.

Code of Federal Regulations, 2010 CFR

2010-07-01

... sale of permit rights. 939.774 Section 939.774 Mineral Resources OFFICE OF SURFACE MINING RECLAMATION... WITHIN EACH STATE RHODE ISLAND § 939.774 Revision; renewal; and transfer, assignment, or sale of permit rights. (a) Part 774 of this chapter, Revision; Renewal; and Transfer, Assignment, or Sale of Permit...

30 CFR 933.774 - Revision; renewal; and transfer, assignment, or sale of permit rights.

Code of Federal Regulations, 2010 CFR

2010-07-01

... sale of permit rights. 933.774 Section 933.774 Mineral Resources OFFICE OF SURFACE MINING RECLAMATION... WITHIN EACH STATE NORTH CAROLINA § 933.774 Revision; renewal; and transfer, assignment, or sale of permit rights. (a) Part 774 of this chapter, Revision; Renewal; and Transfer, Assignment, or Sale of Permit...

30 CFR 912.774 - Revision; renewal; and transfer, assignment, or sale of permit rights.

Code of Federal Regulations, 2010 CFR

2010-07-01

... sale of permit rights. 912.774 Section 912.774 Mineral Resources OFFICE OF SURFACE MINING RECLAMATION... WITHIN EACH STATE IDAHO § 912.774 Revision; renewal; and transfer, assignment, or sale of permit rights. (a) Part 774 of this chapter, Revision; Renewal; and Transfer, Assignment, or Sale of Permit Rights...

30 CFR 947.774 - Revision; renewal; and transfer, assignment, or sale of permit rights.

Code of Federal Regulations, 2010 CFR

2010-07-01

... sale of permit rights. 947.774 Section 947.774 Mineral Resources OFFICE OF SURFACE MINING RECLAMATION... WITHIN EACH STATE WASHINGTON § 947.774 Revision; renewal; and transfer, assignment, or sale of permit rights. (a) Part 774 of this chapter, Revision; Renewal; and Transfer, Assignment, or Sale of Permit...

30 CFR 922.774 - Revision; renewal; and transfer, assignment, or sale of permit rights.

Code of Federal Regulations, 2010 CFR

2010-07-01

... sale of permit rights. 922.774 Section 922.774 Mineral Resources OFFICE OF SURFACE MINING RECLAMATION... WITHIN EACH STATE MICHIGAN § 922.774 Revision; renewal; and transfer, assignment, or sale of permit rights. (a) Part 774 of this chapter, Revision; Renewal; and Transfer, Assignment, or Sale of Permit...

30 CFR 941.774 - Revision; renewal; and transfer, assignment, or sale of permit rights.

Code of Federal Regulations, 2010 CFR

2010-07-01

... sale of permit rights. 941.774 Section 941.774 Mineral Resources OFFICE OF SURFACE MINING RECLAMATION... WITHIN EACH STATE SOUTH DAKOTA § 941.774 Revision; renewal; and transfer, assignment, or sale of permit rights. (a) Part 774 of this chapter, Revision; Renewal; and Transfer, Assignment, or Sale of Permit...

Single-Cell RNA-Seq Reveals the Transcriptional Landscape and Heterogeneity of Aortic Macrophages in Murine Atherosclerosis.

PubMed

Cochain, Clément; Vafadarnejad, Ehsan; Arampatzi, Panagiota; Jaroslav, Pelisek; Winkels, Holger; Ley, Klaus; Wolf, Dennis; Saliba, Antoine-Emmanuel; Zernecke, Alma

2018-03-15

Rationale: It is assumed that atherosclerotic arteries contain several macrophage subsets endowed with specific functions. The precise identity of these subsets is poorly characterized as they ha ve been defined by the expression of a restricted number of markers. Objective: We have applied single-cell RNA-seq as an unbiased profiling strategy to interrogate and classify aortic macrophage heterogeneity at the single-cell level in atherosclerosis. Methods and Results: We performed single-cell RNA sequencing of total aortic CD45 + cells extracted from the non-diseased (chow fed) and atherosclerotic (11 weeks of high fat diet) aorta of Ldlr -/- mice. Unsupervised clustering singled out 13 distinct aortic cell clusters. Among the myeloid cell populations, Resident-like macrophages with a gene expression profile similar to aortic resident macrophages were found in healthy and diseased aortae, whereas monocytes, monocyte-derived dendritic cells (MoDC), and two populations of macrophages were almost exclusively detectable in atherosclerotic aortae, comprising Inflammatory macrophages showing enrichment in I l1b , and previously undescribed TREM2 hi macrophages. Differential gene expression and gene ontology enrichment analyses revealed specific gene expression patterns distinguishing these three macrophage subsets and MoDC, and uncovered putative functions of each cell type. Notably, TREM2 hi macrophages appeared to be endowed with specialized functions in lipid metabolism and catabolism, and presented a gene expression signature reminiscent of osteoclasts, suggesting a role in lesion calcification. TREM2 expression was moreover detected in human lesional macrophages. Importantly, these macrophage populations were present also in advanced atherosclerosis and in Apoe -/- aortae, indicating relevance of our findings in different stages of atherosclerosis and mouse models. Conclusions: These data unprecedentedly uncovered the transcriptional landscape and phenotypic

Antileishmanial and immunomodulatory activity of Xylopia discreta.

PubMed

López, R; Cuca, L E; Delgado, G

2009-10-01

This study aimed at determining the in vitro antileishmanial activity of the essential oil and eight extracts obtained from Xylopia discreta. J774 and U937 macrophages were exposed to the different substances to establish the median lethal concentration (LC(50)). The median effective concentration (EC(50)) was obtained by determining the reduction of Leishmania panamensis-infected cells. A selectivity index (SI) (LC(50)/EC(50)) >or= 20 defined a specific activity for one Xylopia discreta leaf extracts and for the essential oil, being these the two that showed the highest activity (SI = 64.8 and 110, respectively in J774 cells). To assess the substances' immunomodulatory activity, pro- and anti-inflammatory soluble mediators produced after treating infected macrophages were quantified by flow cytometry. The leaf methanol extract and the essential oil induced a differential production of monocyte chemoattractant protein-1, a chemokine associated with a Leishmania-resistant phenotype (Th1).

Mechanisms underlying the redistribution of particles among the lung's alveolar macrophages during alveolar phase clearance

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

Lehnert, B.E.; Oritz, J.B.; Steinkamp, J.A.

1991-01-01

In order to obtain information about the particle redistribution phenomenon following the deposition of inhaled particles, as well as to obtain information about some of the mechanisms that may be operable in the redistribution of particles, lavaged lung free cell analyses and transmission electron microscopic (TEM) analyses of lung tissue and were performed using lungs from rats after they were subchronically exposed to aerosolized dioxide (TiO{sub 2}). TEM analyses indicated that the in situ autolysis of particle-containing Alveolar Macropages (AM) is one important mechanism involved in the redistribution of particles. Evidence was also obtained that indicated that the engulfment ofmore » one particle-containing phagocyte by another phagocyte also occurs. Another prominent mechanism of the particle redistribution phenomenon may be the in situ proliferation of particle-laden AM. We used the macrophage cell line J774A.1 as a surrogate for AM to investigate how different particulate loads in macrophages may affect their abilities to proliferate. These in vitro investigations indicated that the normal rate of proliferation of macrophages is essentially unaffected by the containment of relatively high particulate burdens. Overall, the results of our investigations suggest that in situ autolysis of particle-containing AM and the rephagocytosis of freed particles by other phagocytes, the phagocytosis of effete and disintegrating particle-containing phagocytes by other AM, and the in situ division of particle-containing AM are likely mechanisms that underlie the post-depositional redistribution of particles among the lung's AM during alveolar phase clearance. 19 refs., 8 figs., 2 tabs.« less

Ultrafine particles cause cytoskeletal dysfunctions in macrophages: role of intracellular calcium

PubMed Central

Möller, Winfried; Brown, David M; Kreyling, Wolfgang G; Stone, Vicki

2005-01-01

Background Particulate air pollution is reported to cause adverse health effects in susceptible individuals. Since most of these particles are derived form combustion processes, the primary composition product is carbon with a very small diameter (ultrafine, less than 100 nm in diameter). Besides the induction of reactive oxygen species and inflammation, ultrafine particles (UFP) can cause intracellular calcium transients and suppression of defense mechanisms of alveolar macrophages, such as impaired migration or phagocytosis. Methods In this study the role of intracellular calcium transients caused by UFP was studied on cytoskeleton related functions in J774A.1 macrophages. Different types of fine and ultrafine carbon black particles (CB and ufCB, respectively), such as elemental carbon (EC90), commercial carbon (Printex 90), diesel particulate matter (DEP) and urban dust (UD), were investigated. Phagosome transport mechanisms and mechanical cytoskeletal integrity were studied by cytomagnetometry and cell viability was studied by fluorescence microscopy. Macrophages were exposed in vitro with 100 and 320 μg UFP/ml/million cells for 4 hours in serum free medium. Calcium antagonists Verapamil, BAPTA-AM and W-7 were used to block calcium channels in the membrane, to chelate intracellular calcium or to inhibit the calmodulin signaling pathways, respectively. Results Impaired phagosome transport and increased cytoskeletal stiffness occurred at EC90 and P90 concentrations of 100 μg/ml/million cells and above, but not with DEP or UD. Verapamil and W-7, but not BAPTA-AM inhibited the cytoskeletal dysfunctions caused by EC90 or P90. Additionally the presence of 5% serum or 1% bovine serum albumin (BSA) suppressed the cytoskeletal dysfunctions. Cell viability showed similar results, where co-culture of ufCB together with Verapamil, W-7, FCS or BSA produced less cell dead compared to the particles only. PMID:16202162

Essential role of protein kinase C ζ in transducing a motility signal induced by superoxide and a chemotactic peptide, fMLP

PubMed Central

Kuribayashi, Kageaki; Nakamura, Kiminori; Tanaka, Maki; Sato, Tsutomu; Kato, Junji; Sasaki, Katsunori; Takimoto, Rishu; Kogawa, Katsuhisa; Terui, Takeshi; Takayama, Tetsuji; Onuma, Takayuki; Matsunaga, Takuya; Niitsu, Yoshiro

2007-01-01

Under various pathological conditions, including infection, malignancy, and autoimmune diseases, tissues are incessantly exposed to reactive oxygen species produced by infiltrating inflammatory cells. We show augmentation of motility associated with morphological changes of human squamous carcinoma SASH1 cells, human peripheral monocytes (hPMs), and murine macrophage-like cell line J774.1 by superoxide stimulation. We also disclose that motility of hPMs and J774.1 induced by a chemotactic peptide (N-formyl-methionyl-leucyl-phenylalanine [fMLP]) was inhibited by superoxide dismutase or N-acetylcystein, indicating stimulation of motility by superoxide generated by fMLP stimulation. In these cells, protein kinase C (PKC) ζ was activated to phosphorylate RhoGDI-1, which liberated RhoGTPases, leading to their activation. These events were inhibited by dominant-negative PKCζ in SASH1 cells, myristoylated PKCζ peptides in hPMs and J774.1, or a specific inhibitor of RhoGTPase in SASH1, hPMs, and J774.1. These results suggest a new approach for manipulation of inflammation as well as tumor cell invasion by targeting this novel signaling pathway. PMID:17389234

9 CFR 77.4 - Application for and retention of zones.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 9 Animals and Animal Products 1 2012-01-01 2012-01-01 false Application for and retention of zones. 77.4 Section 77.4 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE INTERSTATE TRANSPORTATION OF ANIMALS (INCLUDING POULTRY) AND ANIMAL PRODUCTS...

9 CFR 77.4 - Application for and retention of zones.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 9 Animals and Animal Products 1 2014-01-01 2014-01-01 false Application for and retention of zones. 77.4 Section 77.4 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE INTERSTATE TRANSPORTATION OF ANIMALS (INCLUDING POULTRY) AND ANIMAL PRODUCTS...

9 CFR 77.4 - Application for and retention of zones.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Application for and retention of zones. 77.4 Section 77.4 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE INTERSTATE TRANSPORTATION OF ANIMALS (INCLUDING POULTRY) AND ANIMAL PRODUCTS...

9 CFR 77.4 - Application for and retention of zones.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 9 Animals and Animal Products 1 2013-01-01 2013-01-01 false Application for and retention of zones. 77.4 Section 77.4 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE INTERSTATE TRANSPORTATION OF ANIMALS (INCLUDING POULTRY) AND ANIMAL PRODUCTS...

9 CFR 77.4 - Application for and retention of zones.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Application for and retention of zones. 77.4 Section 77.4 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE INTERSTATE TRANSPORTATION OF ANIMALS (INCLUDING POULTRY) AND ANIMAL PRODUCTS...

Early Acidification of Phagosomes Containing Brucella suis Is Essential for Intracellular Survival in Murine Macrophages

PubMed Central

Porte, Françoise; Liautard, Jean-Pierre; Köhler, Stephan

1999-01-01

Brucella suis is a facultative intracellular pathogen of mammals, residing in macrophage vacuoles. In this work, we studied the phagosomal environment of these bacteria in order to better understand the mechanisms allowing survival and multiplication of B. suis. Intraphagosomal pH in murine J774 cells was determined by measuring the fluorescence intensity of opsonized, carboxyfluorescein-rhodamine- and Oregon Green 488-rhodamine-labeled bacteria. Compartments containing live B. suis acidified to a pH of about 4.0 to 4.5 within 60 min. Acidification of B. suis-containing phagosomes in the early phase of infection was abolished by treatment of host cells with 100 nM bafilomycin A1, a specific inhibitor of vacuolar proton-ATPases. This neutralization at 1 h postinfection resulted in a 2- to 34-fold reduction of opsonized and nonopsonized viable intracellular bacteria at 4 and 6 h postinfection, respectively. Ammonium chloride and monensin, other pH-neutralizing reagents, led to comparable loss of intracellular viability. Addition of ammonium chloride at 7 h after the beginning of infection, however, did not affect intracellular multiplication of B. suis, in contrast to treatment at 1 h postinfection, where bacteria were completely eradicated within 48 h. Thus, we conclude that phagosomes with B. suis acidify rapidly after infection, and that this early acidification is essential for replication of the bacteria within the macrophage. PMID:10417172

23 CFR 774.13 - Exceptions.

Code of Federal Regulations, 2010 CFR

2010-04-01

..., WILDLIFE AND WATERFOWL REFUGES, AND HISTORIC SITES (SECTION 4(F)) § 774.13 Exceptions. The Administration... result of the consultation under 36 CFR 800.5, that such work will not adversely affect the historic... recreation lands, wildlife and waterfowl refuges, and historic sites that are made, or determinations of...

15 CFR 774.2 - [Reserved

Code of Federal Regulations, 2010 CFR

2010-01-01

...” that “incorporate” commodities or software on the Commerce Control List (Supplement No. 1 to part 774... the practice of medicine (does not include medical research). (2) Commodities or software are considered “incorporated” if the commodity or software is: Essential to the functioning of the medical...

7 CFR 774.18 - Interest rate, terms and security requirements.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 7 Agriculture 7 2010-01-01 2010-01-01 false Interest rate, terms and security requirements. 774.18..., DEPARTMENT OF AGRICULTURE SPECIAL PROGRAMS EMERGENCY LOAN FOR SEED PRODUCERS PROGRAM § 774.18 Interest rate, terms and security requirements. (a) Interest rate. (1) The interest rate on the loan will be zero...

Genome-Wide Survey and Expression Profiling of CCCH-Zinc Finger Family Reveals a Functional Module in Macrophage Activation

PubMed Central

Liang, Jian; Song, Wenjun; Tromp, Gail; Kolattukudy, Pappachan E.; Fu, Mingui

2008-01-01

Previously, we have identified a novel CCCH zinc finger protein family as negative regulators of macrophage activation. To gain an overall insight into the entire CCCH zinc finger gene family and to evaluate their potential role in macrophage activation, here we performed a genome-wide survey of CCCH zinc finger genes in mouse and human. Totally 58 CCCH zinc finger genes in mouse and 55 in human were identified and most of them have not been reported previously. Phylogenetic analysis revealed that the mouse CCCH family was divided into 6 groups. Meanwhile, we employed quantitative real-time PCR to profile their tissue expression patterns in adult mice. Clustering analysis showed that most of CCCH genes were broadly expressed in all of tissues examined with various levels. Interestingly, several CCCH genes Mbnl3, Zfp36l2, Zfp36, Zc3h12a, Zc3h12d, Zc3h7a and Leng9 were enriched in macrophage-related organs such as thymus, spleen, lung, intestine and adipose. Consistently, a comprehensive assessment of changes in expression of the 58 members of the mouse CCCH family during macrophage activation also revealed that these CCCH zinc finger genes were associated with the activation of bone marrow-derived macrophages by lipopolysaccharide. Taken together, this study not only identified a functional module of CCCH zinc finger genes in the regulation of macrophage activation but also provided the framework for future studies to dissect the function of this emerging gene family. PMID:18682727

Medicinal potential from in vivo and acclimatized plants of Cleome rosea.

PubMed

Simões, Claudia; De Mattos, José Carlos P; Sabino, Kátia C C; Caldeira-de-Araújo, Adriano; Coelho, Marsen G P; Albarello, Norma; Figueiredo, Solange F L

2006-02-01

Methanolic extracts obtained from different organs of Cleome rosea, collected from its natural habitat and from in vitro-propagated plants, were submitted to in vitro biological assays. Inhibition of nitric oxide (NO) production by J774 macrophages and antioxidant effects by protecting the plasmid DNA from the SnCl(2)-induced damage were evaluated. Extracts from the stem of both origins and leaf of natural plants inhibited NO production. The plasmid DNA strand breaks induced by SnCl(2) were reduced by extracts from either leaf or stem of both sources. On the other hand, root extracts did not show any kind of effects on plasmid DNA, and presented significant toxic effects to J774 cells. The results showed that C. rosea presents medicinal potential and that the acclimatization process reduces the plant toxicity both to plasmid DNA and to J774 cells, suggesting the use of biotechnology tools to obtain elite plants as source of botanical material for pharmacological and phytochemical studies.

7 CFR 774.24 - Exception.

Code of Federal Regulations, 2010 CFR

2010-01-01

... SPECIAL PROGRAMS EMERGENCY LOAN FOR SEED PRODUCERS PROGRAM § 774.24 Exception. The Agency may grant an exception to any of the requirements of this section, if the proposed change is in the best financial interest of the Government and not inconsistent with the authorizing statute or other applicable law. ...

The in vitro kinetics of the interactions between PEG-ylated magnetic-fluid-loaded liposomes and macrophages.

PubMed

Martina, Marie-Sophie; Nicolas, Valerie; Wilhelm, Claire; Ménager, Christine; Barratt, Gillian; Lesieur, Sylviane

2007-10-01

Binding and uptake kinetics of magnetic-fluid-loaded liposomes (MFL) by endocytotic cells were investigated in vitro on the model cell-line J774. MFL consisted of unilamellar phosphatidylcholine vesicles (mean hydrodynamic diameter close to 200nm) encapsulating 8-nm nanocrystals of maghemite (gamma-Fe(2)O(3)) and sterically stabilized by introducing 5mol% of distearylphosphatidylcholine poly(ethylene glycol)(2,000) (DSPE-PEG(2,000)) in the vesicle bilayer. The association processes with living macrophages were followed at two levels. On one hand, the lipid vesicles were imaged by confocal fluorescence microscopy. For this purpose 1mol% of rhodamine-marked phosphatidylethanolamine was added to the liposome composition. On the other hand, the iron oxide particles associated with cells were independently quantified by magnetophoresis. All the experiments were similarly performed with PEG-ylated or conventional MFL to point out the role of polymer coating. The results showed cell association with both types of liposomes resulting from binding followed by endocytosis. Steric stabilization by PEG chains reduced binding efficiency limiting the amount of MFL internalized by the macrophages. In contrast, PEG coating did not change the kinetics of endocytosis which exhibited the same first-order rate constant for both conventional and PEG-ylated liposomes. Moreover, lipids and iron oxide particle uptakes were perfectly correlated, indicating that MFL vesicle structure and encapsulation rate were preserved upon cell penetration.

30 CFR 774.12 - Post-permit issuance information requirements for permittees.

Code of Federal Regulations, 2010 CFR

2010-07-01

... for permittees. 774.12 Section 774.12 Mineral Resources OFFICE OF SURFACE MINING RECLAMATION AND ENFORCEMENT, DEPARTMENT OF THE INTERIOR SURFACE COAL MINING AND RECLAMATION OPERATIONS PERMITS AND COAL... competent jurisdiction grants a stay of the cessation order and the stay remains in effect. (c) Within 60...

Biochemical Characterization, Action on Macrophages, and Superoxide Anion Production of Four Basic Phospholipases A2 from Panamanian Bothrops asper Snake Venom

PubMed Central

Rueda, Aristides Quintero; Rodríguez, Isela González; Arantes, Eliane C.; Setúbal, Sulamita S.; Calderon, Leonardo de A.; Zuliani, Juliana P.; Stábeli, Rodrigo G.; Soares, Andreimar M.

2013-01-01

Bothrops asper (Squamata: Viperidae) is the most important venomous snake in Central America, being responsible for the majority of snakebite accidents. Four basic PLA2s (pMTX-I to -IV) were purified from crude venom by a single-step chromatography using a CM-Sepharose ion-exchange column (1.5 × 15 cm). Analysis of the N-terminal sequence demonstrated that pMTX-I and III belong to the catalytically active Asp49 phospholipase A2 subclass, whereas pMTX-II and IV belong to the enzymatically inactive Lys49 PLA2s-like subclass. The PLA2s isolated from Panama Bothrops asper venom (pMTX-I, II, III, and IV) are able to induce myotoxic activity, inflammatory reaction mainly leukocyte migration to the muscle, and induce J774A.1 macrophages activation to start phagocytic activity and superoxide production. PMID:23509779

Vpx complementation of 'non-macrophage tropic' R5 viruses reveals robust entry of infectious HIV-1 cores into macrophages.

PubMed

Mlcochova, Petra; Watters, Sarah A; Towers, Greg J; Noursadeghi, Mahdad; Gupta, Ravindra K

2014-03-21

It is now known that clinically derived viruses are most commonly R5 tropic with very low infectivity in macrophages. As these viruses utilize CD4 inefficiently, defective entry has been assumed to be the dominant restriction. The implication is that macrophages are not an important reservoir for the majority of circulating viruses. Macrophage infection by clinical transmitted/founder isolates was 10-100 and 30-450 fold less efficient as compared to YU-2 and BaL respectively. Vpx complementation augmented macrophage infection by non-macrophage tropic viruses to the level of infectivity observed for YU-2 in the absence of Vpx. Augmentation was evident even when Vpx was provided 24 hours post-infection. The entry defect was measured as 2.5-5 fold, with a further 3.5-10 fold block at strong stop and subsequent stages of reverse transcription as compared to YU-2. The overall block to infection was critically dependent on the mechanism of entry as demonstrated by rescue of infection after pseudotyping with VSV-G envelope. Reverse transcription in macrophages could not be enhanced using a panel of cytokines or lipopolysaccharide (LPS). Although the predominant block to clinical transmitted/founder viruses is post-entry, infectivity is determined by Env-CD4 interactions and can be rescued with VSV-G pseudotyping. This suggests a functional link between the optimal entry pathway taken by macrophage tropic viruses and downstream events required for reverse transcription. Consistent with a predominantly post-entry block, replication of R5 using viruses can be greatly enhanced by Vpx. We conclude therefore that entry is not the limiting step and that macrophages represent clinically relevant reservoirs for 'non-macrophage tropic' viruses.

Evaluation of in vitro anti-inflammatory effects of crude ginger and rosemary extracts obtained through supercritical CO2 extraction on macrophage and tumor cell line: the influence of vehicle type.

PubMed

Justo, Oselys Rodriguez; Simioni, Patricia Ucelli; Gabriel, Dirce Lima; Tamashiro, Wirla Maria da Silva Cunha; Rosa, Paulo de Tarso Vieira; Moraes, Ângela Maria

2015-10-29

Numerous plants from have been investigated due to their anti-inflammatory activity and, among then, extracts or components of ginger (Zingiber officinale Roscoe) and rosemary (Rosmarinus officinalis L.), sources of polyphenolic compounds. 6-gingerol from ginger rhizome and carnosic acid and carnosol from rosemary leaves present anti-tumor, anti-inflammatory and antioxidant activities. However, the evaluation of the mechanisms of action of these and other plant extracts is limited due to their high hydrophobicity. Dimethylsulfoxide (DMSO) is commonly used as a vehicle of liposoluble materials to mammalian cells in vitro, presenting enhanced cell penetration. Liposomes are also able to efficiently deliver agents to mammalian cells, being capable to incorporate in their structure not only hydrophobic molecules, but also hydrophilic and amphiphilic compounds. Another strategy is based on the use of Pluronic F-68, a biocompatible low-foaming, non-ionic surfactant, to disperse hydrophobic components. Here, these three delivery approaches were compared to analyze their influence on the in vitro anti-inflammatory effects of ginger and rosemary extracts, at different concentrations, on primary mammalian cells and on a tumor cell line. Ginger and rosemary extracts free of organic solvents were obtained by supercritical fluid extraction and dispersed in DMSO, Pluronic F-68 or liposomes, in variable concentrations. Cell viability, production of inflammatory mediators and nitric oxide (NO) release were measured in vitro on J774 cell line and murine macrophages primary culture stimulated with bacterial lipopolysaccharide and interferon-γ after being exposed or not to these extracts. Ginger and rosemary extracts obtained by supercritical CO2 extraction inhibited the production of pro-inflammatory cytokines and the release of NO by peritoneal macrophages and J774 cells. The delivery vehicles influenced the anti-inflammatory effects. Comparatively, the ginger extract showed the

Dextran loading protects macrophages from lipid peroxidation and induces a Keap1/Nrf2/ARE-dependent antioxidant response.

PubMed

Chechushkov, Anton; Zaitseva, Natalia; Vorontsova, Elena; Kozhin, Petr; Menshchikova, Elena; Shkurupiy, Vyacheslav

2016-12-01

Linear dextrans are often proposed as drug delivery systems with milder adverse effects and lower effective drug concentrations. Linear dextrans are polysaccharides that can potentially be used to load macrophages with drugs to transport them to a site of inflammation. Recently, it was reported that dextrans may exert a protective effect vis-à-vis drug cytotoxicity and during wound healing. The aim of the current work was to evaluate molecular mechanisms of action of dextrans that may be relevant to the cytoprotective effects. We determined the effect of treatment with 40- or 70-kDa dextran on production of reactive oxygen species, lipid peroxidation, and lysosomal pH in the J774 macrophage cell line. In addition, induction of Keap1/Nrf2/ARE and autophagic activity were evaluated. Dextrans of both molecular weights protected the cells from oxidative stress induced by cumene hydroperoxide and from lysosomal stress induced by ammonium chloride. The effect was associated with induction of the Keap1/Nrf2/ARE signaling pathway. Furthermore, dextran stimulated autophagy in a dose-dependent manner but inhibited the autophagosome-lysosome fusion in a time-dependent manner. This study shows possible cytoprotective effects of dextran under oxidative stress, and these findings may be used for the development of novel (dextran-based) drug delivery approaches. Copyright © 2016 Elsevier Inc. All rights reserved.

LL-37 directs macrophage differentiation toward macrophages with a proinflammatory signature.

PubMed

van der Does, Anne M; Beekhuizen, Henry; Ravensbergen, Bep; Vos, Tim; Ottenhoff, Tom H M; van Dissel, Jaap T; Drijfhout, Jan W; Hiemstra, Pieter S; Nibbering, Peter H

2010-08-01

The human cathelicidin LL-37 has broad-spectrum antimicrobial activity. It also participates at the interface of innate and adaptive immunity by chemoattracting immune effector cells, modulating the production of a variety of inflammatory mediators by different cell types, and regulating the differentiation of monocytes into dendritic cells. In this study, we investigated the effects of LL-37 on the differentiation of human monocytes into anti-inflammatory macrophages (MPhi-2; driven by M-CSF) versus proinflammatory macrophages (MPhi-1; driven by GM-CSF) as well as on fully differentiated MPhi-1 and MPhi-2. Results revealed that monocytes cultured with M-CSF in the presence of LL-37 resulted in macrophages displaying a proinflammatory signature, namely, low expression of CD163 and little IL-10 and profound IL-12p40 production on LPS stimulation. The effects of LL-37 on M-CSF-driven macrophage differentiation were dose- and time-dependent with maximal effects observed at 10 microg/ml when the peptide was present from the start of the cultures. The peptide enhanced the GM-CSF-driven macrophage differentiation. Exposure of fully differentiated MPhi-2 to LL-37 for 6 d resulted in macrophages that produced less IL-10 and more IL-12p40 on LPS stimulation than control MPhi-2. In contrast, LL-37 had no effect on fully differentiated MPhi-1. Peptide mapping using a set of 16 overlapping 22-mer peptides covering the complete LL-37 sequence revealed that the C-terminal portion of LL-37 is responsible for directing macrophage differentiation. Our results furthermore indicate that the effects of LL-37 on macrophage differentiation required internalization of the peptide. Together, we conclude that LL-37 directs macrophage differentiation toward macrophages with a proinflammatory signature.

Evaluation of the efficacy of photodynamic antimicrobial therapy using a phenothiazine compound and Laser (λ=660ηm) on the interface: macrophage vs S. aureus

NASA Astrophysics Data System (ADS)

de Oliveira, Susana C. P. S.; Monteiro, Juliana S. C.; Pires-Santos, Gustavo M.; Sampaio, Fernando José P.; Zanin, Fátima Antônia A.; Pinheiro, Antônio L. B.

2015-03-01

Nowadays photodynamic inactivation has been proposed as an alternative treatment for localized bacterial infections as a response to the problem of antibiotic resistance. Much is already known about the photodynamic inactivation of microorganisms: both antibiotic-sensitive and -resistant strains can be successfully photoinactivated and there is the additional advantage that repeated photosensitization of bacterial cells does not induce a selection of resistant strains. Staphylococcus spp. are opportunistic microorganisms known for their capacity to develop resistance against antimicrobial agents. The emergence of resistant strains of bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) poses a major challenge to healthcare. MRSA is a major cause of hospital-acquired infection throughout the world and is now also prevalent in the community as well as nursing and residential homes. The aim of this study was to evaluate the phagocytic function of macrophages J774 against S. aureus in the presence and absence of AmPDT with phenothiazine compound (12.5 μg/mL) and low level laser (λ=660nm, 12 J/cm²). Experimental groups: Control group (L-P-), Phenothiazine group (L-P+) Laser group (L+P-), AmPDT group (L+P+).The tests presented in this study were performed in triplicate. This study showed that AmPDT induced bacterial death in about 80% as well as increasing phagocytic capacity of macrophages by approximately 20% and enhanced the antimicrobial activity by approximately 50% compared to the control group and enabling more intense oxidative burst.

Microparticles prepared with 50-190kDa chitosan as promising non-toxic carriers for pulmonary delivery of isoniazid.

PubMed

Oliveira, Paula M; Matos, Breno N; Pereira, Priscilla A T; Gratieri, Taís; Faccioli, Lucia H; Cunha-Filho, Marcílio S S; Gelfuso, Guilherme M

2017-10-15

Chitosan biocompatibility and mucoadhesiveness make it an ideal polymer for antituberculotic drugs microcapsulation for pulmonary delivery. Yet, previous study indicated toxicity problems to J-774.1-cells treated with some medium molecular weight (190-310kDa) chitosan microparticles. As polymer molecular weight is a crucial factor to be considered, this paper describes the preparation and characterization of chitosan (50-190kDa) microparticles containing isoniazid (INH). Cytotoxicity assays were also performed on murine peritoneal (J-774.1) and alveolar (AMJ2-C11) macrophages cell lines, followed by cytokines detection from AMJ2-C11 cells. Spray-drying process produced mucoadhesive microparticles from 3.2μm to 3.9μm, entrapping more than 89% of the drug and preserving their chemical stability. Drug release behavior could be controlled by the use of cross-linked or uncross-linked chitosan, the latter leading to a rapid drug release. Mucoadhesive potential of the microparticles was characterized following in vitro and ex vivo assays. Finally, a significant reduction on toxicity against peritoneal macrophages and no toxic effect on alveolar macrophages with use of such microparticles were observed. In conclusion, 50-190kDa chitosan microparticles may act as promising non-cytotoxic carriers for pulmonary delivery of INH showing marked alveoli macrophage activation. Copyright © 2017 Elsevier Ltd. All rights reserved.

AMP-Conjugated Quantum Dots: Low Immunotoxicity Both In Vitro and In Vivo

NASA Astrophysics Data System (ADS)

Dai, Tongcheng; Li, Na; Liu, Lu; Liu, Qin; Zhang, Yuanxing

2015-11-01

Quantum dots (QDs) are engineered nanoparticles that possess special optical and electronic properties and have shown great promise for future biomedical applications. In this work, adenosine 5'-monophosphate (AMP), a small biocompatible molecular, was conjugated to organic QDs to produce hydrophilic AMP-QDs. Using macrophage J774A.1 as the cell model, AMP-QDs exhibited both prior imaging property and low toxicity, and more importantly, triggered limited innate immune responses in macrophage, indicating low immunotoxicity in vitro. Using BALB/c mice as the animal model, AMP-QDs were found to be detained in immune organs but did not evoke robust inflammation responses or obvious histopathological abnormalities, which reveals low immunotoxicity in vivo. This work suggests that AMP is an excellent surface ligand with low immunotoxicity, and potentially used in surface modification for more extensive nanoparticles.

MiR-146a modulates macrophage polarization by inhibiting Notch1 pathway in RAW264.7 macrophages.

PubMed

Huang, Cheng; Liu, Xue-Jiao; QunZhou; Xie, Juan; Ma, Tao-Tao; Meng, Xiao-Ming; Li, Jun

2016-03-01

Macrophages are heterogeneous and plastic cells which are able to undergo dynamic transition between M1 and M2 polarized phenotypes in response to the microenvironment signals. However, the underlying molecular mechanisms of macrophage polarization are still obscure. In the current study, it was revealed that miR-146a might play a pivotal role in macrophage polarization. As our results indicated, miR-146a was highly expressed in M2 macrophages rather than M1 macrophages. Over-expression of miR-146a resulted in significantly decreased production of pro-inflammatory cytokines including iNOS and TNF-α in M1 macrophages, while increased production of M2 marker genes such as Arg1 and CD206 in M2 macrophages. In contrast, knockdown of miR-146a promoted M1 macrophage polarization but diminished M2 macrophage polarization. Mechanistically, it was revealed that miR-146a modulated macrophage polarization by targeting Notch1. Of note, PPARγ was responsible as another target for miR-146a-mediated macrophage polarization. Taken together, it was suggested that miR-146a might serve as a molecular regulator in macrophage polarization and is a potential therapeutic target for inflammatory diseases. Copyright © 2016 Elsevier B.V. All rights reserved.

The thymus of the hairless rhino-j (hr/hr-j) mice

PubMed Central

SAN JOSE, I.; GARCÍA-SUÁREZ, O.; HANNESTAD, J.; CABO, R.; GAUNA, L.; REPRESA, J.; VEGA, J. A.

2001-01-01

The hairless (hr) gene is expressed in a large number of tissues, primarily the skin, and a mutation in the hr gene is responsible for the typical cutaneous phenotype of hairless mice. Mutant hr mouse strains show immune defects involving especially T cells and macrophages, as well as an age-related immunodeficiency and an accelerated atrophy of the thymus. These data suggest that the hr mutation causes a defect of this organ, although hr transcripts have not been detected in fetal or adult mice thymus. The present study analyses the thymus of young (3 mo) and adult (9 mo) homozygous hr-rh-j mice (a strain of hairless mice) by means of structural techniques and immunohistochemistry to selectively identify thymic epithelial cells, dendritic cells, and macrophages. There were structural alterations in the thymus of both young and adult rh-rh-j mice, which were more severe in older animals. These alterations consisted of relative cortical atrophy, enlargement of blood vessels, proliferation of perivascular connective tissue, and the appearance of cysts. hr-rh-j mice also showed a decrease in the number of epithelial and dendritic cells, and macrophages. Taken together, present results strongly suggest degeneration and accelerated age-dependent regression of the thymus in hr-rh-j mice, which could explain at least in part the immune defects reported in hairless mouse strains. PMID:11327202

In vitro activity of synthetic tetrahydroindeno[2,1-c]quinolines on Leishmania mexicana.

PubMed

Hernández-Chinea, Concepción; Carbajo, Erika; Sojo, Felipe; Arvelo, Francisco; Kouznetsov, Vladimir V; Romero-Bohórquez, Arnold R; Romero, Pedro J

2015-12-01

New synthetic compounds based on tetrahydroindenoquinoline structure were evaluated for their in vitro antileishmanial activities. The seven compounds assayed have antiproliferative activities against promastigotes of Leishmania mexicana. Compound 1 and 3 were the most active (IC50 1.0 μg/ml) and showed high selectivity towards the parasite. These compounds were selected to evaluate their effect on promastigote morphology and mitochondrial transmembrane potential as well as on the amastigote capability to survive into macrophages J774 cell line. Whereas compound 1 affected the promastigote cell cycle, compound 3 induced morphological changes and the total collapse of the mitochondrial transmembrane potential, a hallmark of apoptosis. Both compounds also affected the amastigote form of the parasite, decreasing their survival rate in J774 macrophages. Due to the greatest selectivity index, the apparent effect as apoptotic inducer and its sustained inhibition on intracellular amastigote replication, compound 3 is the best candidate to be tested in vivo. This compound is worth considering for the development of new antileishmanial drugs. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Uptake and intracellular activity of AM-1155 in phagocytic cells.

PubMed Central

Yamamoto, T; Kusajima, H; Hosaka, M; Fukuda, H; Oomori, Y; Shinoda, H

1996-01-01

The uptake and intracellular activity of AM-1155 in murine J774.1 macrophages and human polymorphonuclear leukocytes were investigated. AM-1155 penetrated phagocytic cells rapidly and reversibly, although the penetration process was not affected by metabolic inhibitors such as sodium fluoride, cyanide m-chlorophenylhydrazone, or ouabain or by nucleoside transport system inhibitors such as adenosine. The intracellular concentration-to-extracellular concentration ratio of AM-1155 in both cell types of phagocytes ranged from 5 to 7. These ratios were almost equal to those for sparfloxacin. The intracellular activity of AM-1155 in J774.1 macrophages, examined with Staphylococcus aureus 209P as a test bacterium, was dependent on the extracellular concentration. AM-1155 at a concentration of 1 microgram/ml reduced the number of viable cells of S. aureus ingested by more than 90%. The intracellular activity of AM-1155 was more potent than those of sparfloxacin, ofloxacin, ciprofloxacin, flomoxef, and erythromycin. These results suggest that the potent intracellular activity of AM-1155 might mainly be due to the high intracellular concentration and its potent in vitro activity. PMID:9124835

15 CFR Supplement No. 1 to Part 774 - The Commerce Control List

Code of Federal Regulations, 2010 CFR

2015-01-01

... 15 Commerce and Foreign Trade 2 2015-01-01 2015-01-01 false The Commerce Control List No. Supplement No. 1 to Part 774 Commerce and Foreign Trade Regulations Relating to Commerce and Foreign Trade (Continued) BUREAU OF INDUSTRY AND SECURITY, DEPARTMENT OF COMMERCE EXPORT ADMINISTRATION REGULATIONS THE COMMERCE CONTROL LIST Pt. 774, Supp. 1...

15 CFR Supplement No. 1 to Part 774 - The Commerce Control List

Code of Federal Regulations, 2010 CFR

2010-01-01

... 15 Commerce and Foreign Trade 2 2010-01-01 2010-01-01 false The Commerce Control List No. Supplement No. 1 to Part 774 Commerce and Foreign Trade Regulations Relating to Commerce and Foreign Trade (Continued) BUREAU OF INDUSTRY AND SECURITY, DEPARTMENT OF COMMERCE EXPORT ADMINISTRATION REGULATIONS THE COMMERCE CONTROL LIST Pt. 774, Supp. 1...

15 CFR Supplement No. 1 to Part 774 - The Commerce Control List

Code of Federal Regulations, 2013 CFR

2013-01-01

... 15 Commerce and Foreign Trade 2 2013-01-01 2013-01-01 false The Commerce Control List No. Supplement No. 1 to Part 774 Commerce and Foreign Trade Regulations Relating to Commerce and Foreign Trade (Continued) BUREAU OF INDUSTRY AND SECURITY, DEPARTMENT OF COMMERCE EXPORT ADMINISTRATION REGULATIONS THE COMMERCE CONTROL LIST Pt. 774, Supp. 1...

15 CFR Supplement No. 1 to Part 774 - The Commerce Control List

Code of Federal Regulations, 2011 CFR

2011-01-01

... 15 Commerce and Foreign Trade 2 2011-01-01 2011-01-01 false The Commerce Control List No. Supplement No. 1 to Part 774 Commerce and Foreign Trade Regulations Relating to Commerce and Foreign Trade (Continued) BUREAU OF INDUSTRY AND SECURITY, DEPARTMENT OF COMMERCE EXPORT ADMINISTRATION REGULATIONS THE COMMERCE CONTROL LIST Pt. 774, Supp. 1...

15 CFR Supplement No. 1 to Part 774 - The Commerce Control List

Code of Federal Regulations, 2012 CFR

2012-01-01

... 15 Commerce and Foreign Trade 2 2012-01-01 2012-01-01 false The Commerce Control List No. Supplement No. 1 to Part 774 Commerce and Foreign Trade Regulations Relating to Commerce and Foreign Trade (Continued) BUREAU OF INDUSTRY AND SECURITY, DEPARTMENT OF COMMERCE EXPORT ADMINISTRATION REGULATIONS THE COMMERCE CONTROL LIST Pt. 774, Supp. 1...

15 CFR Supplement No. 1 to Part 774 - The Commerce Control List

Code of Federal Regulations, 2014 CFR

2014-01-01

... 15 Commerce and Foreign Trade 2 2014-01-01 2014-01-01 false The Commerce Control List No. Supplement No. 1 to Part 774 Commerce and Foreign Trade Regulations Relating to Commerce and Foreign Trade (Continued) BUREAU OF INDUSTRY AND SECURITY, DEPARTMENT OF COMMERCE EXPORT ADMINISTRATION REGULATIONS THE COMMERCE CONTROL LIST Pt. 774, Supp. 1...

In vitro uptake of apoptotic body mimicking phosphatidylserine-quantum dot micelles by monocytic cell line

NASA Astrophysics Data System (ADS)

Maiseyeu, Andrei; Bagalkot, Vaishali

2014-04-01

A new quantum dot (QD) PEGylated micelle laced with phosphatidylserine (PS) for specific scavenger receptor-mediated uptake by macrophages is reported. The size and surface chemistry of PS-QD micelles were characterized by standard methods and the effects of their physicochemical properties on specific targeting and uptake were comprehensively studied in a monocytic cell line (J774A.1).

15 CFR Supplement No. 1 to Part 774 - The Commerce Control List

Code of Federal Regulations, 2010 CFR

2017-01-01

... 15 Commerce and Foreign Trade 2 2017-01-01 2017-01-01 false The Commerce Control List No. Supplement No. 1 to Part 774 Commerce and Foreign Trade Regulations Relating to Commerce and Foreign Trade (Continued) BUREAU OF INDUSTRY AND SECURITY, DEPARTMENT OF COMMERCE EXPORT ADMINISTRATION REGULATIONS THE COMMERCE CONTROL LIST Pt. 774, Supp. No. 1...

15 CFR Supplement No. 1 to Part 774 - The Commerce Control List

Code of Federal Regulations, 2010 CFR

2016-01-01

... 15 Commerce and Foreign Trade 2 2016-01-01 2016-01-01 false The Commerce Control List No. Supplement No. 1 to Part 774 Commerce and Foreign Trade Regulations Relating to Commerce and Foreign Trade (Continued) BUREAU OF INDUSTRY AND SECURITY, DEPARTMENT OF COMMERCE EXPORT ADMINISTRATION REGULATIONS THE COMMERCE CONTROL LIST Pt. 774, Supp. No. 1...

15 CFR Supplement No. 1 to Part 774 - The Commerce Control List

Code of Federal Regulations, 2010 CFR

2018-01-01

... 15 Commerce and Foreign Trade 2 2018-01-01 2018-01-01 false The Commerce Control List No. Supplement No. 1 to Part 774 Commerce and Foreign Trade Regulations Relating to Commerce and Foreign Trade (Continued) BUREAU OF INDUSTRY AND SECURITY, DEPARTMENT OF COMMERCE EXPORT ADMINISTRATION REGULATIONS THE COMMERCE CONTROL LIST Pt. 774, Supp. No. 1...

Quinolactacins A, B and C: novel quinolone compounds from Penicillium sp. EPF-6. I. Taxonomy, production, isolation and biological properties.

PubMed

Kakinuma, N; Iwai, H; Takahashi, S; Hamano, K; Yanagisawa, T; Nagai, K; Tanaka, K; Suzuki, K; Kirikae, F; Kirikae, T; Nakagawa, A

2000-11-01

Quinolactacins A (1), B (2) and C (3), novel quinolone antibiotics have been found from the cultured broth of a fungal strain isolated from the larvae of the mulberry pyralid Margaronia pyloalis Welker). The fungal strain, EPF-6 was identified as Penicillium sp. from its morphological characteristics. Quinolactacins were obtained from the culture medium by solvent extraction and chromatographic purification. Compound 1 showed inhibitory activity against tumor necrosis factor (TNF) production induced by murine peritoneal macrophages and macrophage-like J774.1 cells stimulated with lipopolysaccharide (LPS).

The Effect of Garlic Extract on Expression of INFγ And Inos Genes in Macrophages Infected with Leishmania major

PubMed Central

Gharavi, MJ; Nobakht, M; Khademvatan, SH; Bandani, E; Bakhshayesh, M; Roozbehani, M

2011-01-01

Background The study was aimed to show the effect of molecular mechanism of Aqueous Garlic Extract (AGE) on expression of IFNγ and iNOS genes in Leishmania major. Methods Leishmania major promastigotes (MRHO/IR/75/ER) were added to the in-vitro cultured J774 cell line, the cells were incubated for 72 hours. Various concentrations of garlic extract (9.25, 18.5, 37, 74, 148 mg/ml) were added to the infected cells. MTT assay was applied for cellular proliferation. After 72 hours of incubation, supernatants were collected and total RNA was extracted from the infected cells. The express of IFNγ and iNOS genes were studied by RT-PCR method. Results The colorimetric MTT assay after 3 days of incubation showed cytotoxic effect of garlic extract with an IC50 of 37 mg/ml. In addition, IFNγ and iNOS genes expression by RT-PCR indicated that garlic extract lead to over expression of these genes in J774 cell line infected with L. major. Conclusion Garlic extract exerts cytotoxic effect on infected J774 cell line. In addition, the hypothesis that garlic can improve cellular immunity with raising the expression of IFNγ and of iNOS genes confirmed. PMID:22347300

Manipulation of NF-KappaBetta Activity in the Macrophage Lineage as a Novel Therapeutic Approach

DTIC Science & Technology

2007-05-01

Sadikot, J. W. Christman, and T. S. Blackwell. 2003. Bioluminescent detection of endotoxin effects on HIV-1 LTR-driven transcription in vivo. J...differences in proliferation rates, expression of downstream gene expression and effects mediated by altered macrophages on associated epithelial...kappaB activity within macrophages has significant effects on mammary ductal development. 15. SUBJECT TERMS NF-kappaB, macrophages, mammary ductal

Influence of conversion of penicillin G into a basic derivative on its accumulation and subcellular localization in cultured macrophages

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

Renard, C.; Vanderhaeghe, H.J.; Claes, P.J.

beta-Lactam antibiotics do not accumulate in phagocytes, probably because of their acidic character. We therefore synthesized a basic derivative of penicillin G, namely, /sup 14/C-labeled N-(3-dimethylamino-propyl)benzylpenicillinamide (ABP), and studied its uptake and subcellular localization in J774 macrophages compared with that of /sup 14/C-labeled penicillin G. Whereas the intracellular concentration (Ci) of penicillin G remained lower than its extracellular concentration (Ce), ABP reached a Ci/Ce ratio of 4 to 5. Moreover, approximately 50% of intracellular ABP was found associated with lysosomes after isopycnic centrifugation of cell homogenates in isoosmotic Percoll or hyperosmotic sucrose gradients. The behavior of ABP was thus partlymore » consistent with the model of de Duve et al., in which they described the intralysosomal accumulation of weak organic bases in lysosomes. Although ABP is microbiologically inactive, our results show that beta-lactam antibiotics can be driven into cells by appropriate modification. Further efforts therefore may be warranted in the design of active compounds or prodrugs that may prove useful in the chemotherapy of intracellular infections.« less

Systems Toxicology of Male Reproductive Development: Profiling 774 Chemicals for Molecular Targets and Adverse Outcomes

PubMed Central

Leung, Maxwell C.K.; Phuong, Jimmy; Baker, Nancy C.; Sipes, Nisha S.; Klinefelter, Gary R.; Martin, Matthew T.; McLaurin, Keith W.; Setzer, R. Woodrow; Darney, Sally Perreault; Judson, Richard S.; Knudsen, Thomas B.

2015-01-01

Background: Trends in male reproductive health have been reported for increased rates of testicular germ cell tumors, low semen quality, cryptorchidism, and hypospadias, which have been associated with prenatal environmental chemical exposure based on human and animal studies. Objective: In the present study we aimed to identify significant correlations between environmental chemicals, molecular targets, and adverse outcomes across a broad chemical landscape with emphasis on developmental toxicity of the male reproductive system. Methods: We used U.S. EPA’s animal study database (ToxRefDB) and a comprehensive literature analysis to identify 774 chemicals that have been evaluated for adverse effects on male reproductive parameters, and then used U.S. EPA’s in vitro high-throughput screening (HTS) database (ToxCastDB) to profile their bioactivity across approximately 800 molecular and cellular features. Results: A phenotypic hierarchy of testicular atrophy, sperm effects, tumors, and malformations, a composite resembling the human testicular dysgenesis syndrome (TDS) hypothesis, was observed in 281 chemicals. A subset of 54 chemicals with male developmental consequences had in vitro bioactivity on molecular targets that could be condensed into 156 gene annotations in a bipartite network. Conclusion: Computational modeling of available in vivo and in vitro data for chemicals that produce adverse effects on male reproductive end points revealed a phenotypic hierarchy across animal studies consistent with the human TDS hypothesis. We confirmed the known role of estrogen and androgen signaling pathways in rodent TDS, and importantly, broadened the list of molecular targets to include retinoic acid signaling, vascular remodeling proteins, G-protein coupled receptors (GPCRs), and cytochrome P450s. Citation: Leung MC, Phuong J, Baker NC, Sipes NS, Klinefelter GR, Martin MT, McLaurin KW, Setzer RW, Darney SP, Judson RS, Knudsen TB. 2016. Systems toxicology of male

Pertussis toxin permeabilization enhances the traversal of Escherichia coli K1, macrophages, and monocytes in a cerebral endothelial barrier model in vitro.

PubMed

Seidel, Gabriela; Böcker, Kathrin; Schulte, Jessica; Wewer, Corinna; Greune, Lilo; Humberg, Verena; Schmidt, M Alexander

2011-03-01

The occasionally severe neurological complications following the human respiratory tract infection 'whooping cough' have been attributed to pertussis toxin (PT) expressed by the causative agent Bordetella pertussis. Disruption of the endothelial blood-brain barrier (BBB) by PT might facilitate the translocation of immune cells and of hematogenous microbial pathogens. To test this hypothesis, we investigated whether PT enhances the traversal of bacteria employing human brain microvascular endothelial cells (HBMEC) as an in vitro endothelial barrier model. PT incubation significantly increased the translocation of Escherichia coli K1 across the HBMEC barrier. Only intercellular E. coli K1 bacteria could be identified by electron microscopy suggesting paracellular translocation. In addition, the migration of differentiated HL60-derived macrophages and of human monocytic U937 cells through PT-treated HBMEC barriers was also enhanced. In comparison to E. coli C600, E. coli K1 showed prolonged survival in translocated HL60-derived and J774 macrophages as well as in U937 monocytes which suggested a contribution of the 'Trojan horse' mechanism. In summary, our findings demonstrate that the PT-induced permeabilization of endothelial barriers enhances the paracellular transmigration of microbes and immune cells. In vivo, this activity might lower the threshold of bacteremia facilitating secondary cerebral infections and the subsequent development of brain pathologies. Copyright © 2010 Elsevier GmbH. All rights reserved.

Plexin C1 deficiency permits synaptotagmin 7–mediated macrophage migration and enhances mammalian lung fibrosis

PubMed Central

Peng, Xueyan; Moore, Meagan; Mathur, Aditi; Zhou, Yang; Sun, Huanxing; Gan, Ye; Herazo-Maya, Jose D.; Kaminski, Naftali; Hu, Xinyuan; Pan, Hongyi; Ryu, Changwan; Osafo-Addo, Awo; Homer, Robert J.; Feghali-Bostwick, Carol; Fares, Wassim H.; Gulati, Mridu; Hu, Buqu; Lee, Chun-Geun; Elias, Jack A.; Herzog, Erica L.

2016-01-01

Pulmonary fibrosis is a progressive and often fatal condition that is believed to be partially orchestrated by macrophages. Mechanisms that control migration of these cells into and within the lung remain undefined. We evaluated the contributions of the semaphorin receptor, plexin C1 (PLXNC1), and the exocytic calcium sensor, synaptotagmin 7 (Syt7), in these processes. We evaluated the role of PLXNC1 in macrophage migration by using Boyden chambers and scratch tests, characterized its contribution to experimentally induced lung fibrosis in mice, and defined the mechanism for our observations. Our findings reveal that relative to control participants, patients with idiopathic pulmonary fibrosis demonstrate excessive monocyte migration and underexpression of PLXNC1 in the lungs and circulation, a finding that is recapitulated in the setting of scleroderma-related interstitial lung disease. Relative to wild type, PLXNC1−/− mouse macrophages are excessively migratory, and PLXNC1−/− mice show exacerbated collagen accumulation in response to either inhaled bleomycin or inducible lung targeted TGF-β1 overexpression. These findings are ameliorated by replacement of PLXNC1 on bone marrow–derived cells or by genetic deletion of Syt7. These data demonstrate the previously unrecognized observation that PLXNC1 deficiency permits Syt7-mediated macrophage migration and enhances mammalian lung fibrosis.—Peng, X., Moore, M., Mathur, A., Zhou, Y., Sun, H., Gan, Y., Herazo-Maya, J. D., Kaminski, N., Hu, X., Pan, H., Ryu, C., Osafo-Addo, A., Homer, R. J., Feghali-Bostwick, C., Fares, W. H., Gulati, M., Hu, B., Lee, C.-G., Elias, J. A., Herzog, E. L. Plexin C1 deficiency permits synaptotagmin 7–mediated macrophage migration and enhances mammalian lung fibrosis. PMID:27609773

15 CFR Supplement No. 2 to Part 774 - General Technology and Software Notes

Code of Federal Regulations, 2010 CFR

2010-01-01

... 15 Commerce and Foreign Trade 2 2010-01-01 2010-01-01 false General Technology and Software Notes... REGULATIONS THE COMMERCE CONTROL LIST Pt. 774, Supp. 2 Supplement No. 2 to Part 774—General Technology and Software Notes 1. General Technology Note. The export of “technology” that is “required” for the...

Dynamic activation of basilar membrane macrophages in response to chronic sensory cell degeneration in aging mouse cochleae

PubMed Central

Frye, Mitchell D.; Yang, Weiping; Zhang, Celia; Xiong, Binbin; Hu, Bo Hua

2016-01-01

In the sensory epithelium, macrophages have been identified on the scala tympani side of the basilar membrane. These basilar membrane macrophages are the spatially closest immune cells to sensory cells and are able to directly respond to and influence sensory cell pathogenesis. While basilar membrane macrophages have been studied in acute cochlear stresses, their behavior in response to chronic sensory cell degeneration is largely unknown. Here we report a systematic observation of the variance in phenotypes, the changes in morphology and distribution of basilar membrane tissue macrophages in different age groups of C57BL/6J mice, a mouse model of age-related sensory cell degeneration. This study reveals that mature, fully differentiated tissue macrophages, not recently infiltrated monocytes, are the major macrophage population for immune responses to chronic sensory cell death. These macrophages display dynamic changes in their numbers and morphologies as age increases, and the changes are related to the phases of sensory cell degeneration. Notably, macrophage activation precedes sensory cell pathogenesis, and strong macrophage activity is maintained until sensory cell degradation is complete. Collectively, these findings suggest that mature tissue macrophages on the basilar membrane are a dynamic group of cells that are capable of vigorous adaptation to changes in the local sensory epithelium environment influenced by sensory cell status. PMID:27837652

Dynamic activation of basilar membrane macrophages in response to chronic sensory cell degeneration in aging mouse cochleae.

PubMed

Frye, Mitchell D; Yang, Weiping; Zhang, Celia; Xiong, Binbin; Hu, Bo Hua

2017-02-01

In the sensory epithelium, macrophages have been identified on the scala tympani side of the basilar membrane. These basilar membrane macrophages are the spatially closest immune cells to sensory cells and are able to directly respond to and influence sensory cell pathogenesis. While basilar membrane macrophages have been studied in acute cochlear stresses, their behavior in response to chronic sensory cell degeneration is largely unknown. Here we report a systematic observation of the variance in phenotypes, the changes in morphology and distribution of basilar membrane tissue macrophages in different age groups of C57BL/6J mice, a mouse model of age-related sensory cell degeneration. This study reveals that mature, fully differentiated tissue macrophages, not recently infiltrated monocytes, are the major macrophage population for immune responses to chronic sensory cell death. These macrophages display dynamic changes in their numbers and morphologies as age increases, and the changes are related to the phases of sensory cell degeneration. Notably, macrophage activation precedes sensory cell pathogenesis, and strong macrophage activity is maintained until sensory cell degradation is complete. Collectively, these findings suggest that mature tissue macrophages on the basilar membrane are a dynamic group of cells that are capable of vigorous adaptation to changes in the local sensory epithelium environment influenced by sensory cell status. Copyright © 2016 Elsevier B.V. All rights reserved.

Alternatively activated macrophages (M2 macrophages) in the skin of patient with localized scleroderma.

PubMed

Higashi-Kuwata, Nobuyo; Makino, Takamitsu; Inoue, Yuji; Takeya, Motohiro; Ihn, Hironobu

2009-08-01

Localized scleroderma is a connective tissue disorder that is limited to the skin and subcutaneous tissue. Macrophages have been reported to be particularly activated in patients with skin disease including systemic sclerosis and are potentially important sources for fibrosis-inducing cytokines, such as transforming growth factor beta. To clarify the features of immunohistochemical characterization of the immune cell infiltrates in localized scleroderma focusing on macrophages, skin biopsy specimens were analysed by immunohistochemistry. The number of cells stained with monoclonal antibodies, CD68, CD163 and CD204, was calculated. An evident macrophage infiltrate and increased number of alternatively activated macrophages (M2 macrophages) in their fibrotic areas were observed along with their severity of inflammation. This study revealed that alternatively activated macrophages (M2 macrophages) may be a potential source of fibrosis-inducing cytokines in localized scleroderma, and may play a crucial role in the pathogenesis of localized scleroderma.

NFAT5-Regulated Macrophage Polarization Supports the Proinflammatory Function of Macrophages and T Lymphocytes.

PubMed

Tellechea, Mónica; Buxadé, Maria; Tejedor, Sonia; Aramburu, Jose; López-Rodríguez, Cristina

2018-01-01

Macrophages are exquisite sensors of tissue homeostasis that can rapidly switch between pro- and anti-inflammatory or regulatory modes to respond to perturbations in their microenvironment. This functional plasticity involves a precise orchestration of gene expression patterns whose transcriptional regulators have not been fully characterized. We had previously identified the transcription factor NFAT5 as an activator of TLR-induced responses, and in this study we explore its contribution to macrophage functions in different polarization settings. We found that both in classically and alternatively polarized macrophages, NFAT5 enhanced functions associated with a proinflammatory profile such as bactericidal capacity and the ability to promote Th1 polarization over Th2 responses. In this regard, NFAT5 upregulated the Th1-stimulatory cytokine IL-12 in classically activated macrophages, whereas in alternatively polarized ones it enhanced the expression of the pro-Th1 mediators Fizz-1 and arginase 1, indicating that it could promote proinflammatory readiness by regulating independent genes in differently polarized macrophages. Finally, adoptive transfer assays in vivo revealed a reduced antitumor capacity in NFAT5-deficient macrophages against syngeneic Lewis lung carcinoma and ID8 ovarian carcinoma cells, a defect that in the ID8 model was associated with a reduced accumulation of effector CD8 T cells at the tumor site. Altogether, detailed analysis of the effect of NFAT5 in pro- and anti-inflammatory macrophages uncovered its ability to regulate distinct genes under both polarization modes and revealed its predominant role in promoting proinflammatory macrophage functions. Copyright © 2017 by The American Association of Immunologists, Inc.

Monocyte to macrophage differentiation-associated (MMD) positively regulates ERK and Akt activation and TNF-α and NO production in macrophages.

PubMed

Liu, Qiang; Zheng, Jin; Yin, Dan-Dan; Xiang, Jie; He, Fei; Wang, Yao-Chun; Liang, Liang; Qin, Hong-Yan; Liu, Li; Liang, Ying-Min; Han, Hua

2012-05-01

Macrophage activation is modulated by both environmental cues and endogenous programs. In the present study, we investigated the role of a PAQR family protein, monocyte to macrophage differentiation-associated (MMD), in macrophage activation and unveiled its underlying molecular mechanism. Our results showed that while MMD expression could be detected in all tissues examined, its expression level is significantly up-regulated upon monocyte differentiation. Within cells, EGFP-MMD fusion protein could be co-localized to endoplasmic reticulum, mitochondria, Golgi apparatus, but not lysosomes and cytoplasm. MMD expression is up-regulated in macrophages after LPS stimulation, and this might be modulated by RBP-J, the critical transcription factor of Notch signaling. Overexpression of MMD in macrophages increased the production of TNF-α and NO upon LPS stimulation. We found that MMD overexpression enhanced ERK1/2 and Akt phosphorylation in macrophages after LPS stimulation. Blocking Erk or Akt by pharmacological agent reduced TNF-α or NO production in MMD-overexpressing macrophages, respectively. These results suggested that MMD modulates TNF-α and NO production in macrophages, and this process might involves Erk or Akt.

Cyclosporin A and tacrolimus (FK506) suppress expression of inducible nitric oxide synthase in vitro by different mechanisms

PubMed Central

Dusting, Gregory J; Akita, Kazuhiro; Hickey, Haruyo; Smith, Melanie; Gurevich, Vladimir

1999-01-01

The effects of the immunosuppressant drugs cyclosporin A and tacrolimus (FK506) on nitric oxide synthesis were examined in a murine macrophage cell line (J774) and rat vascular smooth muscle cells (VSMC) in culture for 24 and 48 h, respectively.Cyclosporin A (0.01–10 μM) inhibited by up to 90% accumulation of nitrite induced by lipopolysaccharide (LPS) in both cell lines, but FK506 (0.01–10 μM) had a weaker effect on nitrite accumulation in these cells. Cyclosporin A and FK506 (at 1 μM) also significantly inhibited nitrite production induced by recombinant murine interferon-γ (rIFNγ) and recombinant murine interleukin-1β (rIL-1β) in J774 and VSMC, respectively.In J774 cells, cyclosporin A (but not FK506) at 1 μM was inhibitory when co-incubated with the inducing agents but not when the cells were treated with the immunosuppressant before or after the inducer. In VSMC, nitrite production was inhibited by co-incubation of cyclosporin A or FK506 with the inducer, or when the immunosuppressants were pre-incubated with cells. In contrast, N-monomethyl L-arginine (NMMA) abolished nitrite production when incubated with either cell type during or after addition of inducing agent, but not if cells were preincubated with NMMA.RNA extracted from treated J774 and VSMC was subjected to reverse transcription–polymerase chain reaction (RT–PCR). Cyclosporin A, but not FK506, suppressed expression of mRNA for NOS2 in a concentration-dependent manner when co-incubated with LPS.The fact that the potency difference between cyclosporin A and FK506 for NO suppression is the opposite to that for inhibition of interleukin-2 generation suggests that the immunosuppressants act in J774 macrophages and VSMC through intracellular mechanisms that differ from those elucidated in T-cells. Cyclosporin A suppresses NOS2 gene transcription, but FK506 acts post-transcriptionally to suppress NO generation in VSMC.Taken together the present data suggest that therapeutic

Evaluation of the efficacy of photodynamic antimicrobial therapy using a phenothiazine compound and LED (red-orange) on the interface: macrophage vs S. aureus

NASA Astrophysics Data System (ADS)

Sampaio, Fernando José P.; de Oliveira, Susana C. P. S.; Monteiro, Juliana S. C.; Pires-Santos, Gustavo M.; Gesteira, Maria F. M.; Pinheiro, Antônio L. B.

2015-03-01

Antimicrobial Photodynamic therapy is a technique in which microorganisms are exposed to a photosensitizing drug and then irradiated with low-intensity visible light of the appropriate wavelength. The resulting photochemical reaction generates cytotoxic reactive oxygen species, such as singlet oxygen and free radicals, which are able to exert bactericidal effect. Much is already known about the photodynamic inactivation of microorganisms: both antibiotic-sensitive and - resistant strains can be successfully photo inactivated, and there is the additional advantage that repeated photosensitization of bacterial cells does not induce a selection of resistant strains. Recently, a series of studies have shown that it is possible to kill bacteria with a light source after the microorganisms have been sensitized with low concentration of dye, such as phenothiazines. The aim of this study was to evaluate the phagocytic function of macrophages J774 against S. aureus in the presence and absence of AmPDT with phenothiazine compound (12.5 μg/mL) and red-orange LED. Experimental groups: Control Group (L-F-), Phenothiazine group (L-F+) LED group (L+F-), Photodynamic therapy group (L+F+). The tests presented in this study were carried out in triplicate. This study demonstrated that AmPDT is able to increase about twice the phagocytic ability of macrophages; however, the bactericidal capacity of these cells did not show a substantial improvement, probably because the oxidative burst was less intense.

Endoplasmic reticulum chaperone GRP78 regulates macrophage function and insulin resistance in diet-induced obesity.

PubMed

Kim, Jong Hun; Lee, Eunjung; Friedline, Randall H; Suk, Sujin; Jung, Dae Young; Dagdeviren, Sezin; Hu, Xiaodi; Inashima, Kunikazu; Noh, Hye Lim; Kwon, Jung Yeon; Nambu, Aya; Huh, Jun R; Han, Myoung Sook; Davis, Roger J; Lee, Amy S; Lee, Ki Won; Kim, Jason K

2018-04-01

Obesity-mediated inflammation is a major cause of insulin resistance, and macrophages play an important role in this process. The 78-kDa glucose-regulated protein (GRP78) is a major endoplasmic reticulum chaperone that modulates unfolded protein response (UPR), and mice with GRP78 heterozygosity were resistant to diet-induced obesity. Here, we show that mice with macrophage-selective ablation of GRP78 (Lyz- GRP78 -/- ) are protected from skeletal muscle insulin resistance without changes in obesity compared with wild-type mice after 9 wk of high-fat diet. GRP78-deficient macrophages demonstrated adapted UPR with up-regulation of activating transcription factor (ATF)-4 and M2-polarization markers. Diet-induced adipose tissue inflammation was reduced, and bone marrow-derived macrophages from Lyz- GRP78 -/- mice demonstrated a selective increase in IL-6 expression. Serum IL-13 levels were elevated by >4-fold in Lyz- GRP78 -/- mice, and IL-6 stimulated the myocyte expression of IL-13 and IL-13 receptor. Lastly, recombinant IL-13 acutely increased glucose metabolism in Lyz- GRP78 -/- mice. Taken together, our data indicate that GRP78 deficiency activates UPR by increasing ATF-4, and promotes M2-polarization of macrophages with a selective increase in IL-6 secretion. Macrophage-derived IL-6 stimulates the myocyte expression of IL-13 and regulates muscle glucose metabolism in a paracrine manner. Thus, our findings identify a novel crosstalk between macrophages and skeletal muscle in the modulation of obesity-mediated insulin resistance.-Kim, J. H., Lee, E., Friedline, R. H., Suk, S., Jung, D. Y., Dagdeviren, S., Hu, X., Inashima, K., Noh, H. L., Kwon, J. Y., Nambu, A., Huh, J. R., Han, M. S., Davis, R. J., Lee, A. S., Lee, K. W., Kim, J. K. Endoplasmic reticulum chaperone GRP78 regulates macrophage function and insulin resistance in diet-induced obesity.

The response of macrophages to titanium particles is determined by macrophage polarization.

PubMed

Pajarinen, Jukka; Kouri, Vesa-Petteri; Jämsen, Eemeli; Li, Tian-Fang; Mandelin, Jami; Konttinen, Yrjö T

2013-11-01

Aseptic loosening of total joint replacements is driven by the reaction of macrophages to foreign body particles released from the implant. It was hypothesized that the macrophages' response to these particles is dependent, in addition to particle characteristics and contaminating biomolecules, on the state of macrophage polarization as determined by the local cytokine microenvironment. To test this hypothesis we differentiated M1 and M2 macrophages from human peripheral blood monocytes and compared their responses to titanium particles using genome-wide microarray analysis and a multiplex cytokine assay. In comparison to non-activated M0 macrophages, the overall chemotactic and inflammatory responses to titanium particles were greatly enhanced in M1 macrophages and effectively suppressed in M2 macrophages. In addition, the genome-wide approach revealed several novel, potentially osteolytic, particle-induced mediators, and signaling pathway analysis suggested the involvement of toll-like and nod-like receptor signaling in particle recognition. It is concluded that the magnitude of foreign body reaction caused by titanium particles is dependent on the state of macrophage polarization. Thus, by limiting the action of M1 polarizing factors, e.g. bacterial biofilm formation, in peri-implant tissues and promoting M2 macrophage polarization by biomaterial solutions or pharmacologically, it might be possible to restrict wear-particle-induced inflammation and osteolysis. Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Live imaging of wound angiogenesis reveals macrophage orchestrated vessel sprouting and regression.

PubMed

Gurevich, David B; Severn, Charlotte E; Twomey, Catherine; Greenhough, Alexander; Cash, Jenna; Toye, Ashley M; Mellor, Harry; Martin, Paul

2018-06-04

Wound angiogenesis is an integral part of tissue repair and is impaired in many pathologies of healing. Here, we investigate the cellular interactions between innate immune cells and endothelial cells at wounds that drive neoangiogenic sprouting in real time and in vivo Our studies in mouse and zebrafish wounds indicate that macrophages are drawn to wound blood vessels soon after injury and are intimately associated throughout the repair process and that macrophage ablation results in impaired neoangiogenesis. Macrophages also positively influence wound angiogenesis by driving resolution of anti-angiogenic wound neutrophils. Experimental manipulation of the wound environment to specifically alter macrophage activation state dramatically influences subsequent blood vessel sprouting, with premature dampening of tumour necrosis factor-α expression leading to impaired neoangiogenesis. Complementary human tissue culture studies indicate that inflammatory macrophages associate with endothelial cells and are sufficient to drive vessel sprouting via vascular endothelial growth factor signalling. Subsequently, macrophages also play a role in blood vessel regression during the resolution phase of wound repair, and their absence, or shifted activation state, impairs appropriate vessel clearance. © 2018 The Authors. Published under the terms of the CC BY 4.0 license.

Influence of particle geometry and PEGylation on phagocytosis of particulate carriers.

PubMed

Mathaes, Roman; Winter, Gerhard; Besheer, Ahmed; Engert, Julia

2014-04-25

Particle geometry of micro- and nanoparticles has been identified as an important design parameter to influence the interaction with cells such as macrophages. A head to head comparison of elongated, non-spherical and spherical micro- and nanoparticles with and without PEGylation was carried out to benchmark two phagocytosis inhibiting techniques. J774.A1 macrophages were incubated with fluorescently labeled PLGA micro- and nanoparticles and analyzed by confocal laser scanning microscope (CLSM) and flow cytometry (FACS). Particle uptake into macrophages was significantly reduced upon PEGylation or elongated particle geometry. A combination of both, an elongated shape and PEGylation, had the strongest phagocytosis inhibiting effect for nanoparticles. Copyright © 2014 Elsevier B.V. All rights reserved.

Reactive oxygen species (ROS) induced cytokine production and cytotoxicity of PAMAM dendrimers in J774A.1 cells

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

Naha, Pratap C., E-mail: pratap.naha@dit.i; NanoLab, Focas Research Institute, Dublin Institute of Technology, Kevin Street, Dublin 8; Davoren, Maria

2010-07-15

The immunotoxicity of three generations of polyamidoamine (PAMAM) dendrimers (G-4, G-5 and G-6) was evaluated in mouse macrophage cells in vitro. Using the Alamar blue and MTT assays, a generation dependent cytotoxicity of the PAMAM dendrimers was found whereby G-6 > G-5 > G-4. The toxic response of the PAMAM dendrimers correlated well with the number of surface primary amino groups, with increasing number resulting in an increase in toxic response. An assessment of intracellular ROS generation by the PAMAM dendrimers was performed by measuring the increased fluorescence as a result of intracellular oxidation of Carboxy H{sub 2}DCFDA to DCFmore » both quantitatively using plate reader and qualitatively by confocal laser scanning microscopy. The inflammatory mediators macrophage inflammatory protein-2 (MIP-2), tumour necrosis factor-{alpha} (TNF-{alpha}) and interleukin-6, (IL-6) were measured by the enzyme linked immunosorbant assay (ELISA) following exposure of mouse macrophage cells to PAMAM dendrimers. A generation dependent ROS and cytokine production was found, which correlated well with the cytotoxicological response and therefore number of surface amino groups. A clear time sequence of increased ROS generation (maximum at {approx} 4 h), TNF-{alpha} and IL-6 secretion (maximum at {approx} 24 h), MIP-2 levels and cell death ({approx} 72 h) was observed. The intracellular ROS generation and cytokine production induced cytotoxicity point towards the mechanistic pathway of cell death upon exposure to PAMAM dendrimers.« less

Effect of low power laser irradiation on macrophage phagocytic capacity

NASA Astrophysics Data System (ADS)

Lu, Cuixia; Song, Sheng; Tang, Yu; Zhou, Feifan

2011-03-01

Phagocytosis and subsequent degradation of pathogens by macrophages play a pivotal role in host innate immunity in mammals. Laser irradiation has been found to produce photobiological effects with evidence of interference with immunological functions. However, the effects of laser on the immune response have not been extensively characterized. In this study, we focused our attention on the effects of He-Ne laser on the phagocytic activity of macrophages by using flow cytometry (FCM). After irradiating at fluence of 0, 1, 2 J/cm2 with He-Ne laser (632.8 nm, 3mw), the cells were incubated with microsphere and then subjected to FACS analysis. The results showed that Low-power laser irradiation (LPLI) leads to an increase in phagocytosis on both mouse peritoneal macrophages and the murine macrophage-like cell line RAW264.7. In addition, we demonstrated that LPLI increased phagocytosis of microsphere in a dose-dependent manner, reaching a maximum at fluence of 2 J/cm2. Taken together, our results indicated that Low-power laser irradiation with appropriate dosage can enhance the phagocytosis of macrophage, and provided a theoretical base for the clinical use of the He-Ne laser.

Targeted killing of Leishmania donovani in vivo and in vitro with amphotericin B attached to functionalized carbon nanotubes.

PubMed

Prajapati, Vijay Kumar; Awasthi, Kalpana; Gautam, Shalini; Yadav, Thakur Prasad; Rai, Madhukar; Srivastava, Onkar Nath; Sundar, Shyam

2011-04-01

This study describes the antileishmanial efficacy of the novel drug formulation of amphotericin B (AmB) attached to functionalized carbon nanotubes (f-CNTs) and compares it with AmB. f-CNTs were prepared in a two-step chemical carboxylation and amidation process. The AmB was then attached to make f-CNT-AmB and its construction was confirmed by Fourier transform infrared (FTIR) spectroscopy and transmission electron microscopy (TEM). The cytotoxicity of the constructed compound, f-CNT-AmB, was assessed in vitro using the J774A.1 macrophage cell line and in vivo using healthy BALB/c mice. Antileishmanial activity of AmB and f-CNT-AmB was assessed in vitro using a macrophage (J774A.1 cell line) model of Leishmania donovani infection. Antileishmanial activity was assessed in vivo by comparing the parasite load of hamsters treated with a 5 day course of AmB, f-CNTs or f-CNT-AmB initiated at 30 days after infection with L. donovani parasites. The FTIR spectroscopy and TEM data demonstrate the successful attachment of AmB to f-CNTs. The in vitro cytotoxicity of AmB, f-CNTs and f-CNT-AmB was measured by the cytotoxic concentration required to kill 50% of the cells: 0.48±0.06 μg/mL; 7.31±1.16 μg/mL; 0.66±0.17 μg/mL, respectively, in the J774A.1 cell line. The in vivo toxicity assessment of the compounds in BALB/c mice revealed no hepatic or renal toxicity. Against intracellular amastigotes the in vitro antileishmanial efficacy of f-CNT-AmB was significantly higher than that of AmB (IC50 0.00234±0.00075 μg/mL versus 0.03263±0.00123 μg/mL; P≤0.0001). The percentage inhibition of amastigote replication in hamsters treated with f-CNT-AmB was significantly more than that with AmB (89.85%±2.93% versus 68.97%±1.84%; P=0.0004). The results of these experiments clearly demonstrate that f-CNT-AmB has significantly greater antileishmanial efficacy than AmB and had no significant cytotoxic effects.

The effect of aluminium and sodium impurities on the in vitro toxicity and pro-inflammatory potential of cristobalite

USGS Publications Warehouse

Nattrass, C.; Horwell, Claire J.; Damby, David; Brown, David; Stone, Vicki

2017-01-01

BackgroundExposure to crystalline silica (SiO2), in the form of quartz, tridymite or cristobalite, can cause respiratory diseases, such as silicosis. However, the observed toxicity and pathogenicity of crystalline silica is highly variable. This has been attributed to a number of inherent and external factors, including the presence of impurities. In cristobalite-rich dusts, substitutions of aluminium (Al) for silicon (Si) in the cristobalite structure, and impurities occluding the silica surface, have been hypothesised to decrease its toxicity. This hypothesis is tested here through the characterisation and in vitro toxicological study of synthesised cristobalite with incremental amounts of Al and sodium (Na) dopants. MethodsSamples of synthetic cristobalite with incremental amounts of Al and Na impurities, and tridymite, were produced through heating of a silica sol-gel. Samples were characterised for mineralogy, cristobalite purity and abundance, particle size, surface area and surface charge. In vitro assays assessed the ability of the samples to induce cytotoxicity and TNF-α production in J774 macrophages, and haemolysis of red blood cells. ResultsAl-only doped or Al+Na co-doped cristobalite contained between 1 and 4 oxide wt% Al and Na within its structure. Co-doped samples also contained Al- and Na-rich phases, such as albite. Doping reduced cytotoxicity to J774 macrophages and haemolytic capacity compared to non-doped samples. Al-only doping was more effective at decreasing cristobalite reactivity than Al+Na co-doping. The reduction in the reactivity of cristobalite is attributed to both structural impurities and a lower abundance of crystalline silica in doped samples. Neither non-doped nor doped crystalline silica induced production of the pro-inflammatory cytokine TNF-α in J774 macrophages. ConclusionsImpurities can reduce the toxic potential of cristobalite and may help explain the low reactivity of some cristobalite-rich dusts. Whilst further work

The microbiota metabolite indole inhibits Salmonella virulence: Involvement of the PhoPQ two-component system.

PubMed

Kohli, Nandita; Crisp, Zeni; Riordan, Rebekah; Li, Michael; Alaniz, Robert C; Jayaraman, Arul

2018-01-01

The microbial community present in the gastrointestinal tract is an important component of the host defense against pathogen infections. We previously demonstrated that indole, a microbial metabolite of tryptophan, reduces enterohemorrhagic Escherichia coli O157:H7 attachment to intestinal epithelial cells and biofilm formation, suggesting that indole may be an effector/attenuator of colonization for a number of enteric pathogens. Here, we report that indole attenuates Salmonella Typhimurium (Salmonella) virulence and invasion as well as increases resistance to colonization in host cells. Indole-exposed Salmonella colonized mice less effectively compared to solvent-treated controls, as evident by competitive index values less than 1 in multiple organs. Indole-exposed Salmonella demonstrated 160-fold less invasion of HeLa epithelial cells and 2-fold less invasion of J774A.1 macrophages compared to solvent-treated controls. However, indole did not affect Salmonella intracellular survival in J774A.1 macrophages suggesting that indole primarily affects Salmonella invasion. The decrease in invasion was corroborated by a decrease in expression of multiple Salmonella Pathogenicity Island-1 (SPI-1) genes. We also identified that the effect of indole was mediated by both PhoPQ-dependent and independent mechanisms. Indole also synergistically enhanced the inhibitory effect of a short chain fatty acid cocktail on SPI-1 gene expression. Lastly, indole-treated HeLa cells were 70% more resistant to Salmonella invasion suggesting that indole also increases resistance of epithelial cells to colonization. Our results demonstrate that indole is an important microbiota metabolite that has direct anti-infective effects on Salmonella and host cells, revealing novel mechanisms of pathogen colonization resistance.

The microbiota metabolite indole inhibits Salmonella virulence: Involvement of the PhoPQ two-component system

PubMed Central

Kohli, Nandita; Crisp, Zeni; Riordan, Rebekah; Li, Michael; Alaniz, Robert C.

2018-01-01

The microbial community present in the gastrointestinal tract is an important component of the host defense against pathogen infections. We previously demonstrated that indole, a microbial metabolite of tryptophan, reduces enterohemorrhagic Escherichia coli O157:H7 attachment to intestinal epithelial cells and biofilm formation, suggesting that indole may be an effector/attenuator of colonization for a number of enteric pathogens. Here, we report that indole attenuates Salmonella Typhimurium (Salmonella) virulence and invasion as well as increases resistance to colonization in host cells. Indole-exposed Salmonella colonized mice less effectively compared to solvent-treated controls, as evident by competitive index values less than 1 in multiple organs. Indole-exposed Salmonella demonstrated 160-fold less invasion of HeLa epithelial cells and 2-fold less invasion of J774A.1 macrophages compared to solvent-treated controls. However, indole did not affect Salmonella intracellular survival in J774A.1 macrophages suggesting that indole primarily affects Salmonella invasion. The decrease in invasion was corroborated by a decrease in expression of multiple Salmonella Pathogenicity Island-1 (SPI-1) genes. We also identified that the effect of indole was mediated by both PhoPQ-dependent and independent mechanisms. Indole also synergistically enhanced the inhibitory effect of a short chain fatty acid cocktail on SPI-1 gene expression. Lastly, indole-treated HeLa cells were 70% more resistant to Salmonella invasion suggesting that indole also increases resistance of epithelial cells to colonization. Our results demonstrate that indole is an important microbiota metabolite that has direct anti-infective effects on Salmonella and host cells, revealing novel mechanisms of pathogen colonization resistance. PMID:29342189

Simplified Quantitative Assay System for Measuring Activities of Drugs against Intracellular Legionella pneumophila

PubMed Central

Higa, Futoshi; Kusano, Nobuchika; Tateyama, Masao; Shinzato, Takashi; Arakaki, Noriko; Kawakami, Kazuyoshi; Saito, Atsushi

1998-01-01

We developed a new simple assay for the quantitation of the activities of drugs against intracellular Legionella pneumophila. The cells of a murine macrophage-like cell line (J774.1 cells) allowed the intracellular growth and replication of the bacteria, which ultimately resulted in cell death. The infected J774.1 cell monolayers in 96-well microplates were first treated with antibiotics and were further cultured for 72 h. The number of viable J774.1 cells in each well was quantified by a colorimetric assay with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and an enzyme-linked immunosorbent assay reader. The number of growing bacteria in each well was also determined by counting the numbers of CFU on buffered charcoal yeast extract-α agar plates. Viable J774.1 cell counts, determined by the colorimetric assay, were inversely proportional to the number of intracellular replicating bacteria. The minimum extracellular concentrations (MIECs) of 24 antibiotics causing inhibition of intracellular growth of L. pneumophila were determined by the colorimetric assay system. The MIECs of beta-lactams and aminoglycosides were markedly higher than the MICs in buffered yeast extract-α broth. The MIECs of macrolides, fluoroquinolones, rifampin, and minocycline were similar to the respective MICs. According to their intracellular activities, clarithromycin and sparfloxacin were the most potent among the macrolides or fluoroquinolones tested in this study. Our results indicated that the MTT assay system allows comparative and quantitative evaluations of the intracellular activities of antibiotics and efficient processing of a large number of samples. PMID:9574712

Macrophage polarization in virus-host interactions

USDA-ARS?s Scientific Manuscript database

Macrophage involvement in viral infections and antiviral states is common. However, this involvement has not been well-studied in the paradigm of macrophage polarization, which typically has been categorized by the dichotomy of classical (M1) and alternative (M2) statuses. Recent studies have reveal...

Glutamine Modulates Macrophage Lipotoxicity

PubMed Central

He, Li; Weber, Kassandra J.; Schilling, Joel D.

2016-01-01

Obesity and diabetes are associated with excessive inflammation and impaired wound healing. Increasing evidence suggests that macrophage dysfunction is responsible for these inflammatory defects. In the setting of excess nutrients, particularly dietary saturated fatty acids (SFAs), activated macrophages develop lysosome dysfunction, which triggers activation of the NLRP3 inflammasome and cell death. The molecular pathways that connect lipid stress to lysosome pathology are not well understood, but may represent a viable target for therapy. Glutamine uptake is increased in activated macrophages leading us to hypothesize that in the context of excess lipids glutamine metabolism could overwhelm the mitochondria and promote the accumulation of toxic metabolites. To investigate this question we assessed macrophage lipotoxicity in the absence of glutamine using LPS-activated peritoneal macrophages exposed to the SFA palmitate. We found that glutamine deficiency reduced lipid induced lysosome dysfunction, inflammasome activation, and cell death. Under glutamine deficient conditions mTOR activation was decreased and autophagy was enhanced; however, autophagy was dispensable for the rescue phenotype. Rather, glutamine deficiency prevented the suppressive effect of the SFA palmitate on mitochondrial respiration and this phenotype was associated with protection from macrophage cell death. Together, these findings reveal that crosstalk between activation-induced metabolic reprogramming and the nutrient microenvironment can dramatically alter macrophage responses to inflammatory stimuli. PMID:27077881

Bioelectric modulation of macrophage polarization

NASA Astrophysics Data System (ADS)

Li, Chunmei; Levin, Michael; Kaplan, David L.

2016-02-01

Macrophages play a critical role in regulating wound healing and tissue regeneration by changing their polarization state in response to local microenvironmental stimuli. The native roles of polarized macrophages encompass biomaterials and tissue remodeling needs, yet harnessing or directing the polarization response has been largely absent as a potential strategy to exploit in regenerative medicine to date. Recent data have revealed that specific alteration of cells’ resting potential (Vmem) is a powerful tool to direct proliferation and differentiation in a number of complex tissues, such as limb regeneration, craniofacial patterning and tumorigenesis. In this study, we explored the bioelectric modulation of macrophage polarization by targeting ATP sensitive potassium channels (KATP). Glibenclamide (KATP blocker) and pinacidil (KATP opener) treatment not only affect macrophage polarization, but also influence the phenotype of prepolarized macrophages. Furthermore, modulation of cell membrane electrical properties can fine-tune macrophage plasticity. Glibenclamide decreased the secretion and gene expression of selected M1 markers, while pinacidil augmented M1 markers. More interestingly, glibencalmide promoted macrophage alternative activation by enhancing certain M2 markers during M2 polarization. These findings suggest that control of bioelectric properties of macrophages could offer a promising approach to regulate macrophage phenotype as a useful tool in regenerative medicine.

Heterogeneity of macrophages in injured trigeminal nerves: cytokine/chemokine expressing vs. phagocytic macrophages.

PubMed

Lee, SeungHwan; Zhang, Ji

2012-08-01

Macrophages are important immune effector cells in both innate and adaptive immune responses. Injury to peripheral nerves triggers activation of resident macrophages and infiltration of haematogenous macrophages, which they play critical roles in Wallerian degeneration and neuropathic pain. As macrophages are able to change their phenotypes in response to environment cues, we attempt to identify distinct phenotypes of macrophages in injured nerves and to understand the potential contribution of each macrophage subpopulation to the genesis of neuropathic pain associated with nerve injury. Rat mental nerves (terminal branches of trigeminal nerve) were loosely ligated. Sensitivity to mechanical stimuli at the lower lip area was monitored using calibrated von Frey Hairs. We examined the expression pattern of Iba-1, MAC1 and ED1 which allow us to reveal the immunophenotypes of macrophages at different time points post-injury. Functional status of each macrophage subpopulation was further investigated by colocalization with cytokines/chemokines, myelin basic protein and MHC II antigen, which reflect respectively secretory, phagocytic and antigen presentation properties of activated macrophages. Following nerve injury, a burst of Iba-1(+) macrophages was found in injured mental nerves. Among them, we detected two major immunophenotypes: MAC1(+) cytokines/chemokines secreting macrophages and ED1(+) phagocytic macrophages. Small, round shaped MAC1(+) macrophages were distributed essentially around the lesion site and existed only at early time points. Large, irregular and foamy ED1(+) macrophages were found among damaged nerve fibers and they persisted for at least 3 months post-injury. Although ED1(+) macrophages did not secrete inflammatory mediators, they were able to express neurotransmitter CGRP and MHC II at later time points. In parallel, we observed that mechanical allodynia developed after the nerve ligation was at its lowest level within 1 month. Although slightly

Feedback inhibition of nitric oxide synthase activity by nitric oxide.

PubMed Central

Assreuy, J.; Cunha, F. Q.; Liew, F. Y.; Moncada, S.

1993-01-01

1. A murine macrophage cell line, J774, expressed nitric oxide (NO) synthase activity in response to interferon-gamma (IFN-gamma, 10 u ml-1) plus lipopolysaccharide (LPS, 10 ng ml-1). The enzyme activity was first detectable 6 h after incubation, peaked at 12 h and became undetectable after 48 h. 2. The decline in the NO synthase activity was not due to inhibition by stable substances secreted by the cells into the culture supernatant. 3. The decline in the NO synthase activity was significantly slowed down in cells cultured in a low L-arginine medium or with added haemoglobin, suggesting that NO may be involved in a feedback inhibitory mechanism. 4. The addition of NO generators, S-nitroso-acetyl-penicillamine (SNAP) or S-nitroso-glutathione (GSNO) markedly inhibited the NO synthase activity in a dose-dependent manner. The effect of NO on the enzyme was not due to the inhibition of de novo protein synthesis. 5. SNAP directly inhibited the inducible NO synthase extracted from activated J774 cells, as well as the constitutive NO synthase extracted from the rat brain. 6. The enzyme activity of J774 cells was not restored after the removal of SNAP by gel filtration, suggesting that NO inhibits NO synthase irreversibly. PMID:7682140

Inducible CYP2J2 and its product 11,12-EET promotes bacterial phagocytosis: a role for CYP2J2 deficiency in the pathogenesis of Crohn's disease?

PubMed

Bystrom, Jonas; Thomson, Scott J; Johansson, Jörgen; Edin, Matthew L; Zeldin, Darryl C; Gilroy, Derek W; Smith, Andrew M; Bishop-Bailey, David

2013-01-01

The epoxygenase CYP2J2 has an emerging role in inflammation and vascular biology. The role of CYP2J2 in phagocytosis is not known and its regulation in human inflammatory diseases is poorly understood. Here we investigated the role of CYP2J2 in bacterial phagocytosis and its expression in monocytes from healthy controls and Crohns disease patients. CYP2J2 is anti-inflammatory in human peripheral blood monocytes. Bacterial LPS induced CYP2J2 mRNA and protein. The CYP2J2 arachidonic acid products 11,12-EET and 14,15-EET inhibited LPS induced TNFα release. THP-1 monocytes were transformed into macrophages by 48h incubation with phorbol 12-myristate 13-acetate. Epoxygenase inhibition using a non-selective inhibitor SKF525A or a selective CYP2J2 inhibitor Compound 4, inhibited E. coli particle phagocytosis, which could be specifically reversed by 11,12-EET. Moreover, epoxygenase inhibition reduced the expression of phagocytosis receptors CD11b and CD68. CD11b also mediates L. monocytogenes phagocytosis. Similar, to E. coli bioparticle phagocytosis, epoxygenase inhibition also reduced intracellular levels of L. monocytogenes, which could be reversed by co-incubation with 11,12-EET. Disrupted bacterial clearance is a hallmark of Crohn's disease. Unlike macrophages from control donors, macrophages from Crohn's disease patients showed no induction of CYP2J2 in response to E. coli. These results demonstrate that CYP2J2 mediates bacterial phagocytosis in macrophages, and implicates a defect in the CYP2J2 pathway may regulate bacterial clearance in Crohn's disease.

FcγRI (CD64): an identity card for intestinal macrophages.

PubMed

De Calisto, Jaime; Villablanca, Eduardo J; Mora, J Rodrigo

2012-12-01

Macrophages are becoming increasingly recognized as key cellular players in intestinal immune homeostasis. However, differentiating between macrophages and dendritic cells (DCs) is often difficult, and finding a specific phenotypic signature for intestinal macrophage identification has remained elusive. In this issue of the European Journal of Immunology, Tamoutounour et al. [Eur. J. Immunol. 2012. 42: 3150-3166] identify CD64 as a specific macrophage marker that can be used to discriminate DCs from macrophages in the murine small and large intestine, under both steady-state and inflammatory conditions. The authors also propose a sequential 'monocyte-waterfall' model for intestinal macrophage differentiation, with implications for immune tolerance and inflammation at the gut mucosal interface. This Commentary will discuss the advantages and potential limitations of CD64 as a marker for intestinal macrophages. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Swift obervation of PSN J10250739+1709146 in NGC 3239

NASA Astrophysics Data System (ADS)

Xu, D.

2012-01-01

The optical transient, PSN J10250739+1709146, was discovered by Moore, Newton and Puckett at 2012/01/07.387 (ref. http://www.cbat.eps.harvard.edu/unconf/followups/J10250739+1709146.html), and localised in NGC 3239 (z=0.002512). Spectroscopy at 2012/01/10.275 confirms this source as a young SN II (Cao et al., ATel #3855). A Swift-ToO was executed to observe the field of this SN starting from 2012/01/10.774 (exposure time: 2390s), with the primary aim to constrain any accompanying X-ray emission.

A solar super-flare as cause for the 14C variation in AD 774/5 ?

NASA Astrophysics Data System (ADS)

Neuhäuser, R.; Hambaryan, V. V.

2014-11-01

We present further considerations regarding the strong 14C variation in AD 774/5. For its cause, either a solar super-flare or a short gamma-ray burst were suggested. We show that all kinds of stellar or neutron star flares would be too weak for the observed energy input at Earth in AD 774/5. Even though Maehara et al. (2012) present two super-flares with {˜ 1035} erg of presumably solar-type stars, we would like to caution: These two stars are poorly studied and may well be close binaries, and/or having a M-type dwarf companion, and/or may be much younger and/or much more magnetic than the Sun - in any such case, they might not be true solar analog stars. From the frequency of large stellar flares averaged over all stellar activity phases (maybe obtained only during grand activity maxima), one can derive (a limit of) the probability for a large solar flare at a random time of normal activity: We find the probability for one flare within 3000 years to be possibly as low as 0.3 to 0.008 considering the full 1σ error range. Given the energy estimate in Miyake et al. (2012) for the AD 774/5 event, it would need to be {˜ 2000} stronger than the Carrington event as solar super-flare. If the AD 774/5 event as solar flare would be beamed (to an angle of only {˜ 24°}), 100 times lower energy would be needed. A new AD 774/5 energy estimate by Usoskin et al. (2013) with a different carbon cycle model, yielding 4 ot 6 time lower 14C production, predicts 4-6 times less energy. If both reductions are applied, the AD 774/5 event would need to be only ˜ 4 times stronger than the Carrington event in 1859 (if both had similar spectra). However, neither 14C nor 10Be peaks were found around AD 1859. Hence, the AD 774/5 event (as solar flare) either was not beamed that strongly, and/or it would have been much more than 4-6 times stronger than Carrington, and/or the lower energy estimate (Usoskin et al. 2013) is not correct, and/or such solar flares cannot form (enough) 14C and

Inducible CYP2J2 and Its Product 11,12-EET Promotes Bacterial Phagocytosis: A Role for CYP2J2 Deficiency in the Pathogenesis of Crohn’s Disease?

PubMed Central

Bystrom, Jonas; Thomson, Scott J.; Johansson, Jörgen; Edin, Matthew L.; Zeldin, Darryl C.; Gilroy, Derek W.; Smith, Andrew M.; Bishop-Bailey, David

2013-01-01

The epoxygenase CYP2J2 has an emerging role in inflammation and vascular biology. The role of CYP2J2 in phagocytosis is not known and its regulation in human inflammatory diseases is poorly understood. Here we investigated the role of CYP2J2 in bacterial phagocytosis and its expression in monocytes from healthy controls and Crohns disease patients. CYP2J2 is anti-inflammatory in human peripheral blood monocytes. Bacterial LPS induced CYP2J2 mRNA and protein. The CYP2J2 arachidonic acid products 11,12-EET and 14,15-EET inhibited LPS induced TNFα release. THP-1 monocytes were transformed into macrophages by 48h incubation with phorbol 12-myristate 13-acetate. Epoxygenase inhibition using a non-selective inhibitor SKF525A or a selective CYP2J2 inhibitor Compound 4, inhibited E. coli particle phagocytosis, which could be specifically reversed by 11,12-EET. Moreover, epoxygenase inhibition reduced the expression of phagocytosis receptors CD11b and CD68. CD11b also mediates L. monocytogenes phagocytosis. Similar, to E. coli bioparticle phagocytosis, epoxygenase inhibition also reduced intracellular levels of L. monocytogenes, which could be reversed by co-incubation with 11,12-EET. Disrupted bacterial clearance is a hallmark of Crohn’s disease. Unlike macrophages from control donors, macrophages from Crohn’s disease patients showed no induction of CYP2J2 in response to E. coli. These results demonstrate that CYP2J2 mediates bacterial phagocytosis in macrophages, and implicates a defect in the CYP2J2 pathway may regulate bacterial clearance in Crohn’s disease. PMID:24058654

Expression of M2-polarized macrophages is associated with poor prognosis for advanced epithelial ovarian cancer.

PubMed

Lan, Chunyan; Huang, Xin; Lin, Suxia; Huang, Huiqiang; Cai, Qichun; Wan, Ting; Lu, Jiabin; Liu, Jihong

2013-06-01

Macrophages are polarized into two functionally distinct forms, M1 and M2, in response to different microenvironment. Tumor-associated macrophages (TAMs) generally have M2 phenotype and promote tumor progression. Few studies to date have described the infiltration of M2-polarized macrophages in ovarian cancer. We used two macrophages markers, CD68 and CD163, to analyze the expression of TAMs and to clarify the relationship between the M2 form and survival in advanced ovarian cancer. Clinical data of 110 patients with stages III-IV epithelial ovarian cancer at Sun Yat-sen University Cancer Center between 1999 and 2007 were retrospectively reviewed. Immunohistochemical staining of CD68 and CD163 was performed. Correlations between macrophage density and patient survival were analyzed. Our data showed that no significant difference was observed in survival between patients in the high- and the low-CD68 expression groups. In contrast, the progression-free survival (PFS) rates (p = 0.003) and overall survival (OS) rates (p = 0.004) were significantly higher in the low-CD163 expression group than in the high-CD163 expression group, respectively. Similarly, we also observed significantly improved 3-year PFS (49.8% vs. 11.0%, p < 0.001) and OS (77.4% vs. 45.0%, p < 0.001) rates in patients in the low-CD163/CD68 ratio group when compared with the high-CD163/CD68 ratio group. Multivariate analysis identified the density of CD163-positive cells as well as the ratio of CD163/CD68 as negative predictors for PFS and OS, respectively. Our results show that the infiltration of CD163-positive M2 macrophages as well as activation of macrophages towards the M2 phenotype may contribute to poor survival in advanced ovarian cancer.

Does carbonation of steel slag particles reduce their toxicity? An in vitro approach.

PubMed

Ibouraadaten, Saloua; van den Brule, Sybille; Lison, Dominique

2015-06-01

Mineral carbonation can stabilize industrial residues and, in the steel industry, may contribute to simultaneously valorize CO2 emissions and slag. We hypothesized that, by restricting the leaching of metals of toxicological concern such as Cr and V, carbonation can suppress the toxicity of these materials. The cytotoxic activity (WST1 assay) of slag dusts collected from a stainless and a Linz-Donawitz (LD) steel plant, before and after carbonation, was examined in J774 macrophages. The release of Cr, V, Fe, Mn and Ni was measured after incubation in artificial lung fluids mimicking the extracellular and phagolysosomal milieu to which particles are confronted after inhalation. LD slag had the higher Fe, Mn and V content, and was more cytotoxic than stainless steel slag. The cytotoxic activity of LD but not of stainless dusts was reduced after carbonation. The cytotoxic activity of the dusts toward J774 macrophages necessitated a direct contact with the cells and was reduced in the presence of inhibitors of phagocytosis (cytochalasin D) or phagolysosome acidification (bafilomycin), pointing to a key role of metallic constituents released in phagolysosomes. This in vitro study supports a limited reduction of the cytotoxic activity of LD, but not of stainless, steel dusts upon carbonation. Copyright © 2015 Elsevier Ltd. All rights reserved.

Potential Eye Drop Based on a Calix[4]arene Nanoassembly for Curcumin Delivery: Enhanced Drug Solubility, Stability, and Anti-Inflammatory Effect.

PubMed

Granata, Giuseppe; Paterniti, Irene; Geraci, Corrada; Cunsolo, Francesca; Esposito, Emanuela; Cordaro, Marika; Blanco, Anna Rita; Cuzzocrea, Salvatore; Consoli, Grazia M L

2017-05-01

Curcumin is an Indian spice with a wide spectrum of biological and pharmacological activities but poor aqueous solubility, rapid degradation, and low bioavailability that affect medical benefits. To overcome these limits in ophthalmic application, curcumin was entrapped in a polycationic calix[4]arene-based nanoaggregate by a simple and reproducible method. The calix[4]arene-curcumin supramolecular assembly (Calix-Cur) appeared as a clear colloidal solution consisting in micellar nanoaggregates with size, polydispersity index, surface potential, and drug loading percentage meeting the requirements for an ocular drug delivery system. The encapsulation in the calix[4]arene nanoassembly markedly enhanced the solubility, reduced the degradation, and improved the anti-inflammatory effects of curcumin compared to free curcumin in both in vitro and in vivo experiments. Calix-Cur did not compromise the viability of J774A.1 macrophages and suppressed pro-inflammatory marker expression in J774A.1 macrophages subjected to LPS-induced oxidative stress. Histological and immunohistochemical analyses showed that Calix-Cur reduced signs of inflammation in a rat model of LPS-induced uveitis when topically administrated in the eyes. Overall, the results supported the calix[4]arene nanoassembly as a promising nanocarrier for delivering curcumin to anterior ocular tissues.

Macrophages and cellular immunity in Drosophila melanogaster

PubMed Central

Gold, Katrina S.; Brückner, Katja

2016-01-01

The invertebrate Drosophila melanogaster has been a powerful model for understanding blood cell development and immunity. Drosophila is a holometabolous insect, which transitions through a series of life stages from embryo, larva and pupa to adulthood. In spite of this, remarkable parallels exist between Drosophila and vertebrate macrophages, both in terms of development and function. More than 90% of Drosophila blood cells (hemocytes) are macrophages (plasmatocytes), making this highly tractable genetic system attractive for studying a variety of questions in macrophage biology. In vertebrates, recent findings revealed that macrophages have two independent origins: self-renewing macrophages, which reside and proliferate in local microenvironments in a variety of tissues, and macrophages of the monocyte lineage, which derive from hematopoietic stem or progenitor cells. Like vertebrates, Drosophila possesses two macrophage lineages with a conserved dual ontogeny. These parallels allow us to take advantage of the Drosophila model when investigating macrophage lineage specification, maintenance and amplification, and the induction of macrophages and their progenitors by local microenvironments and systemic cues. Beyond macrophage development, Drosophila further serves as a paradigm for understanding the mechanisms underlying macrophage function and cellular immunity in infection, tissue homeostasis and cancer, throughout development and adult life. PMID:27117654

Transcription factor NFAT5 promotes macrophage survival in rheumatoid arthritis

PubMed Central

Choi, Susanna; Choi, Soo Youn; Kwon, H. Moo; Hwang, Daehee; Park, Yune-Jung; Cho, Chul-Soo

2017-01-01

Defective apoptotic death of activated macrophages has been implicated in the pathogenesis of rheumatoid arthritis (RA). However, the molecular signatures defining apoptotic resistance of RA macrophages are not fully understood. Here, global transcriptome profiling of RA macrophages revealed that the osmoprotective transcription factor nuclear factor of activated T cells 5 (NFAT5) critically regulates diverse pathologic processes in synovial macrophages including the cell cycle, apoptosis, and proliferation. Transcriptomic analysis of NFAT5-deficient macrophages revealed the molecular networks defining cell survival and proliferation. Proinflammatory M1-polarizing stimuli and hypoxic conditions were responsible for enhanced NFAT5 expression in RA macrophages. An in vitro functional study demonstrated that NFAT5-deficient macrophages were more susceptible to apoptotic death. Specifically, CCL2 secretion in an NFAT5-dependent fashion bestowed apoptotic resistance to RA macrophages in vitro. Injection of recombinant CCL2 into one of the affected joints of Nfat5+/– mice increased joint destruction and macrophage infiltration, demonstrating the essential role of the NFAT5/CCL2 axis in arthritis progression in vivo. Moreover, after intra-articular injection, NFAT5-deficient macrophages were more susceptible to apoptosis and less efficient at promoting joint destruction than were NFAT5-sufficient macrophages. Thus, NFAT5 regulates macrophage survival by inducing CCL2 secretion. Our results provide evidence that NFAT5 expression in macrophages enhances chronic arthritis by conferring apoptotic resistance to activated macrophages. PMID:28192374

Native low-density lipoprotein uptake by macrophage colony-stimulating factor-differentiated human macrophages is mediated by macropinocytosis and micropinocytosis.

PubMed

Anzinger, Joshua J; Chang, Janet; Xu, Qing; Buono, Chiara; Li, Yifu; Leyva, Francisco J; Park, Bum-Chan; Greene, Lois E; Kruth, Howard S

2010-10-01

To examine the pinocytotic pathways mediating native low-density lipoprotein (LDL) uptake by human macrophage colony-stimulating factor-differentiated macrophages (the predominant macrophage phenotype in human atherosclerotic plaques). We identified the kinase inhibitor SU6656 and the Rho GTPase inhibitor toxin B as inhibitors of macrophage fluid-phase pinocytosis of LDL. Assessment of macropinocytosis by time-lapse microscopy revealed that both drugs almost completely inhibited macropinocytosis, although LDL uptake and cholesterol accumulation by macrophages were only partially inhibited (approximately 40%) by these agents. Therefore, we investigated the role of micropinocytosis in mediating LDL uptake in macrophages and identified bafilomycin A1 as an additional partial inhibitor (approximately 40%) of macrophage LDL uptake that targeted micropinocytosis. When macrophages were incubated with both bafilomycin A1 and SU6656, inhibition of LDL uptake was additive (reaching 80%), showing that these inhibitors target different pathways. Microscopic analysis of fluid-phase uptake pathways in these macrophages confirmed that LDL uptake occurs through both macropinocytosis and micropinocytosis. Our findings show that human macrophage colony-stimulating factor-differentiated macrophages take up native LDL by macropinocytosis and micropinocytosis, underscoring the importance of both pathways in mediating LDL uptake by these cells.

A broken krebs cycle in macrophages.

PubMed

O'Neill, Luke A J

2015-03-17

Macrophages undergo metabolic rewiring during polarization but details of this process are unclear. In this issue of Immunity, Jha et al. (2015) report a systems approach for unbiased analysis of cellular metabolism that reveals key metabolites and metabolic pathways required for distinct macrophage polarization states. Copyright © 2015 Elsevier Inc. All rights reserved.

Effects of macrophage colony-stimulating factor on macrophages and their related cell populations in the osteopetrosis mouse defective in production of functional macrophage colony-stimulating factor protein.

PubMed Central

Umeda, S.; Takahashi, K.; Shultz, L. D.; Naito, M.; Takagi, K.

1996-01-01

The development of macrophage populations in osteopetrosis (op) mutant mice defective in production of functional macrophage colony-stimulating factor (M-CSF) and the response of these cell populations to exogenous M-CSF were used to classify macrophages into four groups: 1) monocytes, monocyte-derived macrophages, and osteoclasts, 2) MOMA-1-positive macrophages, 3) ER-TR9-positive macrophages, and 4) immature tissue macrophages. Monocytes, monocyte-derived macrophages, osteoclasts in bone, microglia in brain, synovial A cells, and MOMA-1- or ER-TR9-positive macrophages were deficient in op/op mice. The former three populations expanded to normal levels in op/op mice after daily M-CSF administration, indicating that they are developed and differentiated due to the effect of M-CSF supplied humorally. In contrast, the other cells did not respond or very slightly responded to M-CSF, and their development seems due to either M-CSF produced in situ or expression of receptor for M-CSF. Macrophages present in tissues of the mutant mice were immature and appear to be regulated by either granulocyte/macrophage colony-stimulating factor and/or interleukin-3 produced in situ or receptor expression. Northern blot analysis revealed different expressions of GM-CSF and IL-3 mRNA in various tissues of the op/op mice. However, granulocyte/macrophage colony-stimulating factor and interleukin-3 in serum were not detected by enzyme-linked immunosorbent assay. The immature macrophages differentiated and matured into resident macrophages after M-CSF administration, and some of these cells proliferated in response to M-CSF. Images Figure 4 Figure 6 Figure 8 Figure 10 Figure 11 PMID:8701995

Laboratory Aspects of Biological Warfare Agents

DTIC Science & Technology

2016-01-01

Embryonated chicken egg yolk sacs have typically been the method of choice for culture. They are inoculated when the embryos are 5-7 days old. The... chicken or mouse embryo fibroblasts, J774.16 mouse macrophages, L929 murine fibroblasts, HEL (human embryonic lung) or vero cells are more commonly...the family, Poxviridae, is a legacy of the original grouping of viruses associated with diseases that produced poxes in the skin, however, if

19 CFR 10.774 - Direct costs of processing operations.

Code of Federal Regulations, 2014 CFR

2014-04-01

... manufacture of the specific good, including fringe benefits, on-the-job training, and the costs of engineering..., design, engineering, and blueprint costs, to the extent that they are allocable to the specific good; (4... 19 Customs Duties 1 2014-04-01 2014-04-01 false Direct costs of processing operations. 10.774...

Cytokine production of the neutrophils and macrophages in time of phagocytosis under influence of infrared low-level laser irradiation

NASA Astrophysics Data System (ADS)

Rudik, Dmitry V.; Tikhomirova, Elena I.; Tuchina, Elena S.

2006-08-01

Influence of infrared low-level laser irradiation (LLLI) on induction of synthesis of some cytokines such as interleykin-1 (Il-1), tumor necrosis factor-α (TNF-α), interferon-γ (INF-γ), interleykin-8 (Il-8) and interleykin-4 (Il-4) by the neutrophils and macrophages in time of bacterial cells phagocytosis that was searched. As the object of analysis we used peritoneal macrophages from white mice and neutrophils from peripheral blood of healthy donors. We used the laser diod with spectrum maximum of 850 nm with doses 300, 900 and 1500 mJ (exposition -60, 180 and 300 s respectively; capacity - 5 mW). We carried out the Enzyme-Linked Immunospot Assay (ELISA) to determine cytokine content during phagocytosis after 3 h and 6 h. We found dynamics in production of the cytokines, which was different for the neutrophils and macrophages. We showed that the infrared LLLI has significant stimulating activity on the proinflammatory cytokines production by neutrophils and macrophages. Moreover we revealed dynamics changing in the Il-8 and Il-4 production.

Proteomic Analysis Reveals Distinct Metabolic Differences Between Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) and Macrophage Colony Stimulating Factor (M-CSF) Grown Macrophages Derived from Murine Bone Marrow Cells.

PubMed

Na, Yi Rang; Hong, Ji Hye; Lee, Min Yong; Jung, Jae Hun; Jung, Daun; Kim, Young Won; Son, Dain; Choi, Murim; Kim, Kwang Pyo; Seok, Seung Hyeok

2015-10-01

Macrophages are crucial in controlling infectious agents and tissue homeostasis. Macrophages require a wide range of functional capabilities in order to fulfill distinct roles in our body, one being rapid and robust immune responses. To gain insight into macrophage plasticity and the key regulatory protein networks governing their specific functions, we performed quantitative analyses of the proteome and phosphoproteome of murine primary GM-CSF and M-CSF grown bone marrow derived macrophages (GM-BMMs and M-BMMs, respectively) using the latest isobaric tag based tandem mass tag (TMT) labeling and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Strikingly, metabolic processes emerged as a major difference between these macrophages. Specifically, GM-BMMs show significant enrichment of proteins involving glycolysis, the mevalonate pathway, and nitrogen compound biosynthesis. This evidence of enhanced glycolytic capability in GM-BMMs is particularly significant regarding their pro-inflammatory responses, because increased production of cytokines upon LPS stimulation in GM-BMMs depends on their acute glycolytic capacity. In contrast, M-BMMs up-regulate proteins involved in endocytosis, which correlates with a tendency toward homeostatic functions such as scavenging cellular debris. Together, our data describes a proteomic network that underlies the pro-inflammatory actions of GM-BMMs as well as the homeostatic functions of M-BMMs. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Macrophages and cellular immunity in Drosophila melanogaster.

PubMed

Gold, Katrina S; Brückner, Katja

2015-12-01

The invertebrate Drosophila melanogaster has been a powerful model for understanding blood cell development and immunity. Drosophila is a holometabolous insect, which transitions through a series of life stages from embryo, larva and pupa to adulthood. In spite of this, remarkable parallels exist between Drosophila and vertebrate macrophages, both in terms of development and function. More than 90% of Drosophila blood cells (hemocytes) are macrophages (plasmatocytes), making this highly tractable genetic system attractive for studying a variety of questions in macrophage biology. In vertebrates, recent findings revealed that macrophages have two independent origins: self-renewing macrophages, which reside and proliferate in local microenvironments in a variety of tissues, and macrophages of the monocyte lineage, which derive from hematopoietic stem or progenitor cells. Like vertebrates, Drosophila possesses two macrophage lineages with a conserved dual ontogeny. These parallels allow us to take advantage of the Drosophila model when investigating macrophage lineage specification, maintenance and amplification, and the induction of macrophages and their progenitors by local microenvironments and systemic cues. Beyond macrophage development, Drosophila further serves as a paradigm for understanding the mechanisms underlying macrophage function and cellular immunity in infection, tissue homeostasis and cancer, throughout development and adult life. Copyright © 2016. Published by Elsevier Ltd.

AMP-activated protein kinase enhances the phagocytic ability of macrophages and neutrophils

PubMed Central

Bae, Hong-Beom; Zmijewski, Jaroslaw W.; Deshane, Jessy S.; Tadie, Jean-Marc; Chaplin, David D.; Takashima, Seiji; Abraham, Edward

2011-01-01

Although AMPK plays well-established roles in the modulation of energy balance, recent studies have shown that AMPK activation has potent anti-inflammatory effects. In the present experiments, we examined the role of AMPK in phagocytosis. We found that ingestion of Escherichia coli or apoptotic cells by macrophages increased AMPK activity. AMPK activation increased the ability of neutrophils or macrophages to ingest bacteria (by 46±7.8 or 85±26%, respectively, compared to control, P<0.05) and the ability of macrophages to ingest apoptotic cells (by 21±1.4%, P<0.05 compared to control). AMPK activation resulted in cytoskeletal reorganization, including enhanced formation of actin and microtubule networks. Activation of PAK1/2 and WAVE2, which are downstream effectors of Rac1, accompanied AMPK activation. AMPK activation also induced phosphorylation of CLIP-170, a protein that participates in microtubule synthesis. The increase in phagocytosis was reversible by the specific AMPK inhibitor compound C, siRNA to AMPKα1, Rac1 inhibitors, or agents that disrupt actin or microtubule networks. In vivo, AMPK activation resulted in enhanced phagocytosis of bacteria in the lungs by 75 ± 5% vs. control (P<0.05). These results demonstrate a novel function for AMPK in enhancing the phagocytic activity of neutrophils and macrophages.—Bae, H. -B., Zmijewski, J. W., Deshane, J. S., Tadie, J. -M., Chaplin, D. D., Takashima, S., Abraham, E. AMP-activated protein kinase enhances the phagocytic ability of macrophages and neutrophils. PMID:21885655

Inhibition of inducible nitric oxide synthase expression by novel nonsteroidal anti-inflammatory derivatives with gastrointestinal-sparing properties.

PubMed Central

Cirino, G.; Wheeler-Jones, C. P.; Wallace, J. L.; Del Soldato, P.; Baydoun, A. R.

1996-01-01

1. The effects of novel nitric oxide-releasing nonsteroidal anti-inflammatory compounds (NO-NSAIDs) on induction of nitric oxide (NO) synthase by bacterial lipopolysaccharide (LPS) were examined in a murine cultured macrophage cell line, J774. 2. LPS-induced nitrite production was markedly attenuated by the nitroxybutylester derivatives of flurbiprofen (FNBE), aspirin, ketoprofen, naproxen, diclofenac and ketorolac, with each compound reducing accumulated nitrite levels by > 40% at the maximum concentrations (100 micrograms ml-1) used. 3. Further examination revealed that nitrite production was inhibited in a concentration-dependent (1-100 micrograms ml-1) manner by FNBE which at 100 micrograms ml-1 decreased LPS-stimulated levels by 63.3 +/- 8.6% (n = 7). The parent compound flurbiprofen was relatively ineffective over the same concentration-range, inhibiting nitrite accumulation by 24 +/- 0.9% (n = 3) at the maximum concentration used (100 micrograms ml-1). 4. FNBE reduced LPS-induced nitrite production when added to cells up to 4 h after LPS. Thereafter, FNBE caused very little or no reduction in nitrite levels. Furthermore NO-NSAIDs (100 micrograms ml-1) did not inhibit the metabolism of L-[3H]-arginine to citrulline by NO synthase isolated from LPS-activated macrophages. 5. Western blot analysis demonstrated that NO synthase expression was markedly attenuated following co-incubation of J774 cell with LPS (1 microgram ml-1; 24 h) and FNBE (100 micrograms ml-1; 24 h). Thus taken together, these findings indicate that NO-NSAIDs inhibit induction of NO synthase without directly affecting enzyme activity. 6. In conclusion our results indicate that NO-NSAIDs can inhibit the inducible L-arginine-NO pathway, and are capable of suppressing NO synthesis by inhibiting expression of NO synthase. The clinical implications of these findings remain to be established. Images Figure 4 PMID:8730734

Kinetics of tumor necrosis factor production by photodynamic-therapy-activated macrophages

NASA Astrophysics Data System (ADS)

Pass, Harvey I.; Evans, Steven; Perry, Roger; Matthews, Wilbert

1990-07-01

The ability of photodynamic therapy (PDT) to activate macrophages and produce cytokines, specifically tumor necrosis factor (TNF), is unknown. Three day thioglycolate elicited macrophages were incubated with 25 ug/mi Photofrin II (P11) for 2 hour, after which they were subjected to 630 nm light with fluences of 0-1800 J/m. The amount of TNF produced in the system as well as macrophage viability was measured 1, 3, 6, and 18 hours after POT. The level of TNF produced by the macrophages was significantly elevated over control levels 6 hours after POT and the absolute level of tumor necrosis factor production was influenced by the treatment energy and the resulting macrophage cytotoxicity. These data suggest that POT therapy induced cytotoxicity in vivo may be amplified by macrophage stimulation to secrete cytokines and these cytokines may also participate in other direct/indirect photodynamic therapy effects, i.e. immunosuppression, vascular effects.

Quantitative Profiling of Protein S-Glutathionylation Reveals Redox-Dependent Regulation of Macrophage Function During Nanoparticle-Induced Oxidative Stress

DOE PAGES

Duan, Jicheng; Kodali, Vamsi K.; Gaffrey, Matthew J.; ...

2015-12-23

Engineered nanoparticles (ENPs) are emerging functional materials increasingly utilized for commercial and medical applications. Due to the potential hazard effects of ENPs to human health, it is significant to assess and understand the underlying mechanisms of nanotoxicity. Here, we investigate protein S-glutathionylation (SSG) as an underlying regulatory mechanism for ENP-induced oxidative stress in macrophages by applying a recently developed quantitative redox proteomics approach for site-specific measurements of SSG. Three high-volume production ENPs (SiO 2, Fe 3O 4 and CoO) were selected as representative ENPs with low, moderate, and high reactive oxygen species (ROS) activity, respectively. Among these nanoparticles, we observemore » that CoO led to the most significant dose-dependent oxidative stress and increase of protein SSG modifications in macrophages. Our site-specific SSG changes highlighted a broad set of redox sensitive proteins and their specific Cys residues potentially implicated in stress response. Functional analysis revealed that the most significantly enriched functional categories for SSG-modified proteins were stress response, cellular structure change, and cell death or survival. Moreover, ENPs-induce oxidative stress levels (CoO > Fe 3O 4 > SiO 2) were found to correlate well with the levels of impairment of macrophage phagocytic activity and the overall degrees of increases in SSG. RNA silencing knockdown experiment of glutaredoxin 1 (Grx1) also led to a decreased phagocytic activity in macrophages, which suggested a regulatory role of SSG in phagocytosis. Together, the results provided valuable insights of protein SSG as a potential regulatory mechanism in response to nanomaterial-induced oxidative stress and immunity dysfunction.« less

Quantitative Profiling of Protein S-Glutathionylation Reveals Redox-Dependent Regulation of Macrophage Function During Nanoparticle-Induced Oxidative Stress

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

Duan, Jicheng; Kodali, Vamsi K.; Gaffrey, Matthew J.

Engineered nanoparticles (ENPs) are emerging functional materials increasingly utilized for commercial and medical applications. Due to the potential hazard effects of ENPs to human health, it is significant to assess and understand the underlying mechanisms of nanotoxicity. Here, we investigate protein S-glutathionylation (SSG) as an underlying regulatory mechanism for ENP-induced oxidative stress in macrophages by applying a recently developed quantitative redox proteomics approach for site-specific measurements of SSG. Three high-volume production ENPs (SiO 2, Fe 3O 4 and CoO) were selected as representative ENPs with low, moderate, and high reactive oxygen species (ROS) activity, respectively. Among these nanoparticles, we observemore » that CoO led to the most significant dose-dependent oxidative stress and increase of protein SSG modifications in macrophages. Our site-specific SSG changes highlighted a broad set of redox sensitive proteins and their specific Cys residues potentially implicated in stress response. Functional analysis revealed that the most significantly enriched functional categories for SSG-modified proteins were stress response, cellular structure change, and cell death or survival. Moreover, ENPs-induce oxidative stress levels (CoO > Fe 3O 4 > SiO 2) were found to correlate well with the levels of impairment of macrophage phagocytic activity and the overall degrees of increases in SSG. RNA silencing knockdown experiment of glutaredoxin 1 (Grx1) also led to a decreased phagocytic activity in macrophages, which suggested a regulatory role of SSG in phagocytosis. Together, the results provided valuable insights of protein SSG as a potential regulatory mechanism in response to nanomaterial-induced oxidative stress and immunity dysfunction.« less

Acrolein increases 5-lipoxygenase expression in murine macrophages through activation of ERK pathway

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

Kim, Chae E.; Lee, Seung J.; Seo, Kyo W.

2010-05-15

Episodic exposure to acrolein-rich pollutants has been linked to acute myocardial infarction, and 5-lipoxygenase (5-LO) is involved in the production of matrix metalloproteinase-9 (MMP-9), which destabilizes atherosclerotic plaques. Thus, the present study determined the effect of acrolein on 5-LO/leukotriene B{sub 4} (LTB{sub 4}) production in murine macrophages. Stimulation of J774A.1 cells with acrolein led to increased LTB{sub 4} production in association with increased 5-LO expression. Acrolein-evoked 5-LO expression was blocked by pharmacological inhibition of the ERK pathway, but not by inhibitors for JNK and p38 MAPK pathways. In line with these results, acrolein exclusively increased the phosphorylation of ERK amongmore » these MAPK, suggesting a role for the ERK pathway in acrolein-induced 5-LO expression with subsequent production of LTB{sub 4}. Among the receptor tyrosine kinases including epidermal growth factor receptor (EGFR) and platelet derived growth factor receptor (PDGFR), acrolein-evoked ERK phosphorylation was attenuated by AG1478, an EGFR inhibitor, but not by AG1295, a PDGFR inhibitor. In addition, acrolein-evoked 5-LO expression was also inhibited by inhibition of EGFR pathway, but not by inhibition of PDGFR pathway. These observations suggest that acrolein has a profound effect on the 5-LO pathway via an EGFR-mediated activation of ERK pathway, leading to acute ischemic syndromes through the generation of LTB{sub 4}, subsequent MMP-9 production and plaque rupture.« less

Capsaicin, a novel inhibitor of the NorA efflux pump, reduces the intracellular invasion of Staphylococcus aureus.

PubMed

Kalia, Nitin Pal; Mahajan, Priya; Mehra, Rukmankesh; Nargotra, Amit; Sharma, Jai Parkash; Koul, Surrinder; Khan, Inshad Ali

2012-10-01

To delineate the role of capsaicin (8-methyl-N-vanillyl-6-nonenamide) as an inhibitor of the NorA efflux pump and its impact on invasion of macrophages by Staphylococcus aureus. Capsaicin in combination with ciprofloxacin was tested for activity against S. aureus SA-1199B (NorA overproducing), SA-1199 (wild-type) and SA-K1758 (norA knockout). The role of NorA in the intracellular invasion of S. aureus and the ability of capsaicin to inhibit this invasion was established in J774 macrophage cell lines. The three-dimensional structure of NorA was predicted using an in silico approach and docking studies of capsaicin were performed. Capsaicin significantly reduced the MIC of ciprofloxacin for S. aureus SA-1199 and SA-1199B. Furthermore, capsaicin also extended the post-antibiotic effect of ciprofloxacin by 1.1 h at MIC concentration. There was a decrease in mutation prevention concentration of ciprofloxacin when combined with capsaicin. Inhibition of ethidium bromide efflux by NorA-overproducing S. aureus SA-1199B confirmed the role of capsaicin as a NorA efflux pump inhibitor (EPI). The most significant finding of this study was the ability of capsaicin to reduce the intracellular invasion of S. aureus SA-1199B (NorA overproducing) in J774 macrophage cell lines by 2 log(10). This study, for the first time, has shown that capsaicin, a novel EPI, not only inhibits the NorA efflux pump of S. aureus but also reduces the invasiveness of S. aureus, thereby reducing its virulence.

Regulation of iNOS function and cellular redox state by macrophage Gch1 reveals specific requirements for tetrahydrobiopterin in NRF2 activation

PubMed Central

McNeill, Eileen; Crabtree, Mark J.; Sahgal, Natasha; Patel, Jyoti; Chuaiphichai, Surawee; Iqbal, Asif J.; Hale, Ashley B.; Greaves, David R.; Channon, Keith M.

2015-01-01

Inducible nitric oxide synthase (iNOS) is a key enzyme in the macrophage inflammatory response, which is the source of nitric oxide (NO) that is potently induced in response to proinflammatory stimuli. However, the specific role of NO production, as distinct from iNOS induction, in macrophage inflammatory responses remains unproven. We have generated a novel mouse model with conditional deletion of Gch1, encoding GTP cyclohydrolase 1 (GTPCH), an essential enzyme in the biosynthesis of tetrahydrobiopterin (BH4) that is a required cofactor for iNOS NO production. Mice with a floxed Gch1 allele (Gch1fl/fl) were crossed with Tie2cre transgenic mice, causing Gch1 deletion in leukocytes (Gch1fl/flTie2cre). Macrophages from Gch1fl/flTie2cre mice lacked GTPCH protein and de novo biopterin biosynthesis. When activated with LPS and IFNγ, macrophages from Gch1fl/flTie2cre mice induced iNOS protein in a manner indistinguishable from wild-type controls, but produced no detectable NO, as judged by L-citrulline production, EPR spin trapping of NO, and by nitrite accumulation. Incubation of Gch1fl/flTie2cre macrophages with dihydroethidium revealed significantly increased production of superoxide in the presence of iNOS expression, and an iNOS-independent, BH4-dependent increase in other ROS species. Normal BH4 levels, nitric oxide production, and cellular redox state were restored by sepiapterin, a precursor of BH4 production by the salvage pathway, demonstrating that the effects of BH4 deficiency were reversible. Gch1fl/flTie2cre macrophages showed only minor alterations in cytokine production and normal cell migration, and minimal changes in basal gene expression. However, gene expression analysis after iNOS induction identified 78 genes that were altered between wild-type and Gch1fl/flTie2cre macrophages. Pathway analysis identified decreased NRF2 activation, with reduced induction of archetypal NRF2 genes (gclm, prdx1, gsta3, nqo1, and catalase) in BH4-deficient Gch1fl

Celastrol nanomicelles attenuate cytokine secretion in macrophages and inhibit macrophage-induced corneal neovascularization in rats.

PubMed

Li, Zhanrong; Li, Jingguo; Zhu, Lei; Zhang, Ying; Zhang, Junjie; Yao, Lin; Liang, Dan; Wang, Liya

The aim of the present study was to investigate the inhibitory effects of celastrol-loaded nanomicelles (CNMs) on activated macrophage-induced corneal neovascularization (CNV) in rats and cytokine secretion in macrophages. Using an angiogenesis assay in vitro, we detected the effects of CNMs on human umbilical vein endothelial cell (HUVEC) migration and invasion. In addition, the expression levels of cytokines secreted from hypoxia-induced macrophages were assessed through cytokine array analysis. The expression of hypoxia-inducible factors-1α (HIF-1α), nuclear factor-kappa B p65 (NF-κB p65), phospho-nuclear factor-kappa B p65 (phospho-NF-κB p65), p38 mitogen-activated protein kinase (p38 MAPK), phospho-p38 MAPK, extracellular signal-regulated kinase 1/2 (ERK1/2), and phospho-ERK1/2 was analyzed by western blotting. Activated macrophages were elicited through mineral oil lumbar injection, labeled with 1,19-dioctadecyl-3-3-39,39-tetramethylindocarbocyanine (DiI) and implanted into the corneal micro-pocket to induce CNV and to assess the antiangiogenic effect in rats. CNV was morphometrically analyzed using ImageJ software. Histopathological features were evaluated by immunofluorescence immunostaining for vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP-9) on day 2 after surgery. In the present study, the results indicated that CNMs significantly inhibited the migration and invasion of HUVECs; remarkably attenuated the expression of VEGF, tumor necrosis factor-α, interleukin-1α, monocyte chemoattractant protein 1, cytokine-induced neutrophil chemoattractant 3, and MMP-9 protein; and downregulated ERK1/2, p38 MAPK, NF-κB activation, and HIF-1α expression in macrophages. The peritoneal cells elicited using mineral oil were highly purified macrophages, and the length and area of CNV were significantly decreased in the CNMs group compared with the control group. There was a significant reduction in the expression of VEGF and MMP-9 in

Estrogen receptor-alpha promotes alternative macrophage activation during cutaneous repair.

PubMed

Campbell, Laura; Emmerson, Elaine; Williams, Helen; Saville, Charis R; Krust, Andrée; Chambon, Pierre; Mace, Kimberly A; Hardman, Matthew J

2014-09-01

Efficient local monocyte/macrophage recruitment is critical for tissue repair. Recruited macrophages are polarized toward classical (proinflammatory) or alternative (prohealing) activation in response to cytokines, with tight temporal regulation crucial for efficient wound repair. Estrogen acts as a potent anti-inflammatory regulator of cutaneous healing. However, an understanding of estrogen/estrogen receptor (ER) contribution to macrophage polarization and subsequent local effects on wound healing is lacking. Here we identify, to our knowledge previously unreported, a role whereby estrogen receptor α (ERα) signaling preferentially polarizes macrophages from a range of sources to an alternative phenotype. Cell-specific ER ablation studies confirm an in vivo role for inflammatory cell ERα, but not ERβ, in poor healing associated with an altered cytokine profile and fewer alternatively activated macrophages. Furthermore, we reveal intrinsic changes in ERα-deficient macrophages, which are unable to respond to alternative activation signals in vitro. Collectively, our data reveal that inflammatory cell-expressed ERα promotes alternative macrophage polarization, which is beneficial for timely healing. Given the diverse physiological roles of ERs, these findings will likely be of relevance to many pathologies involving excessive inflammation.

7 CFR 774.18 - Interest rate, terms and security requirements.

Code of Federal Regulations, 2014 CFR

2014-01-01

..., DEPARTMENT OF AGRICULTURE SPECIAL PROGRAMS EMERGENCY LOAN FOR SEED PRODUCERS PROGRAM § 774.18 Interest rate... percent for 36 months or until the date of settlement of, completion of, or final distribution of assets... bankruptcy claim or 36 months from the date of the note. (2) However, any principal remaining thereafter will...

7 CFR 774.18 - Interest rate, terms and security requirements.

Code of Federal Regulations, 2011 CFR

2011-01-01

..., DEPARTMENT OF AGRICULTURE SPECIAL PROGRAMS EMERGENCY LOAN FOR SEED PRODUCERS PROGRAM § 774.18 Interest rate... percent for 36 months or until the date of settlement of, completion of, or final distribution of assets... bankruptcy claim or 36 months from the date of the note. (2) However, any principal remaining thereafter will...

7 CFR 774.18 - Interest rate, terms and security requirements.

Code of Federal Regulations, 2013 CFR

2013-01-01

..., DEPARTMENT OF AGRICULTURE SPECIAL PROGRAMS EMERGENCY LOAN FOR SEED PRODUCERS PROGRAM § 774.18 Interest rate... percent for 36 months or until the date of settlement of, completion of, or final distribution of assets... bankruptcy claim or 36 months from the date of the note. (2) However, any principal remaining thereafter will...

7 CFR 774.18 - Interest rate, terms and security requirements.

Code of Federal Regulations, 2012 CFR

2012-01-01

..., DEPARTMENT OF AGRICULTURE SPECIAL PROGRAMS EMERGENCY LOAN FOR SEED PRODUCERS PROGRAM § 774.18 Interest rate... percent for 36 months or until the date of settlement of, completion of, or final distribution of assets... bankruptcy claim or 36 months from the date of the note. (2) However, any principal remaining thereafter will...

SAA drives proinflammatory heterotypic macrophage differentiation in the lung via CSF-1R-dependent signaling

PubMed Central

Anthony, Desiree; McQualter, Jonathan L.; Bishara, Maria; Lim, Ee X.; Yatmaz, Selcuk; Seow, Huei Jiunn; Hansen, Michelle; Thompson, Michelle; Hamilton, John A.; Irving, Louis B.; Levy, Bruce D.; Vlahos, Ross; Anderson, Gary P.; Bozinovski, Steven

2014-01-01

Serum amyloid A (SAA) is expressed locally in chronic inflammatory conditions such as chronic obstructive pulmonary disease (COPD), where macrophages that do not accord with the classic M1/M2 paradigm also accumulate. In this study, the role of SAA in regulating macrophage differentiation was investigated in vitro using human blood monocytes from healthy subjects and patients with COPD and in vivo using an airway SAA challenge model in BALB/c mice. Differentiation of human monocytes with SAA stimulated the proinflammatory monokines IL-6 and IL-1β concurrently with the M2 markers CD163 and IL-10. Furthermore, SAA-differentiated macrophages stimulated with lipopolysaccharide (LPS) expressed markedly higher levels of IL-6 and IL-1β. The ALX/FPR2 antagonist WRW4 reduced IL-6 and IL-1β expression but did not significantly inhibit phagocytic and efferocytic activity. In vivo, SAA administration induced the development of a CD11chighCD11bhigh macrophage population that generated higher levels of IL-6, IL-1β, and G-CSF following ex vivo LPS challenge. Blocking CSF-1R signaling effectively reduced the number of CD11chighCD11bhigh macrophages by 71% and also markedly inhibited neutrophilic inflammation by 80%. In conclusion, our findings suggest that SAA can promote a distinct CD11chighCD11bhigh macrophage phenotype, and targeting this population may provide a novel approach to treating chronic inflammatory conditions associated with persistent SAA expression.—Anthony, D., McQualter, J. L., Bishara, M., Lim, E. X., Yatmaz, S., Seow, H. J., Hansen, M., Thompson, M., Hamilton, J. A., Irving, L. B., Levy, B. D., Vlahos, R., Anderson, G. P., Bozinovski, S. SAA drives proinflammatory heterotypic macrophage differentiation in the lung via CSF-1R-dependent signaling. PMID:24846388

Bioavailable Citrus sinensis Extract: Polyphenolic Composition and Biological Activity.

PubMed

Pepe, Giacomo; Pagano, Francesco; Adesso, Simona; Sommella, Eduardo; Ostacolo, Carmine; Manfra, Michele; Chieppa, Marcello; Sala, Marina; Russo, Mariateresa; Marzocco, Stefania; Campiglia, Pietro

2017-04-15

Citrus plants contain large amounts of flavonoids with beneficial effects on human health. In the present study, the antioxidant and anti-inflammatory potential of bioavailable polyphenols from Citrus sinensis was evaluated in vitro and ex vivo, using the murine macrophages cell line J774A.1 and primary peritoneal macrophages. Following simulated gastro-intestinal digestion, the in vitro bioavailability of Citrus sinensis polyphenolic extract was assessed using the human cell line Caco-2 grown as monolayers on a transwell membrane. Data demonstrated a relative permeation of its compounds (8.3%). Thus, the antioxidant and anti-inflammatory effect of polyphenolic Citrus sinensis fraction (Cs) was compared to the bioavailable one (CsB). Results revealed that Citrus extract were able to reduce macrophages pro-inflammatory mediators, including nitric oxide, iNOS, COX-2 and different cytokines. Moreover, the effect of Citrus sinensis polyphenols was associated with antioxidant effects, such as a reduction of reactive oxygen species (ROS) and heme-oxygenase-1 (HO-1) increased expression. Our results provide evidence that the bioavailable polyphenolic constituents of the Citrus sinensis extract accumulate prevalently at intestinal level and could reach systemic circulation exerting their effect. The bioavailable fraction showed a higher anti-inflammatory and antioxidant potential compared to the initial extract, thus highlighting its potential nutraceutical value.

Regulation of macrophage development and function in peripheral tissues

PubMed Central

Lavin, Yonit; Mortha, Arthur; Rahman, Adeeb; Merad, Miriam

2015-01-01

Macrophages are immune cells of haematopoietic origin that provide crucial innate immune defence and have tissue-specific functions in the regulation and maintenance of organ homeostasis. Recent studies of macrophage ontogeny, as well as transcriptional and epigenetic identity, have started to reveal the decisive role of the tissue stroma in the regulation of macrophage function. These findings suggest that most macrophages seed the tissues during embryonic development and functionally specialize in response to cytokines and metabolites that are released by the stroma and drive the expression of unique transcription factors. In this Review, we discuss how recent insights into macrophage ontogeny and macrophage–stroma interactions contribute to our understanding of the crosstalk that shapes macrophage function and the maintenance of organ integrity. PMID:26603899

Surfing along Filopodia: A Particle Transport Revealed by Molecular-Scale Fluctuation Analyses

PubMed Central

Kohler, Felix; Rohrbach, Alexander

2015-01-01

Filopodia perform cellular functions such as environmental sensing or cell motility, but they also grab for particles and withdraw them leading to an increased efficiency of phagocytic uptake. Remarkably, withdrawal of micron-sized particles is also possible without noticeable movements of the filopodia. Here, we demonstrate that polystyrene beads connected by optical tweezers to the ends of adherent filopodia of J774 macrophages, are transported discontinuously toward the cell body. After a typical resting time of 1–2 min, the cargo is moved with alternating velocities, force constants, and friction constants along the surface of the filopodia. This surfing-like behavior along the filopodium is recorded by feedback-controlled interferometric three-dimensional tracking of the bead motions at 10–100 kHz. We measured transport velocities of up to 120 nm/s and transport forces of ∼70 pN. Small changes in position, fluctuation width, and temporal correlation, which are invisible in conventional microscopy, indicate molecular reorganization of transport-relevant proteins in different phases of the entire transport process. A detailed analysis implicates a controlled particle transport with fingerprints of a nanoscale unbinding/binding behavior. The manipulation and analysis methods presented in our study may also be helpful in other fields of cellular biophysics. PMID:25954870

Brazilian propolis ethanol extract and its component kaempferol induce myeloid-derived suppressor cells from macrophages of mice in vivo and in vitro.

PubMed

Kitamura, Hiroshi; Saito, Natsuko; Fujimoto, Junpei; Nakashima, Ken-Ichi; Fujikura, Daisuke

2018-05-02

Brazilian green propolis is produced by mixing secretions from Africanized honey bees with exudate, mainly from Baccharis dracunculifolia. Brazilian propolis is especially rich in flavonoids and cinammic acid derivatives, and it has been widely used in folk medicine owing to its anti-inflammatory, anti-viral, tumoricidal, and analgesic effects. Moreover, it is applied to prevent metabolic disorders, such as type 2 diabetes and arteriosclerosis. Previously, we demonstrated that propolis ethanol extract ameliorated type 2 diabetes in a mouse model through the resolution of adipose tissue inflammation. The aims of this study were to identify the immunosuppressive cells directly elicited by propolis extract and to evaluate the flavonoids that induce such cells. Ethanol extract of Brazilian propolis (PEE; 100 mg/kg i.p., twice a week) was injected into lean or high fat-fed obese C57BL/6 mice or C57BL/6 ob/ob mice for one month. Subsequently, immune cells in visceral adipose tissue and the peritoneal cavity were monitored using FACS analysis. Isolated macrophages and the macrophage-like cell line J774.1 were treated with PEE and its constituent components, and the expression of immune suppressive myeloid markers were evaluated. Finally, we injected one of the identified compounds, kaempferol, into C57BL/6 mice and performed FACS analysis on the adipose tissue. Intraperitoneal treatment of PEE induces CD11b + , Gr-1 + myeloid-derived suppressor cells (MDSCs) in visceral adipose tissue and the peritoneal cavity of lean and obese mice. PEE directly stimulates cultured M1 macrophages to transdifferentiate into MDSCs. Among twelve compounds isolated from PEE, kaempferol has an exclusive effect on MDSCs induction in vitro. Accordingly, intraperitoneal injection of kaempferol causes accumulation of MDSCs in the visceral adipose tissue of mice. Brazilian PEE and its compound kaempferol strongly induce MDSCs in visceral adipose tissue at a relatively early phase of inflammation

Report: Over $774 Million of Puerto Rico State Revolving Funds at Risk

EPA Pesticide Factsheets

Report #17-P-0186, April 26, 2017. Over $774 million of state revolving funds is at risk due to Puerto Rico’s ongoing financial crisis. Restoration of the revolving funds in the near future is highly unlikely.

Broad NE 8 lambda 774 emission from quasars in the HST-FOS snapshot survey (ABSNAP)

NASA Technical Reports Server (NTRS)

Hamann, Fred; Zuo, Lin; Tytler, David

1995-01-01

We discuss the strength and frequency of broad Ne VIII lambda 774 emission from quasars measured in the Hubble Space Telescope Faint Object Spectrograph (HST-FOS) snapshot survey (Absnap). Five sources in the survey have suitable redshifts (0.86 less than or equal to Z(sub em) less than or equal to 1.31), signal-to-noise ratios and no Lyman limit absorptions. Three of the five sources have a strong broad emission line near 774 A (rest), and the remaining two sources have a less securely measured line near this wavelength. We identify these lines with Ne VIII lambda 774 based on the measured wavelengths and theoretical estimates of various line fluxes (Hamann et al. 1995a). Secure Ne VIII detections occur in both radio-loud and radio-quiet sources. We tentatively conclude that broad Ne VIII lambda 774 emission is common in quasars, with typical strengths between approximately 25% and approximately 200% of O VI lambda 1034. These Ne VIII lambda 774 measurements imply that the broad emission line regions have a much hotter and more highly ionized component than previously recognized. They also suggest that quasar continua have substantial ionizing flux out to energies greater than 207 eV (greater than 15.2 ryd, lambda less than 60 A). Photoionization calculations using standard incident spectra indicate that the Ne VIII emission requires ionization parameters U greater than or = 5, total column densities N(sub H) greater than or = 10(sub 22)/sq cm and covering factors greater than or = 25%. The temperatures could be as high as approximately 10(exp 5) K. If the gas is instead collisionally ionized, strong Ne VIII would imply equilibrium temperatures in the range approximately 400,000 less than or approximately = T(sub e) less than or approximately = 10(exp 6) K. In either case, the highly ionized Ne VIII emission regions would appear as X-ray 'warm absorbers' if they lie along our line of sight to the X-ray continuum source.

30 CFR 774.17 - Transfer, assignment, or sale of permit rights.

Code of Federal Regulations, 2010 CFR

2010-07-01

... 30 Mineral Resources 3 2010-07-01 2010-07-01 false Transfer, assignment, or sale of permit rights... SYSTEMS UNDER REGULATORY PROGRAMS REVISION; RENEWAL; TRANSFER, ASSIGNMENT, OR SALE OF PERMIT RIGHTS; POST... § 774.17 Transfer, assignment, or sale of permit rights. (a) General. No transfer, assignment, or sale...

30 CFR 774.17 - Transfer, assignment, or sale of permit rights.

Code of Federal Regulations, 2011 CFR

2011-07-01

... 30 Mineral Resources 3 2011-07-01 2011-07-01 false Transfer, assignment, or sale of permit rights... SYSTEMS UNDER REGULATORY PROGRAMS REVISION; RENEWAL; TRANSFER, ASSIGNMENT, OR SALE OF PERMIT RIGHTS; POST... § 774.17 Transfer, assignment, or sale of permit rights. (a) General. No transfer, assignment, or sale...

15 CFR Supplement No. 3 to Part 774 - Statements of Understanding

Code of Federal Regulations, 2011 CFR

2011-01-01

...” that “incorporate” commodities or software on the Commerce Control List (Supplement No. 1 to part 774... medical research). (2) Commodities or software are considered “incorporated” if the commodity or software... medical equipment; and exported or reexported with the medical equipment. (3) Except for such software...

TNF signaling and macrophages govern fin regeneration in zebrafish larvae.

PubMed

Nguyen-Chi, Mai; Laplace-Builhé, Béryl; Travnickova, Jana; Luz-Crawford, Patricia; Tejedor, Gautier; Lutfalla, Georges; Kissa, Karima; Jorgensen, Christian; Djouad, Farida

2017-08-10

Macrophages are essential for appendage regeneration after amputation in regenerative species. The molecular mechanisms through which macrophages orchestrate blastema formation and regeneration are still unclear. Here, we use the genetically tractable and transparent zebrafish larvae to study the functions of polarized macrophage subsets during caudal fin regeneration. After caudal fin amputation, we show an early and transient accumulation of pro-inflammatory macrophages concomitant with the accumulation of non-inflammatory macrophages which, in contrast to pro-inflammatory macrophages, remain associated to the fin until the end of the regeneration. Chemical and genetic depletion of macrophages suggested that early recruited macrophages that express TNFα are critical for blastema formation. Combining parabiosis and morpholino knockdown strategies, we show that TNFα/TNFR1 signaling pathway is required for the fin regeneration. Our study reveals that TNFR1 has a necessary and direct role in blastema cell activation suggesting that macrophage subset balance provides the accurate TNFα signal to prime regeneration in zebrafish.

Structure-function analysis of the auxilin J-domain reveals an extended Hsc70 interaction interface.

PubMed

Jiang, Jianwen; Taylor, Alexander B; Prasad, Kondury; Ishikawa-Brush, Yumiko; Hart, P John; Lafer, Eileen M; Sousa, Rui

2003-05-20

J-domains are widespread protein interaction modules involved in recruiting and stimulating the activity of Hsp70 family chaperones. We have determined the crystal structure of the J-domain of auxilin, a protein which is involved in uncoating clathrin-coated vesicles. Comparison to the known structures of J-domains from four other proteins reveals that the auxilin J-domain is the most divergent of all J-domain structures described to date. In addition to the canonical J-domain features described previously, the auxilin J-domain contains an extra N-terminal helix and a long loop inserted between helices I and II. The latter loop extends the positively charged surface which forms the Hsc70 binding site, and is shown by directed mutagenesis and surface plasmon resonance to contain side chains important for binding to Hsc70.

[Distribution diversity of integrins and calcium channels on major human and mouse host cells of Leptospira species].

PubMed

Li, Cheng-xue; Zhao, Xin; Qian, Jing; Yan, Jie

2012-07-01

To determine the distribution of integrins and calcium channels on major human and mouse host cells of Leptospira species. The expression of β1, β2 and β3 integrins was detected with immunofluorescence assay on the surface of human monocyte line THP-1, mouse mononuclear-macrophage-like cell line J774A.1, human vascular endothelial cell line HUVEC, mouse vascular endothelial cell EOMA, human hepatocyte line L-02, mouse hepatocyte line Hepa1-6, human renal tubular epithelial cell line HEK-293, mouse glomerular membrane epithelial cell line SV40-MES13, mouse collagen blast line NIH/3T3, human and mouse platelets. The distribution of voltage gate control calcium channels Cav3.1, Cav3.2, Cav3.3 and Cav2.3, and receptor gate calcium channels P(2)X(1), P(2)2X(2), P(2)X(3), P(2)X(4), P(2)X(5), P(2)X(6) and P(2)X(7) were determined with Western blot assay. β1 integrin proteins were positively expressed on the membrane surface of J774A.1, THP-1, HUVEC, EOMA, L-02, Hepa1-6 and HEK-239 cells as well as human and mouse platelets. β2 integrin proteins were expressed on the membrane surface of J774A.1, THP-1, HUVEC, EOMA, and NIH/3T3 cells. β3 integrin proteins were expressed on the membrane surface of J774A.1, THP-1, HUVEC, EOMA, Hepa1-6, HEK-239 and NIH/3T3 cells as well as human and mouse platelets. P(2)X(1) receptor gate calcium channel was expressed on the membrane surface of human and mouse platelets, while P(2)X(5) receptor gate calcium channel was expressed on the membrane surface of J774A.1, THP-1, L-02, Hepa1-6, HEK-239 and HUVEC cells. However, the other calcium channels were not detected on the tested cell lines or platelets. There is a large distribution diversity of integrins and calcium channel proteins on the major human and mouse host cells of Leptospira species, which may be associated with the differences of leptospira-induced injury in different host cells.

The Increase in Mannose Receptor Recycling Favors Arginase Induction and Trypanosoma Cruzi Survival in Macrophages

PubMed Central

Garrido, Vanina V.; Dulgerian, Laura R.; Stempin, Cinthia C.; Cerbán, Fabio M.

2011-01-01

The macrophage mannose receptor (MR) is a pattern recognition receptor of the innate immune system that binds to microbial structures bearing mannose, fucose and N-acetylglucosamine on their surface. Trypanosoma cruzi antigen cruzipain (Cz) is found in the different developmental forms of the parasite. This glycoprotein has a highly mannosylated C-terminal domain that participates in the host-antigen contact. Our group previously demonstrated that Cz-macrophage (Mo) interaction could modulate the immune response against T. cruzi through the induction of a preferential metabolic pathway. In this work, we have studied in Mo the role of MR in arginase induction and in T. cruzi survival using different MR ligands. We have showed that pre-incubation of T. cruzi infected cells with mannose-Bovine Serum Albumin (Man-BSA, MR specific ligand) biased nitric oxide (NO)/urea balance towards urea production and increased intracellular amastigotes growth. The study of intracellular signals showed that pre-incubation with Man-BSA in T. cruzi J774 infected cells induced down-regulation of JNK and p44/p42 phosphorylation and increased of p38 MAPK phosphorylation. These results are coincident with previous data showing that Cz also modifies the MAPK phosphorylation profile induced by the parasite. In addition, we have showed by confocal microscopy that Cz and Man-BSA enhance MR recycling. Furthermore, we studied MR behavior during T. cruzi infection in vivo. MR was up-regulated in F4/80+ cells from T. cruzi infected mice at 13 and 15 days post infection. Besides, we investigated the effect of MR blocking antibody in T. cruzi infected peritoneal Mo. Arginase activity and parasite growth were decreased in infected cells pre-incubated with anti-MR antibody as compared with infected cells treated with control antibody. Therefore, we postulate that during T. cruzi infection, Cz may contact with MR, increasing MR recycling which leads to arginase activity up-regulation and intracellular

Macrophage heterogeneity in liver injury and fibrosis.

PubMed

Tacke, Frank; Zimmermann, Henning W

2014-05-01

Hepatic macrophages are central in the pathogenesis of chronic liver injury and have been proposed as potential targets in combatting fibrosis. Recent experimental studies in animal models revealed that hepatic macrophages are a remarkably heterogeneous population of immune cells that fulfill diverse functions in homeostasis, disease progression, and regression from injury. These range from clearance of pathogens or cellular debris and maintenance of immunological tolerance in steady state conditions; central roles in initiating and perpetuating inflammation in response to injury; promoting liver fibrosis via activating hepatic stellate cells in chronic liver damage; and, finally, resolution of inflammation and fibrosis by degradation of extracellular matrix and release of anti-inflammatory cytokines. Cellular heterogeneity in the liver is partly explained by the origin of macrophages. Hepatic macrophages can either arise from circulating monocytes, which are recruited to the injured liver via chemokine signals, or from self-renewing embryo-derived local macrophages, termed Kupffer cells. Kupffer cells appear essential for sensing tissue injury and initiating inflammatory responses, while infiltrating Ly-6C(+) monocyte-derived macrophages are linked to chronic inflammation and fibrogenesis. In addition, proliferation of local or recruited macrophages may possibly further contribute to their accumulation in injured liver. During fibrosis regression, monocyte-derived cells differentiate into Ly-6C (Ly6C, Gr1) low expressing 'restorative' macrophages and promote resolution from injury. Understanding the mechanisms that regulate hepatic macrophage heterogeneity, either by monocyte subset recruitment, by promoting restorative macrophage polarization or by impacting distinctive macrophage effector functions, may help to develop novel macrophage subset-targeted therapies for liver injury and fibrosis. Copyright © 2014 European Association for the Study of the Liver

Optimization of cell receptor-specific targeting through multivalent surface decoration of polymeric nanocarriers

PubMed Central

D’Addio, Suzanne M.; Baldassano, Steven; Shi, Lei; Cheung, Lila; Adamson, Douglas H.; Bruzek, Matthew; Anthony, John E.; Laskin, Debra L.; Sinko, Patrick J.; Prud’homme, Robert K.

2013-01-01

Treatment of tuberculosis is impaired by poor drug bioavailability, systemic side effects, patient non-compliance, and pathogen resistance to existing therapies. The mannose receptor (MR) is known to be involved in the recognition and internalization of Mycobacterium tuberculosis. We present a new assembly process to produce nanocarriers with variable surface densities of mannose targeting ligands in a single step, using kinetically-controlled, block copolymer-directed assembly. Nanocarrier association with murine macrophage J774 cells expressing the MR is examined as a function of incubation time and temperature, nanocarrier size, dose, and PEG corona properties. Amphiphilic diblock copolymers are prepared with terminal hydroxyl, methoxy, or mannoside functionality and incorporated into nanocarrier formulations at specific ratios by Flash NanoPrecipitation. Association of nanocarriers protected by a hydroxyl-terminated PEG corona with J774 cells is size dependent, while nanocarriers with methoxy-terminated PEG coronas do not associate with cells, regardless of size. Specific targeting of the MR is investigated using nanocarriers having 0-75% mannoside-terminated PEG chains in the PEG corona. This is a wider range of mannose densities than has been previously studied. Maximum nanocarrier association is attained with 9% mannoside-terminated PEG chains, increasing uptake more than 3-fold compared to non-targeted nanocarriers with a 5 kg mol−1 methoxy-terminated PEG corona. While a 5 kg mol−1 methoxy-terminated PEG corona prevents non-specific uptake, a 1.8 kg mol−1 methoxy-terminated PEG corona does not sufficiently protect the nanocarriers from nonspecific association. There is continuous uptake of MR-targeted nanocarriers at 37°C, but a saturation of association at 4°C. The majority of targeted nanocarriers associate with J774E cells are internalized at 37°C and uptake is receptor-dependent, diminishing with competitive inhibition by dextran. This

Tissue Inhibitor of Metalloproteinases–3 Moderates the Proinflammatory Status of Macrophages

PubMed Central

Gharib, Sina A.; Bench, Eli M.; Sussman, Samuel W.; Wang, Roy T.; Rims, Cliff; Birkland, Timothy P.; Wang, Ying; Manicone, Anne M.; McGuire, John K.; Parks, William C.

2013-01-01

Tissue inhibitor of metalloproteinases–3 (TIMP-3) has emerged as a key mediator of inflammation. Recently, we reported that the resolution of inflammation is impaired in Timp3−/− mice after bleomycin-induced lung injury. Here, we demonstrate that after LPS instillation (another model of acute lung injury), Timp3−/− mice demonstrate enhanced and persistent neutrophilia, increased numbers of infiltrated macrophages, and delayed weight gain, compared with wild-type (WT) mice. Because macrophages possess broad immune functions and can differentiate into cells that either stimulate inflammation (M1 macrophages) or are immunosuppressive (M2 macrophages), we examined whether TIMP-3 influences macrophage polarization. Comparisons of the global gene expression of unstimulated or LPS-stimulated bone marrow–derived macrophages (BMDMs) from WT and Timp3−/− mice revealed that Timp3−/− BMDMs exhibited an increased expression of genes associated with proinflammatory (M1) macrophages, including Il6, Il12, Nos2, and Ccl2. Microarray analyses also revealed a baseline difference in gene expression between WT and Timp3−/− BMDMs, suggesting altered macrophage differentiation. Furthermore, the treatment of Timp3−/− BMDMs with recombinant TIMP-3 rescued this altered gene expression. We also examined macrophage function, and found that Timp3−/− M1 cells exhibit significantly more neutrophil chemotactic activity and significantly less soluble Fas ligand–induced caspase-3/7 activity, a marker of apoptosis, compared with WT M1 cells. Macrophage differentiation into immunosuppressive M2 cells is mediated by exposure to IL-4/IL-13, and we found that Timp3−/− M2 macrophages demonstrated a lower expression of genes associated with an anti-inflammatory phenotype, compared with WT M2 cells. Collectively, these findings indicate that TIMP-3 functions to moderate the differentiation of macrophages into proinflammatory (M1) cells. PMID:23742180

Polysaccharide of Dendrobium huoshanense activates macrophages via toll-like receptor 4-mediated signaling pathways.

PubMed

Xie, Song-Zi; Hao, Ran; Zha, Xue-Qiang; Pan, Li-Hua; Liu, Jian; Luo, Jian-Ping

2016-08-01

The present work aimed at investigating the pattern recognition receptor (PRR) and immunostimulatory mechanism of a purified Dendrobium huoshanense polysaccharide (DHP). We found that DHP could bind to the surface of macrophages and stimulate macrophages to secrete NO, TNF-α and IL-1β. To unravel the mechanism for the binding of DHP to macrophages, flow cytometry, confocal laser-scanning microscopy, affinity electrophoresis, SDS-PAGE and western blotting were employed to verify the type of PRR responsible for the recognition of DHP by RAW264.7 macrophages and peritoneal macrophages of C3H/HeN and C3H/HeJ macrophages. Results showed that toll-like receptor 4 (TLR4) was an essential receptor for macrophages to directly bind DHP. Further, the phosphorylation of ERK, JNK, Akt and p38 were observed to be time-dependently promoted by DHP, as well as the nuclear translocation of NF-κB p65. These results suggest that DHP activates macrophages via its direct binding to TLR4 to trigger TLR4 signaling pathways. Copyright © 2016 Elsevier Ltd. All rights reserved.

Macrophage Mitochondrial Oxidative Stress Promotes Atherosclerosis and NF-κB-Mediated Inflammation in Macrophages

PubMed Central

Wang, Ying; Wang, Gary Z.; Rabinovitch, Peter S.; Tabas, Ira

2014-01-01

Rationale Mitochondrial oxidative stress (mitoOS) has been shown to correlate with the progression of human atherosclerosis. However, definitive cell-type specific causation studies in vivo are lacking, and the molecular mechanisms of potential pro-atherogenic effects remain to be determined. Objective To assess the importance of macrophage mitoOS in atherogenesis and explore the underlying molecular mechanisms. Methods & Results We first validated Western-type diet-fed Ldlr-/- mice as a model of human mitoOS-atherosclerosis association by showing that a marker of mitoOS in lesional macrophages, non-nuclear oxidative DNA damage, correlates with aortic root lesion development. To investigate the importance of macrophage-mitoOS, we used a genetic engineering strategy in which the OS suppressor catalase was ectopically expressed in mitochondria (mCAT) in macrophages. MitoOS in lesional macrophages was successfully suppressed in these mice, and this led to a significant reduction in aortic root lesional area. The mCAT lesions had less monocyte-derived cells, less Ly6chi monocyte infiltration into lesions, and lower levels of the monocyte chemotactic protein-1 (MCP-1). The decrease in lesional MCP-1 was associated with suppression of other markers of inflammation and with decreased phosphorylation of RelA (NF-κB p65), indicating decreased activation of the pro-inflammatory NF-κB pathway. Using models of mitoOS in cultured macrophages, we showed that mCAT suppressed MCP-1 expression by decreasing activation of the Iκ-kinase-RelA NF-κB pathway. Conclusions MitoOS in lesional macrophages amplifies atherosclerotic lesion development by promoting NF-κB-mediated entry of monocytes and other inflammatory processes. In view of the mitoOS-atherosclerosis link in human atheromata, these findings reveal a potentially new therapeutic target to prevent the progression of atherosclerosis. PMID:24297735

A critical role for macrophages in neovessel formation and the development of stenosis in tissue-engineered vascular grafts

PubMed Central

Hibino, Narutoshi; Yi, Tai; Duncan, Daniel R.; Rathore, Animesh; Dean, Ethan; Naito, Yuji; Dardik, Alan; Kyriakides, Themis; Madri, Joseph; Pober, Jordan S.; Shinoka, Toshiharu; Breuer, Christopher K.

2011-01-01

The primary graft-related complication during the first clinical trial evaluating the use of tissue-engineered vascular grafts (TEVGs) was stenosis. We investigated the role of macrophages in the formation of TEVG stenosis in a murine model. We analyzed the natural history of TEVG macrophage infiltration at critical time points and evaluated the role of cell seeding on neovessel formation. To assess the function of infiltrating macrophages, we implanted TEVGs into mice that had been macrophage depleted using clodronate liposomes. To confirm this, we used a CD11b-diphtheria toxin-receptor (DTR) transgenic mouse model. Monocytes infiltrated the scaffold within the first few days and initially transformed into M1 macrophages. As the scaffold degraded, the macrophage infiltrate disappeared. Cell seeding decreased the incidence of stenosis (32% seeded, 64% unseeded, P=0.024) and the degree of macrophage infiltration at 2 wk. Unseeded TEVGs demonstrated conversion from M1 to M2 phenotype, whereas seeded grafts did not. Clodronate and DTR inhibited macrophage infiltration and decreased stenosis but blocked formation of vascular neotissue, evidenced by the absence of endothelial and smooth muscle cells and collagen. These findings suggest that macrophage infiltration is critical for neovessel formation and provides a strategy for predicting, detecting, and inhibiting stenosis in TEVGs.—Hibino, N., Yi, T., Duncan, D. R., Rathore, A., Dean, E., Naito, Y., Dardik, A., Kyriakides, T., Madri, J., Pober, J. S., Shinoka, T., Breuer, C. K. A critical role for macrophages in neovessel formation and the development of stenosis in tissue-engineered vascular grafts. PMID:21865316

CD74-Downregulation of Placental Macrophage-Trophoblastic Interactions in Preeclampsia.

PubMed

Przybyl, Lukasz; Haase, Nadine; Golic, Michaela; Rugor, Julianna; Solano, Maria Emilia; Arck, Petra Clara; Gauster, Martin; Huppertz, Berthold; Emontzpohl, Christoph; Stoppe, Christian; Bernhagen, Jürgen; Leng, Lin; Bucala, Richard; Schulz, Herbert; Heuser, Arnd; Weedon-Fekjær, M Susanne; Johnsen, Guro M; Peetz, Dirk; Luft, Friedrich C; Staff, Anne Cathrine; Müller, Dominik N; Dechend, Ralf; Herse, Florian

2016-06-24

We hypothesized that cluster of differentiation 74 (CD74) downregulation on placental macrophages, leading to altered macrophage-trophoblast interaction, is involved in preeclampsia. Preeclamptic pregnancies feature hypertension, proteinuria, and placental anomalies. Feto-placental macrophages regulate villous trophoblast differentiation during placental development. Disturbance of this well-balanced regulation can lead to pathological pregnancies. We performed whole-genome expression analysis of placental tissue. CD74 was one of the most downregulated genes in placentas from preeclamptic women. By reverse transcriptase-polymerase chain reaction, we confirmed this finding in early-onset (<34 gestational week, n=26) and late-onset (≥34 gestational week, n=24) samples from preeclamptic women, compared with healthy pregnant controls (n=28). CD74 protein levels were analyzed by Western blot and flow cytometry. We identified placental macrophages to express CD74 by immunofluorescence, flow cytometry, and RT-PCR. CD74-positive macrophages were significantly reduced in preeclamptic placentas compared with controls. CD74-silenced macrophages showed that the adhesion molecules ALCAM, ICAM4, and Syndecan-2, as well as macrophage adhesion to trophoblasts were diminished. Naive and activated macrophages lacking CD74 showed a shift toward a proinflammatory signature with an increased secretion of tumor necrosis factor-α, chemokine (C-C motif) ligand 5, and monocyte chemotactic protein-1, when cocultured with trophoblasts compared with control macrophages. Trophoblasts stimulated by these factors express more CYP2J2, sFlt1, TNFα, and IL-8. CD74-knockout mice showed disturbed placental morphology, reduced junctional zone, smaller placentas, and impaired spiral artery remodeling with fetal growth restriction. CD74 downregulation in placental macrophages is present in preeclampsia. CD74 downregulation leads to altered macrophage activation toward a proinflammatory signature and

Comprehensive proteome analysis of lysosomes reveals the diverse function of macrophages in immune responses.

PubMed

Gao, Yanpan; Chen, Yanyu; Zhan, Shaohua; Zhang, Wenhao; Xiong, Feng; Ge, Wei

2017-01-31

Phagocytosis and autophagy in macrophages have been shown to be essential to both innate and adaptive immunity. Lysosomes are the main catabolic subcellular organelles responsible for degradation and recycling of both extracellular and intracellular material, which are the final steps in phagocytosis and autophagy. However, the molecular mechanisms underlying lysosomal functions after infection remain obscure. In this study, we conducted a quantitative proteomics analysis of the changes in constitution and glycosylation of proteins in lysosomes derived from murine RAW 264.7 macrophage cells treated with different types of pathogens comprising examples of bacteria (Listeria monocytogenes, L. m), DNA viruses (herpes simplex virus type-1, HSV-1) and RNA viruses (vesicular stomatitis virus, VSV). In total, 3,704 lysosome-related proteins and 300 potential glycosylation sites on 193 proteins were identified. Comparative analysis showed that the aforementioned pathogens induced distinct alterations in the proteome of the lysosome, which is closely associated with the immune functions of macrophages, such as toll-like receptor activation, inflammation and antigen-presentation. The most significant changes in proteins and fluctuations in glycosylation were also determined. Furthermore, Western blot analysis showed that the changes in expression of these proteins were undetectable at the whole cell level. Thus, our study provides unique insights into the function of lysosomes in macrophage activation and immune responses.

Comprehensive proteome analysis of lysosomes reveals the diverse function of macrophages in immune responses

PubMed Central

Zhan, Shaohua; Zhang, Wenhao; Xiong, Feng; Ge, Wei

2017-01-01

Phagocytosis and autophagy in macrophages have been shown to be essential to both innate and adaptive immunity. Lysosomes are the main catabolic subcellular organelles responsible for degradation and recycling of both extracellular and intracellular material, which are the final steps in phagocytosis and autophagy. However, the molecular mechanisms underlying lysosomal functions after infection remain obscure. In this study, we conducted a quantitative proteomics analysis of the changes in constitution and glycosylation of proteins in lysosomes derived from murine RAW 264.7 macrophage cells treated with different types of pathogens comprising examples of bacteria (Listeria monocytogenes, L. m), DNA viruses (herpes simplex virus type-1, HSV-1) and RNA viruses (vesicular stomatitis virus, VSV). In total, 3,704 lysosome-related proteins and 300 potential glycosylation sites on 193 proteins were identified. Comparative analysis showed that the aforementioned pathogens induced distinct alterations in the proteome of the lysosome, which is closely associated with the immune functions of macrophages, such as toll-like receptor activation, inflammation and antigen-presentation. The most significant changes in proteins and fluctuations in glycosylation were also determined. Furthermore, Western blot analysis showed that the changes in expression of these proteins were undetectable at the whole cell level. Thus, our study provides unique insights into the function of lysosomes in macrophage activation and immune responses. PMID:28088779

Src family kinase expression and subcellular localization in macrophages: implications for their role in CSF-1-induced macrophage migration.

PubMed

Dwyer, Amy R; Mouchemore, Kellie A; Steer, James H; Sunderland, Andrew J; Sampaio, Natalia G; Greenland, Eloise L; Joyce, David A; Pixley, Fiona J

2016-07-01

A major role of colony-stimulating factor-1 is to stimulate the differentiation of mononuclear phagocytic lineage cells into adherent, motile, mature macrophages. The colony-stimulating factor-1 receptor transduces colony-stimulating factor-1 signaling, and we have shown previously that phosphatidylinositol 3-kinase p110δ is a critical mediator of colony-stimulating factor-1-stimulated motility through the colony-stimulating factor-1 receptor pY721 motif. Src family kinases are also implicated in the regulation of macrophage motility and in colony-stimulating factor-1 receptor signaling, although functional redundancy of the multiple SFKs expressed in macrophages makes it challenging to delineate their specific functions. We report a comprehensive analysis of individual Src family kinase expression in macrophage cell lines and primary macrophages and demonstrate colony-stimulating factor-1-induced changes in Src family kinase subcellular localization, which provides clues to their distinct and redundant functions in macrophages. Moreover, expression of individual Src family kinases is both species specific and dependent on colony-stimulating factor-1-induced macrophage differentiation. Hck associated with the activated colony-stimulating factor-1 receptor, whereas Lyn associated with the receptor in a constitutive manner. Consistent with this, inhibitor studies revealed that Src family kinases were important for both colony-stimulating factor-1 receptor activation and colony-stimulating factor-1-induced macrophage spreading, motility, and invasion. Distinct colony-stimulating factor-1-induced changes in the subcellular localization of individual SFKs suggest specific roles for these Src family kinases in the macrophage response to colony-stimulating factor-1. © Society for Leukocyte Biology.

Association with AflR in Endosomes Reveals New Functions for AflJ in Aflatoxin Biosynthesis

PubMed Central

Ehrlich, Kenneth C.; Mack, Brian M.; Wei, Qijian; Li, Ping; Roze, Ludmila V.; Dazzo, Frank; Cary, Jeffrey W.; Bhatnagar, Deepak; Linz, John E.

2012-01-01

Aflatoxins are the most potent naturally occurring carcinogens of fungal origin. Biosynthesis of aflatoxin involves the coordinated expression of more than 25 genes. The function of one gene in the aflatoxin gene cluster, aflJ, is not entirely understood but, because previous studies demonstrated a physical interaction between the Zn2Cys6 transcription factor AflR and AflJ, AflJ was proposed to act as a transcriptional co-activator. Image analysis revealed that, in the absence of aflJ in A. parasiticus, endosomes cluster within cells and near septa. AflJ fused to yellow fluorescent protein complemented the mutation in A. parasiticus ΔaflJ and localized mainly in endosomes. We found that AflJ co-localizes with AflR both in endosomes and in nuclei. Chromatin immunoprecipitation did not detect AflJ binding at known AflR DNA recognition sites suggesting that AflJ either does not bind to these sites or binds to them transiently. Based on these data, we hypothesize that AflJ assists in AflR transport to or from the nucleus, thus controlling the availability of AflR for transcriptional activation of aflatoxin biosynthesis cluster genes. AflJ may also assist in directing endosomes to the cytoplasmic membrane for aflatoxin export. PMID:23342682

A transcriptomic analysis of Yersinia enterocolitica biovar 1B infecting murine macrophages reveals new mechanisms of intracellular survival

DOE PAGES

Bent, Zachary W.; Poorey, Kunal; Brazel, David M.; ...

2015-04-20

Yersinia enterocolitica is typically considered an extracellular pathogen; however, during the course of an infection, a significant number of bacteria are stably maintained within host cell vacuoles. Little is known about this population and the role it plays during an infection. To address this question and to elucidate the spatially and temporally dynamic gene expression patterns of Y. enterocoliticabiovar 1B through the course of an in vitro infection, transcriptome sequencing and differential gene expression analysis of bacteria infecting murine macrophage cells were performed under four distinct conditions. Bacteria were first grown in a nutrient-rich medium at 26°C to establish amore » baseline of gene expression that is unrelated to infection. The transcriptomes of these bacteria were then compared to bacteria grown in a conditioned cell culture medium at 37°C to identify genes that were differentially expressed in response to the increased temperature and medium but not in response to host cells. Infections were then performed, and the transcriptomes of bacteria found on the extracellular surface and intracellular compartments were analyzed individually. The upregulated genes revealed potential roles for a variety of systems in promoting intracellular virulence, including the Ysa type III secretion system, the Yts2 type II secretion system, and the Tad pilus. It was further determined that mutants of each of these systems had decreased virulence while infecting macrophages. Overall, these results reveal the complete set of genes expressed by Y. enterocolitica in response to infection and provide the groundwork for future virulence studies.« less

Intragenic recombination has a critical role on the evolution of Legionella pneumophila virulence-related effector sidJ.

PubMed

Costa, Joana; Teixeira, Paulo Gonçalves; d'Avó, Ana Filipa; Júnior, Célio Santos; Veríssimo, António

2014-01-01

SidJ is a Dot/Icm effector involved in the trafficking or retention of ER-derived vesicles to Legionella pneumophila vacuoles whose mutation causes an observable growth defect, both in macrophage and amoeba hosts. Given the crucial role of this effector in L. pneumophila virulence we investigated the mechanisms shaping its molecular evolution. The alignment of SidJ sequences revealed several alleles with amino acid variations that may influence the protein properties. The identification of HGT events and the detection of balancing selection operating on sidJ evolution emerge as a clear result. Evidence suggests that intragenic recombination is an important strategy in the evolutionary adaptive process playing an active role on sidJ genetic plasticity. This pattern of evolution is in accordance with the life style of L. pneumophila as a broad host-range pathogen by preventing host-specialization and contributing to the resilience of the species.

Macrophage mitochondrial oxidative stress promotes atherosclerosis and nuclear factor-κB-mediated inflammation in macrophages.

PubMed

Wang, Ying; Wang, Gary Z; Rabinovitch, Peter S; Tabas, Ira

2014-01-31

Mitochondrial oxidative stress (mitoOS) has been shown to correlate with the progression of human atherosclerosis. However, definitive cell type-specific causation studies in vivo are lacking, and the molecular mechanisms of potential proatherogenic effects remain to be determined. Our aims were to assess the importance of macrophage mitoOS in atherogenesis and to explore the underlying molecular mechanisms. We first validated Western diet-fed Ldlr(-/-) mice as a model of human mitoOS-atherosclerosis association by showing that non-nuclear oxidative DNA damage, a marker of mitoOS in lesional macrophages, correlates with aortic root lesion development. To investigate the importance of macrophage mitoOS, we used a genetic engineering strategy in which the OS suppressor catalase was ectopically expressed in mitochondria (mCAT) in macrophages. MitoOS in lesional macrophages was successfully suppressed in these mice, and this led to a significant reduction in aortic root lesional area. The mCAT lesions had less monocyte-derived cells, less Ly6c(hi) monocyte infiltration into lesions, and lower levels of monocyte chemotactic protein-1. The decrease in lesional monocyte chemotactic protein-1 was associated with the suppression of other markers of inflammation and with decreased phosphorylation of RelA (NF-κB p65), indicating decreased activation of the proinflammatory NF-κB pathway. Using models of mitoOS in cultured macrophages, we showed that mCAT suppressed monocyte chemotactic protein-1 expression by decreasing the activation of the IκB-kinase β-RelA NF-κB pathway. MitoOS in lesional macrophages amplifies atherosclerotic lesion development by promoting NF-κB-mediated entry of monocytes and other inflammatory processes. In view of the mitoOS-atherosclerosis link in human atheromata, these findings reveal a potentially new therapeutic target to prevent the progression of atherosclerosis.

Morphologic, phenotypic, and transcriptomic characterization of classically and alternatively activated canine blood-derived macrophages in vitro

PubMed Central

Heinrich, Franziska; Lehmbecker, Annika; Raddatz, Barbara B.; Kegler, Kristel; Tipold, Andrea; Stein, Veronika M.; Kalkuhl, Arno; Deschl, Ulrich; Baumgärtner, Wolfgang; Ulrich, Reiner

2017-01-01

Macrophages are a heterogeneous cell population playing a pivotal role in tissue homeostasis and inflammation, and their phenotype strongly depends on the micromilieu. Despite its increasing importance as a translational animal model for human diseases, there is a considerable gap of knowledge with respect to macrophage polarization in dogs. The present study comprehensively investigated the morphologic, phenotypic, and transcriptomic characteristics of unstimulated (M0), M1- (GM-CSF, LPS, IFNγ-stimulated) and M2- (M-CSF, IL-4-stimulated)-polarized canine blood-derived macrophages in vitro. Scanning electron microscopy revealed distinct morphologies of polarized macrophages with formation of multinucleated cells in M2-macrophages, while immunofluorescence employing literature-based prototype-antibodies against CD16, CD32, iNOS, MHC class II (M1-markers), CD163, CD206, and arginase-1 (M2-markers) demonstrated that only CD206 was able to discriminate M2-macrophages from both other phenotypes, highlighting this molecule as a promising marker for canine M2-macrophages. Global microarray analysis revealed profound changes in the transcriptome of polarized canine macrophages. Functional analysis pointed out that M1-polarization was associated with biological processes such as “respiratory burst”, whereas M2-polarization was associated with processes such as “mitosis”. Literature-based marker gene selection revealed only minor overlaps in the gene sets of the dog compared to prototype markers of murine and human macrophages. Biomarker selection using supervised clustering suggested latexin (LXN) and membrane-spanning 4-domains, subfamily A, member 2 (MS4A2) to be the most powerful predicting biomarkers for canine M1- and M2-macrophages, respectively. Immunofluorescence for both markers demonstrated expression of both proteins by macrophages in vitro but failed to reveal differences between canine M1 and M2-macrophages. The present study provides a solid basis for

The Prolonged Life-Span of Alveolar Macrophages

PubMed Central

Murphy, Jaime; Summer, Ross; Wilson, Andrew A.; Kotton, Darrell N.; Fine, Alan

2008-01-01

To further examine the half-life of alveolar macrophages, chimeric CD 45.2 mice were generated through bone marrow transplantation of donor CD 45.1 cells. Before administration of donor cells, recipient mice were divided into two cohorts: the first cohort received total body irradiation; the second cohort also received irradiation—however, the thorax, head, and upper extremities were shielded with lead. Flow cytometric analysis was then performed on blood, peritoneal, and bronchoalveolar lavage cells over time to quantify engraftment. The data generated for the unshielded cohort of mice revealed a macrophage half-life of 30 days. In the shielded cohort, however, we found that by 8 months there was negligible replacement of recipient alveolar macrophages by donor cells, despite reconstitution of the blood and peritoneum by donor bone marrow. Consistent with these findings, the mean fluorescent intensity of alveolar macrophages remained stable over a 4-week period after in vivo PKH26 dye loading. Together, these data show that previous alveolar macrophage half-life studies were confounded by the fact that they did not account for the toxic effects of irradiation conditioning regimens, and demonstrate that the bone marrow does not significantly contribute to the alveolar macrophage compartment during steady-state conditions. PMID:18192503

Silica-induced initiation of circular ZC3H4 RNA/ZC3H4 pathway promotes the pulmonary macrophage activation.

PubMed

Yang, Xiyue; Wang, Jing; Zhou, Zewei; Jiang, Rong; Huang, Jie; Chen, Lulu; Cao, Zhouli; Chu, Han; Han, Bing; Cheng, Yusi; Chao, Jie

2018-06-01

Phagocytosis of silicon dioxide (SiO 2 ) into lung cells causes an inflammatory cascade that results in fibroblast proliferation and migration, followed by fibrosis. Circular RNAs (circRNAs) are a subclass of noncoding RNAs that are present within mammalian cells; however, researchers have not determined whether circRNAs are involved in the pathophysiologic process of silicosis. To elucidate the role of these RNAs in SiO 2 -induced inflammation in pulmonary macrophages, we investigated the upstream molecular mechanisms and functional effects of circRNAs on cell apoptosis, proliferation, and migration. Primary cultures of alveolar macrophages from healthy donors and from patients and the RAW264.7 macrophage cell line were used to explore the functions of circZC3H4 RNA in macrophage activation. The experimental results indicated the following: 1) SiO 2 concomitantly increased circZC3H4 RNA expression and increased ZC3H4 protein levels; 2) circular ZC3H4 (circZC3H4) RNA and ZC3H4 protein participated in SiO 2 -induced macrophage activation; and 3) SiO 2 -activated macrophages promoted fibroblast proliferation and migration via the circZC3H4 RNA/ZC3H4 pathway. The up-regulation of the ZC3H4 protein was confirmed in tissue samples from patients with silicosis. Our study elucidates a link between SiO 2 -induced macrophage activation and the circZC3H4 RNA/ZC3H4 pathway, thereby providing novel insight into the potential use of ZC3H4 to develop novel therapeutic strategies for silicosis.-Yang, X., Wang, J., Zhou, Z., Jiang, R., Huang, J., Chen, L., Cao, Z., Chu, H., Han, B., Cheng, Y., Chao, J. Silica-induced initiation of circular ZC3H4 RNA/ZC3H4 pathway promotes the pulmonary macrophage activation.

Cardiac resident macrophages are involved in hypoxia-induced postnatal cardiomyocyte proliferation

PubMed Central

Liu, Bo; Zhang, Hua-Gang; Zhu, Yun; Jiang, Yun-Han; Luo, Gui-Ping; Tang, Fu-Qin; Jian, Zhao; Xiao, Ying-Bin

2017-01-01

Induction of cardiomyocyte proliferation, the most promising approach to reverse myocardial attrition, has been gaining importance as a therapy for cardiovascular disease. Hypoxia and macrophages were previously independently reported to promote cardiomyocyte proliferation in mice. However, whether hypoxia promotes cardiomyocyte proliferation in humans, and the association between hypoxia and macrophages in cardiomyocyte proliferation, have not to the best of our knowledge been previously investigated. The present study investigated the cardiomyocyte proliferation in 22 acyanotic and 29 cyanotic patients. Cardiomyocyte proliferation in a hypoxic mouse model (15% O2) was subsequently performed and the macrophage subsets were analyzed. A C-C chemokine receptor type 2 (CCR2) inhibitor was used to increase the number of resident macrophages in order to investigate the effect of macrophages on cardiomyocyte proliferation. The results demonstrated that cardiomyocyte proliferation in the cyanotic infant group was significantly increased compared with the acyanotic infant group and the hypoxia-treated C57BL/6J neonates confirmed the hypoxia-induced cardiomyocyte proliferation. However, hypoxia did not induce the proliferation of isolated cardiomyocytes. Notably, hypoxia treatment increased the number of cardiac resident macrophages in neonate hearts. Furthermore, increasing the number of resident macrophages significantly enhanced cardiomyocyte proliferation. In conclusion, postnatal hypoxia promoted cardiomyocyte proliferation in humans and animals, and cardiac resident macrophages may be involved in this process. Therefore, this novel mechanism may provide a promising strategy for cardiovascular disease treatment. PMID:28393210

Embryonic and adult-derived resident cardiac macrophages are maintained through distinct mechanisms at steady state and during inflammation

PubMed Central

Epelman, Slava; Lavine, Kory J.; Beaudin, Anna E.; Sojka, Dorothy K.; Carrero, Javier A.; Calderon, Boris; Brija, Thaddeus; Gautier, Emmanuel L.; Ivanov, Stoyan; Satpathy, Ansuman T.; Schilling, Joel D.; Schwendener, Reto; Sergin, Ismail; Razani, Babak; Forsberg, E. Camilla; Yokoyama, Wayne; Unanue, Emil R.; Colonna, Marco; Randolph, Gwendalyn J.; Mann, Douglas L.

2014-01-01

Summary Cardiac macrophages are crucial for tissue repair after cardiac injury but have not been well characterized. Here we identify four populations of cardiac macrophages. At steady state, resident macrophages were primarily maintained through local proliferation. However, after macrophage depletion or during cardiac inflammation, Ly6chi monocytes contributed to all four macrophage populations, whereas resident macrophages also expanded numerically through proliferation. Genetic fate mapping revealed that yolk-sac and fetal monocyte progenitors gave rise to the majority of cardiac macrophages, and the heart was among a minority of organs in which substantial numbers of yolk-sac macrophages persisted in adulthood. CCR2 expression and dependence distinguished cardiac macrophages of adult monocyte versus embryonic origin. Transcriptional and functional data revealed that monocyte-derived macrophages coordinate cardiac inflammation, while playing redundant but lesser roles in antigen sampling and efferocytosis. These data highlight the presence of multiple cardiac macrophage subsets, with different functions, origins and strategies to regulate compartment. PMID:24439267

Investigation of Dracocephalum kostchyi plant extract on the effective inflammatory transcription factors and mediators in activated macrophages.

PubMed

Kalantar, Kurosh; Gholijani, Nasser; Mousaei, Nashmin; Yazdani, Malihe; Amirghofran, Zahra

2018-06-07

Dracocephalum kotschyi is traditionally used for its anti-inflammatory effects. We aimed to investigate the effects of ethyl acetate extract of D. kotschyi on the expression of key inflammatory mediators and main signaling molecules involved in regulation of inflammation. Lipopolysaccharide (LPS)-stimulated J774.1 mouse macrophages were cultured in the presence of the plant extract and examined by the real time-PCR for gene expressions of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2. Cytokine levels and phosphorylated forms of stress-activated protein kinases/c-Jun N-terminal kinase (SAPK/JNK), signal transducer and activator of transcription (STAT)-3, p38, IκB-α and nuclear factor (NF)-κB p65 were determined using ELISA. The extract significantly reduced the expression of key mediators of inflammation. iNOS expression level decreased from 138±8.5 fold in LPS-only treated cells to 6.5±2.6 fold after treatment with 25 ug/ml of the extract (p<0.001). Similarly, COX-2 expression decreased from 632 ±98.8 fold in control to 124 ±24.6 fold (p<0.01). Treatment of cells with the extract significantly reduced IL-1β and TNF-α cytokines at both gene and protein expression levels. The extract at 25 µg/ml caused significant decreases in phospho-SAPK/JNK and phospho-STAT3 levels in macrophages (p<0.01). Proteins of phospho-p38, NFκB-p65 and phospho-NF-κB p65 had a reduced levels in treated cells (p<0.05). No significant change in phospho-IĸB level was observed. These findings suggested that D. kotschyi with inhibition of NF-κB, SAPK/JNK, STAT-3 and p-38 might have reduced the expression levels of key inflammatory mediators and thus possibly have potential beneficial impact on inflammatory diseases. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Differential Expression of Inward and Outward Potassium Currents in the Macrophage-like Cell Line J774.1

DTIC Science & Technology

1985-04-02

skeletal muscle (Adrian, Hodgkin & Chandler, 1970), and nerve (Goldman & Schauf , 1973; Thompson, 1977). The general mathematical formalism describes the...594-609. Goldman, L. & Schauf , C. L. (1973) Quantitative description of sodium and potassium currents and computed action potentials in Myxicola giant

ALV-J infection induces chicken monocyte death accompanied with the production of IL-1β and IL-18.

PubMed

Dai, Manman; Feng, Min; Xie, Tingting; Li, Yuanfang; Ruan, Zhuohao; Shi, Meiqing; Liao, Ming; Zhang, Xiquan

2017-11-21

Immunosuppression induced by avian leukosis virus subgroup J (ALV-J) causes serious reproduction problems and secondary infections in chickens. Given that monocytes are important precursors of immune cells including macrophages and dendritic cells, we investigated the fate of chicken monocytes after ALV-J infection. Our results indicated that most monocytes infected with ALV-J including field or laboratory strains could not successfully differentiate into macrophages due to cells death. And cells death was dependent upon viral titer and accompanied with increased IL-1β and IL-18 mRNA levels. In addition, ALV-J infection up-regulated caspase-1 and caspase-3 activity in monocytes. Collectively, we found that ALV-J could cause cell death in chicken monocytes, especially pyroptosis, which may be a significant reason for ALV-J induced immunosuppression.

ALV-J infection induces chicken monocyte death accompanied with the production of IL-1β and IL-18

PubMed Central

Dai, Manman; Feng, Min; Xie, Tingting; Li, Yuanfang; Ruan, Zhuohao; Shi, Meiqing; Liao, Ming; Zhang, Xiquan

2017-01-01

Immunosuppression induced by avian leukosis virus subgroup J (ALV-J) causes serious reproduction problems and secondary infections in chickens. Given that monocytes are important precursors of immune cells including macrophages and dendritic cells, we investigated the fate of chicken monocytes after ALV-J infection. Our results indicated that most monocytes infected with ALV-J including field or laboratory strains could not successfully differentiate into macrophages due to cells death. And cells death was dependent upon viral titer and accompanied with increased IL-1β and IL-18 mRNA levels. In addition, ALV-J infection up-regulated caspase-1 and caspase-3 activity in monocytes. Collectively, we found that ALV-J could cause cell death in chicken monocytes, especially pyroptosis, which may be a significant reason for ALV-J induced immunosuppression. PMID:29245947

Cytoskeleton-centric protein transportation by exosomes transforms tumor-favorable macrophages.

PubMed

Chen, Zhipeng; Yang, Lijuan; Cui, Yizhi; Zhou, Yanlong; Yin, Xingfeng; Guo, Jiahui; Zhang, Gong; Wang, Tong; He, Qing-Yu

2016-10-11

The exosome is a key initiator of pre-metastatic niche in numerous cancers, where macrophages serve as primary inducers of tumor microenvironment. However, the proteome that can be exosomally transported from cancer cells to macrophages has not been sufficiently characterized so far. Here, we used colorectal cancer (CRC) exosomes to educate tumor-favorable macrophages. With a SILAC-based mass spectrometry strategy, we successfully traced the proteome transported from CRC exosomes to macrophages. Such a proteome primarily focused on promoting cytoskeleton rearrangement, which was biologically validated with multiple cell lines. We reproduced the exosomal transportation of functional vimentin as a proof-of-concept example. In addition, we found that some CRC exosomes could be recognized by macrophages via Fc receptors. Therefore, we revealed the active and necessary role of exosomes secreted from CRC cells to transform cancer-favorable macrophages, with the cytoskeleton-centric proteins serving as the top functional unit.

Mononuclear-macrophages but not neutrophils act as major infiltrating anti-leptospiral phagocytes during leptospirosis.

PubMed

Chen, Xu; Li, Shi-Jun; Ojcius, David M; Sun, Ai-Hua; Hu, Wei-Lin; Lin, Xu'ai; Yan, Jie

2017-01-01

To identify the major infiltrating phagocytes during leptospirosis and examine the killing mechanism used by the host to eliminate Leptospira interrogans. Major infiltrating phagocytes in Leptospira-infected C3H/HeJ mice were detected by immunohistochemistry. Chemokines and vascular endothelial cell adhesion molecules (VECAMs) of Leptospira-infected mice and leptospirosis patients were detected by microarray and immunohistochemistry. Leptospira-phagocytosing and -killing abilities of human or mouse macrophages and neutrophils, and the roles of intracellular ROS, NO and [Ca2+]i in Leptospira-killing process were evaluated by confocal microscopy and spectrofluorimetry. Peripheral blood mononuclear-macrophages rather than neutrophils were the main infiltrating phagocytes in the lungs, liver and kidneys of infected mice. Levels of macrophage- but not neutrophil-specific chemokines and VECAMs were significantly increased in the samples from infected mice and patients. All macrophages tested had a higher ability than neutrophils to phagocytose and kill leptospires. Higher ROS and NO levels and [Ca2+]i in the macrophages were involved in killing leptospires. Human macrophages displayed more phagolysosome formation and a stronger leptospire-killing ability to than mouse macrophages. Mononuclear-macrophages but not neutrophils represent the main infiltrating and anti-leptospiral phagocytes during leptospirosis. A lower level of phagosome-lysosome fusion may be responsible for the lower Leptospira-killing ability of human macrophages.

Macrophage origin limits functional plasticity in helminth-bacterial co-infection

PubMed Central

Campbell, Sharon M.; Duncan, Sheelagh; Hewitson, James P.; Barr, Tom A.; Jackson-Jones, Lucy H.; Maizels, Rick M.

2017-01-01

Rapid reprogramming of the macrophage activation phenotype is considered important in the defense against consecutive infection with diverse infectious agents. However, in the setting of persistent, chronic infection the functional importance of macrophage-intrinsic adaptation to changing environments vs. recruitment of new macrophages remains unclear. Here we show that resident peritoneal macrophages expanded by infection with the nematode Heligmosomoides polygyrus bakeri altered their activation phenotype in response to infection with Salmonella enterica ser. Typhimurium in vitro and in vivo. The nematode-expanded resident F4/80high macrophages efficiently upregulated bacterial induced effector molecules (e.g. MHC-II, NOS2) similarly to newly recruited monocyte-derived macrophages. Nonetheless, recruitment of blood monocyte-derived macrophages to Salmonella infection occurred with equal magnitude in co-infected animals and caused displacement of the nematode-expanded, tissue resident-derived macrophages from the peritoneal cavity. Global gene expression analysis revealed that although nematode-expanded resident F4/80high macrophages made an anti-bacterial response, this was muted as compared to newly recruited F4/80low macrophages. However, the F4/80high macrophages adopted unique functional characteristics that included enhanced neutrophil-stimulating chemokine production. Thus, our data provide important evidence that plastic adaptation of MΦ activation does occur in vivo, but that cellular plasticity is outweighed by functional capabilities specific to the tissue origin of the cell. PMID:28334040

Conditional deletion of caspase-8 in macrophages alters macrophage activation in a RIPK-dependent manner.

PubMed

Cuda, Carla M; Misharin, Alexander V; Khare, Sonal; Saber, Rana; Tsai, FuNien; Archer, Amy M; Homan, Philip J; Haines, G Kenneth; Hutcheson, Jack; Dorfleutner, Andrea; Budinger, G R Scott; Stehlik, Christian; Perlman, Harris

2015-10-16

Although caspase-8 is a well-established initiator of apoptosis and suppressor of necroptosis, recent evidence suggests that this enzyme maintains functions beyond its role in cell death. As cells of the innate immune system, and in particular macrophages, are now at the forefront of autoimmune disease pathogenesis, we examined the potential involvement of caspase-8 within this population. Cre (LysM) Casp8 (fl/fl) mice were bred via a cross between Casp8 (fl/fl) mice and Cre (LysM) mice, and RIPK3 (-/-) Cre (LysM) Casp8 (fl/fl) mice were generated to assess the contribution of receptor-interacting serine-threonine kinase (RIPK)3. Immunohistochemical and immunofluorescence analyses were used to examine renal damage. Flow cytometric analysis was employed to characterize splenocyte distribution and activation. Cre (LysM) Casp8 (fl/fl) mice were treated with either Toll-like receptor (TLR) agonists or oral antibiotics to assess their response to TLR activation or TLR agonist removal. Luminex-based assays and enzyme-linked immunosorbent assays were used to measure cytokine/chemokine and immunoglobulin levels in serum and cytokine levels in cell culture studies. In vitro cell culture was used to assess macrophage response to cell death stimuli, TLR activation, and M1/M2 polarization. Data were compared using the Mann-Whitney U test. Loss of caspase-8 expression in macrophages promotes onset of a mild systemic inflammatory disease, which is preventable by the deletion of RIPK3. In vitro cell culture studies reveal that caspase-8-deficient macrophages are prone to a caspase-independent death in response to death receptor ligation; yet, caspase-8-deficient macrophages are not predisposed to unchecked survival, as analysis of mixed bone marrow chimeric mice demonstrates that caspase-8 deficiency does not confer preferential expansion of myeloid populations. Loss of caspase-8 in macrophages dictates the response to TLR activation, as injection of TLR ligands upregulates

Novel interactions between erythroblast macrophage protein and cell migration.

PubMed

Javan, Gulnaz T; Can, Ismail; Yeboah, Fred; Lee, Youngil; Soni, Shivani

2016-09-01

Erythroblast macrophage protein is a novel protein known to mediate attachment of erythroid cells to macrophages to form erythroblastic islands in bone marrow during erythropoiesis. Emp-null macrophages are small with round morphologies, and lack cytoplasmic projections which imply immature structure. The role of Emp in macrophage development and function is not fully elucidated. Macrophages perform varied functions (e.g. homeostasis, erythropoiesis), and are implicated in numerous pathophysiological conditions such as cellular malignancy. The objective of the current study is to investigate the interaction of Emp with cytoskeletal- and cell migration-associated proteins involved in macrophage functions. A short hairpin RNA lentiviral system was use to down-regulate the expression of Emp in macrophage cells. A cell migration assay revealed that the relocation of macrophages was significantly inhibited when Emp expression was decreased. To further analyze changes in gene expression related to cell motility, PCR array was performed by down-regulating Emp expression. The results indicated that expression of mitogen-activated protein kinase 1 and thymoma viral proto-oncogene 1 were significantly higher when Emp was down-regulated. The results implicate Emp in abnormal cell motility, thus, warrants to assess its role in cancer where tumor cell motility is required for invasion and metastasis. Copyright © 2016 Elsevier Inc. All rights reserved.

Primitive macrophages control HSPC mobilization and definitive haematopoiesis.

PubMed

Travnickova, Jana; Tran Chau, Vanessa; Julien, Emmanuelle; Mateos-Langerak, Julio; Gonzalez, Catherine; Lelièvre, Etienne; Lutfalla, Georges; Tavian, Manuela; Kissa, Karima

2015-02-17

In vertebrates, haematopoietic stem/progenitor cells (HSPCs) first emerge in the aorta-gonad-mesonephros (AGM) before colonizing transitory and subsequently definitive haematopoietic organs allowing haematopoiesis throughout adult life. Here we identify an unexpected primitive macrophage population accumulated in the dorsal mesenteric mesoderm surrounding the dorsal aorta of the human embryo and study its function in the transparent zebrafish embryo. Our study reveals dynamic interactions occurring between the HSPCs and primitive macrophages in the AGM. Specific chemical and inducible genetic depletion of macrophages or inhibition of matrix metalloproteinases (Mmps) leads to an accumulation of HSPCs in the AGM and a decrease in the colonization of haematopoietic organs. Finally, in vivo zymography demonstrates the function of primitive macrophages in extracellular matrix degradation, which allows HSPC migration through the AGM stroma, their intravasation, leading to the colonization of haematopoietic organs and the establishment of definitive haematopoiesis.

The endoplasmic reticulum stress inducer thapsigargin enhances the toxicity of ZnO nanoparticles to macrophages and macrophage-endothelial co-culture.

PubMed

Chen, Gui; Shen, Yuexin; Li, Xiyue; Jiang, Qin; Cheng, Shanshan; Gu, Yuxiu; Liu, Liangliang; Cao, Yi

2017-03-01

It was recently shown that exposure to ZnO nanoparticles (NPs) could induce endoplasmic reticulum (ER) stress both in vivo and in vitro, but the role of ER stress in ZnO NP induced toxicity remains unclear. Because macrophages are sensitive to ER stress, we hypothesized that stressing macrophages with ER stress inducer could enhance the toxicity of ZnO NPs. In this study, the effects of ER stress inducer thapsigargin (TG) on the toxicity of ZnO NPs to THP-1 macrophages were investigated. The results showed that TG enhanced ZnO NP induced cytotoxicity as revealed by water soluble tetrazolium-1 (WST-1) and neutral red uptake assays, but not lactate dehydrogenase (LDH) assay. ZnO NPs dose-dependently enhanced the accumulation of intracellular Zn ions without the induction of reactive oxygen species (ROS), and the presence of TG did not significantly affect these effects. In the co-culture, exposure of THP-1 macrophages in the upper chamber to ZnO NPs and TG significantly reduced the viability of human umbilical vein endothelial cells (HUVECs) in the lower chamber, but the release of tumor necrosis factor α (TNFα) was not induced. In summary, our data showed that stressing THP-1 macrophages with TG enhanced the cytotoxicity of ZnO NPs to macrophages and macrophage-endothelial co-cultures. Copyright © 2017 Elsevier B.V. All rights reserved.

Culture of Macrophage Colony-stimulating Factor Differentiated Human Monocyte-derived Macrophages.

PubMed

Jin, Xueting; Kruth, Howard S

2016-06-30

A protocol is presented for cell culture of macrophage colony-stimulating factor (M-CSF) differentiated human monocyte-derived macrophages. For initiation of experiments, fresh or frozen monocytes are cultured in flasks for 1 week with M-CSF to induce their differentiation into macrophages. Then, the macrophages can be harvested and seeded into culture wells at required cell densities for carrying out experiments. The use of defined numbers of macrophages rather than defined numbers of monocytes to initiate macrophage cultures for experiments yields macrophage cultures in which the desired cell density can be more consistently attained. Use of cryopreserved monocytes reduces dependency on donor availability and produces more homogeneous macrophage cultures.

Involvement of hepcidin in iron metabolism dysregulation in Gaucher disease.

PubMed

Lefebvre, Thibaud; Reihani, Niloofar; Daher, Raed; de Villemeur, Thierry Billette; Belmatoug, Nadia; Rose, Christian; Colin-Aronovicz, Yves; Puy, Hervé; Le Van Kim, Caroline; Franco, Mélanie; Karim, Zoubida

2018-04-01

Gaucher disease (GD) is an inherited deficiency of glucocerebrosidase leading to accumulation of glucosylceramide in tissues such as the spleen, liver, and bone marrow. The resulting lipid-laden macrophages lead to the appearance of "Gaucher cells". Anemia associated with an unexplained hyperferritinemia is a frequent finding in GD, but whether this pathogenesis is related to an iron metabolism disorder has remained unclear. To investigate this issue, we explored the iron status of a large cohort of 90 type I GD patients, including 66 patients treated with enzyme replacement therapy. Ten of the patients treated with enzyme replacement were followed up before and during treatment. Serum levels of hepcidin, the iron regulatory peptide, remained within the physiological range, while the transferrin saturation was slightly decreased in children. Inflammation-independent hyperferritinemia was found in 65% of the patients, and Perl's staining of the spleen and marrow smear revealed iron accumulation in Gaucher cells. Treated patients exhibited reduced hyperferritinemia, increased transferrin saturation and transiently increased systemic hepcidin. In addition, the hepcidin and ferritin correlation was markedly improved, and, in most patients, the hemoglobin level was normalized. To further explore eventual iron sequestration in macrophages, we produce a Gaucher cells model by treating the J774 macrophage cell line with a glucocerebrosidase inhibitor and showed induced local hepcidin and membrane retrieval of the iron exporter, ferroportin. These data reveal the involvement of Gaucher cells in abnormal iron sequestration, which may explain the mechanism of hyperferritinemia in GD patients. Local hepcidin-ferroportin interaction was involved in this pathogenesis. Copyright© 2018 Ferrata Storti Foundation.

Soluble factors from biofilm of Candida albicans and Staphylococcus aureus promote cell death and inflammatory response.

PubMed

de Carvalho Dias, Kassia; Barbugli, Paula Aboud; de Patto, Fernanda; Lordello, Virginia Barreto; de Aquino Penteado, Letícia; Medeiros, Alexandra Ivo; Vergani, Carlos Eduardo

2017-06-30

The objective of this study was to better understand the effects of soluble factors from biofilm of single- and mixed-species Candida albicans (C. albicans) and methicillin-sensitive Staphylococcus aureus (MSSA) cultures after 36 h in culture on keratinocytes (NOK-si and HaCaT) and macrophages (J774A.1). Soluble factors from biofilms of C. albicans and MSSA were collected and incubated with keratinocytes and macrophages, which were subsequently evaluated by cell viability assays (MTT). Lactate dehydrogenase (LDH) enzyme release was measured to assess cell membrane damage to keratinocytes. Cells were analysed by brightfield microscopy after 2 and 24 h of exposure to the soluble factors from biofilm. Cell death was detected by labelling apoptotic cells with annexin V and necrotic cells with propidium iodide (PI) and was visualized via fluorescence microscopy. Soluble factors from biofilm were incubated with J774A.1 cells for 24 h; the subsequent production of NO and the cytokines IL-6 and TNF-α was measured by ELISA. The cell viability assays showed that the soluble factors of single-species C. albicans cultures were as toxic as the soluble factors from biofilm of mixed cultures, whereas the soluble factors of MSSA cultures were less toxic than those of C. albicans or mixed cultures. The soluble factors from biofilm of mixed cultures were the most toxic to the NOK-si and HaCaT cells, as confirmed by analyses of PI labelling and cell morphology. Soluble factors from biofilm of single-species MSSA and mixed-species cultures induced the production of IL-6, NO and TNF-α by J744A.1 macrophages. The production of IL-6 and NO induced by the soluble factors from biofilm of mixed cultures was lower than that induced by the soluble factors from biofilm of single-species MSSA cultures, whereas the soluble factors from biofilm of C. albicans cultures induced only low levels of NO. Soluble factors from 36-h-old biofilm of C. albicans and MSSA cultures promoted cell death and

VIEW OF THE INTERIOR OF BUILDING 774, THE ORIGINAL LIQUID ...

Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

VIEW OF THE INTERIOR OF BUILDING 774, THE ORIGINAL LIQUID PROCESS WASTEWATER TREATMENT FACILITY. THE PHOTOGRAPH SHOWS STORAGE TANKS AND ASSOCIATED PLUTONIUM-CONTAMINATED SOLUTIONS. THE GLOVE BOX IS USED BY OPERATORS TO MANUALLY OPERATE PUMPS AND VALVES THAT REQUIRE PERIODIC ADJUSTMENT. OTHER VALVES IN THE ROOM WERE INFREQUENTLY ADJUSTED, AND ARE SEALED IN PLASTIC WRAP - Rocky Flats Plant, Waste Treatment Facility, Adjacent to bldg 771C, in northern portion of protected area, Golden, Jefferson County, CO

IGF1 Shapes Macrophage Activation in Response to Immunometabolic Challenge.

PubMed

Spadaro, Olga; Camell, Christina D; Bosurgi, Lidia; Nguyen, Kim Y; Youm, Yun-Hee; Rothlin, Carla V; Dixit, Vishwa Deep

2017-04-11

In concert with their phagocytic activity, macrophages are thought to regulate the host immunometabolic responses primarily via their ability to produce specific cytokines and metabolites. Here, we show that IL-4-differentiated, M2-like macrophages secrete IGF1, a hormone previously thought to be exclusively produced from liver. Ablation of IGF1 receptors from myeloid cells reduced phagocytosis, increased macrophages in adipose tissue, elevated adiposity, lowered energy expenditure, and led to insulin resistance in mice fed a high-fat diet. The investigation of adipose macrophage phenotype in obese myeloid IGF1R knockout (MIKO) mice revealed a reduction in transcripts associated with M2-like macrophage activation. Furthermore, the MIKO mice infected with helminth Nippostrongylus brasiliensis displayed delayed resolution from infection with normal insulin sensitivity. Surprisingly, cold challenge did not trigger an overt M2-like state and failed to induce tyrosine hydroxylase expression in adipose tissue macrophages of control or MIKO mice. These results show that IGF1 signaling shapes the macrophage-activation phenotype. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Increased 13-hydroxyoctadecadienoic acid content in lipopolysaccharide stimulated macrophages.

PubMed

Schade, U F; Burmeister, I; Engel, R

1987-09-15

Endotoxin-stimulated mouse peritoneal macrophages were found to contain 13-hydroxyoctadecadienoic acid, which was released upon alkaline hydrolysis of the cells. Compared to untreated cells, incubation with LPS increased the content of 13-hydroxyoctadecadienoic acid in macrophage hydrolysates to about 8-fold. Analysis of the material on chiralphase HPLC revealed that it consisted prevalently of 13(S)-hydroxyoctadecadienoic acid. This indicates its enzymatic origine.

Cot/tpl2 participates in the activation of macrophages by adiponectin.

PubMed

Sanz-Garcia, Carlos; Nagy, Laura E; Lasunción, Miguel A; Fernandez, Margarita; Alemany, Susana

2014-06-01

Whereas the main function of APN is to enhance insulin activity, it is also involved in modulating the macrophage phenotype. Here, we demonstrate that at physiological concentrations, APN activates Erk1/2 via the IKKβ-p105/NF-κΒ1-Cot/tpl2 intracellular signal transduction cassette in macrophages. In peritoneal macrophages stimulated with APN, Cot/tpl2 influences the ability to phagocytose beads. However, Cot/tpl2 did not modulate the known capacity of APN to decrease lipid content in peritoneal macrophages in response to treatment with oxLDL or acLDL. A microarray analysis of gene-expression profiles in BMDMs exposed to APN revealed that APN modulated the expression of ∼3300 genes; the most significantly affected biological functions were the inflammatory and the infectious disease responses. qRT-PCR analysis of WT and Cot/tpl2 KO macrophages stimulated with APN for 0, 3, and 18 h revealed that Cot/tpl2 participated in the up-regulation of APN target inflammatory mediators included in the cytokine-cytokine receptor interaction pathway (KEGG ID 4060). In accordance with these data, macrophages stimulated with APN increased secretion of cytokines and chemokines, including IL-1β, IL-1α, TNF-α, IL-10, IL-12, IL-6, and CCL2. Moreover, Cot/tpl2 also played an important role in the production of these inflammatory mediators upon stimulation of macrophages with APN. It has been reported that different types of signals that stimulate TLRs, IL-1R, TNFR, FcγR, and proteinase-activated receptor-1 activate Cot/tpl2. Here, we demonstrate that APN is a new signal that activates the IKKβ-p105/NF-κΒ1-Cot/tpl2-MKK1/2-Erk1/2 axis in macrophages. Furthermore, this signaling cassette modulates the biological functions triggered by APN in macrophages. © 2014 Society for Leukocyte Biology.

Cot/tpl2 participates in the activation of macrophages by adiponectin

PubMed Central

Sanz-Garcia, Carlos; Nagy, Laura E.; Lasunción, Miguel A.; Fernandez, Margarita; Alemany, Susana

2014-01-01

Whereas the main function of APN is to enhance insulin activity, it is also involved in modulating the macrophage phenotype. Here, we demonstrate that at physiological concentrations, APN activates Erk1/2 via the IKKβ-p105/NF-κΒ1-Cot/tpl2 intracellular signal transduction cassette in macrophages. In peritoneal macrophages stimulated with APN, Cot/tpl2 influences the ability to phagocytose beads. However, Cot/tpl2 did not modulate the known capacity of APN to decrease lipid content in peritoneal macrophages in response to treatment with oxLDL or acLDL. A microarray analysis of gene-expression profiles in BMDMs exposed to APN revealed that APN modulated the expression of ∼3300 genes; the most significantly affected biological functions were the inflammatory and the infectious disease responses. qRT-PCR analysis of WT and Cot/tpl2 KO macrophages stimulated with APN for 0, 3, and 18 h revealed that Cot/tpl2 participated in the up-regulation of APN target inflammatory mediators included in the cytokine–cytokine receptor interaction pathway (KEGG ID 4060). In accordance with these data, macrophages stimulated with APN increased secretion of cytokines and chemokines, including IL-1β, IL-1α, TNF-α, IL-10, IL-12, IL-6, and CCL2. Moreover, Cot/tpl2 also played an important role in the production of these inflammatory mediators upon stimulation of macrophages with APN. It has been reported that different types of signals that stimulate TLRs, IL-1R, TNFR, FcγR, and proteinase-activated receptor-1 activate Cot/tpl2. Here, we demonstrate that APN is a new signal that activates the IKKβ-p105/NF-κΒ1-Cot/tpl2-MKK1/2-Erk1/2 axis in macrophages. Furthermore, this signaling cassette modulates the biological functions triggered by APN in macrophages. PMID:24532642

Stimulation of complement component C3 synthesis in macrophagelike cell lines by group B streptococci.

PubMed Central

Goodrum, K J

1987-01-01

Complement levels and complement activation are key determinants in streptococcus-induced inflammatory responses. Activation of macrophage functions, such as complement synthesis, by group B streptococci (GBS) was examined as a possible component of GBS-induced chronic inflammation. Using an enzyme-linked immunosorbent assay, secreted C3 from mouse macrophagelike cell lines (PU5-1.8 and J774A.1) was monitored after cultivation with GBS. Whole, heat-killed GBS (1 to 10 CFU per macrophage) of both type Ia and III strains induced 25 to 300% increases in secreted C3 in both cell lines after a 24-h cultivation. GBS-treated cell lines exhibited increases in secreted lysozyme (10%) and in cellular protein (25 to 50%). Inhibition of macrophage phagocytosis by cytochalasin B inhibited GBS stimulation of C3. Purified cell walls of GBS type III strain 603-79 (1 to 10 micrograms/ml) also enhanced C3 synthesis. Local enhancement of macrophage C3 production by ingested streptococci or by persistent cell wall antigens may serve to promote chronic inflammatory responses. PMID:3552987

Macrophage Activation Mechanisms in Human Monocytic Cell Line-derived Macrophages.

PubMed

Sumiya, Yu; Ishikawa, Mami; Inoue, Takahiro; Inui, Toshio; Kuchiike, Daisuke; Kubo, Kentaro; Uto, Yoshihiro; Nishikata, Takahito

2015-08-01

Although the mechanisms of macrophage activation are important for cancer immunotherapy, they are poorly understood. Recently, easy and robust assay systems for assessing the macrophage-activating factor (MAF) using monocytic cell line-derived macrophages were established. Gene-expression profiles of U937- and THP-1-derived macrophages were compared using gene expression microarray analysis and their responses against several MAFs were examined by in vitro experiments. Activated states of these macrophages could not be assigned to a specific sub-type but showed, however, different unique characteristics. The unique of monocytic cell line-derived macrophages could provide clues to understand the activation mechanism of macrophages and, therefore, help to develop effective cancer immunotherapy with MAFs. Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Emodin Bidirectionally Modulates Macrophage Polarization and Epigenetically Regulates Macrophage Memory.

PubMed

Iwanowycz, Stephen; Wang, Junfeng; Altomare, Diego; Hui, Yvonne; Fan, Daping

2016-05-27

Macrophages are pleiotropic cells capable of performing a broad spectrum of functions. Macrophage phenotypes are classified along a continuum between the extremes of proinflammatory M1 macrophages and anti-inflammatory M2 macrophages. The seemingly opposing functions of M1 and M2 macrophages must be tightly regulated for an effective and proper response to foreign molecules or damaged tissue. Excessive activation of either M1 or M2 macrophages contributes to the pathology of many diseases. Emodin is a Chinese herb-derived compound and has shown potential to inhibit inflammation in various settings. In this study, we tested the ability of emodin to modulate the macrophage response to both M1 and M2 stimuli. Primary mouse macrophages were stimulated with LPS/IFNγ or IL4 with or without emodin, and the effects of emodin on gene transcription, cell signaling pathways, and histone modifications were examined by a variety of approaches, including microarray, quantitative real-time PCR, Western blotting, chromatin immunoprecipitation, and functional assays. We found that emodin bidirectionally tunes the induction of LPS/IFNγ- and IL4-responsive genes through inhibiting NFκB/IRF5/STAT1 signaling and IRF4/STAT6 signaling, respectively. Thereby, emodin modulates macrophage phagocytosis, migration, and NO production. Furthermore, emodin inhibited the removal of H3K27 trimethylation (H3K27m3) marks and the addition of H3K27 acetylation (H3K27ac) marks on genes required for M1 or M2 polarization of macrophages. In conclusion, our data suggest that emodin is uniquely able to suppress the excessive response of macrophages to both M1 and M2 stimuli and therefore has the potential to restore macrophage homeostasis in various pathologies. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Emodin Bidirectionally Modulates Macrophage Polarization and Epigenetically Regulates Macrophage Memory*

PubMed Central

Iwanowycz, Stephen; Wang, Junfeng; Altomare, Diego; Hui, Yvonne; Fan, Daping

2016-01-01

Macrophages are pleiotropic cells capable of performing a broad spectrum of functions. Macrophage phenotypes are classified along a continuum between the extremes of proinflammatory M1 macrophages and anti-inflammatory M2 macrophages. The seemingly opposing functions of M1 and M2 macrophages must be tightly regulated for an effective and proper response to foreign molecules or damaged tissue. Excessive activation of either M1 or M2 macrophages contributes to the pathology of many diseases. Emodin is a Chinese herb-derived compound and has shown potential to inhibit inflammation in various settings. In this study, we tested the ability of emodin to modulate the macrophage response to both M1 and M2 stimuli. Primary mouse macrophages were stimulated with LPS/IFNγ or IL4 with or without emodin, and the effects of emodin on gene transcription, cell signaling pathways, and histone modifications were examined by a variety of approaches, including microarray, quantitative real-time PCR, Western blotting, chromatin immunoprecipitation, and functional assays. We found that emodin bidirectionally tunes the induction of LPS/IFNγ- and IL4-responsive genes through inhibiting NFκB/IRF5/STAT1 signaling and IRF4/STAT6 signaling, respectively. Thereby, emodin modulates macrophage phagocytosis, migration, and NO production. Furthermore, emodin inhibited the removal of H3K27 trimethylation (H3K27m3) marks and the addition of H3K27 acetylation (H3K27ac) marks on genes required for M1 or M2 polarization of macrophages. In conclusion, our data suggest that emodin is uniquely able to suppress the excessive response of macrophages to both M1 and M2 stimuli and therefore has the potential to restore macrophage homeostasis in various pathologies. PMID:27008857

Effects of BCG infection on the susceptibility of mouse macrophages to endotoxin.

PubMed Central

Peavy, D L; Baughn, R E; Musher, D M; Musher, D M

1979-01-01

Mice infected intravenously with Mycobacterium bovis (BCG) are 100 to 1,000 times more sensitive to the lethal effects of bacterial lipopolysaccharides (LPS). Since BCG infection results in macrophage activation and LPS may cause pathophysiological effects through interaction with this cell type, it was of interest to determine whether macrophages from BCG-infected animals were more susceptible to the toxic effects of LPS in vitro. When LPS-susceptible, C57BL/6 mice were infected with BCG, a significant reduction in the 50% lethal dose of LPS was first observed after 7 days and persisted for several weeks. Macrophages from these animals had greatly increased susceptibility to LPS in vitro, which correlated with the development of acquired cellular resistance as determined by their ability to inhibit the growth of Listeria monocytogenes. In contrast, BCG infection of C3H/HeJ mice, a strain resistant to LPS, did not alter the 50% lethal dose of LPS for these animals or increase the sensitivity of their peritoneal macrophages to LPS in vitro. These results indicate that susceptibility of BCG-infected mice to the lethal effects of LPS parallels the susceptibility of their macrophages in vitro; release of vasoactive substances from LPS-susceptible activated macrophages in vivo may be, in part, responsible for lethality. PMID:378847

Characterization of Macrophage/Microglial Activation and Effect of Photobiomodulation in the Spared Nerve Injury Model of Neuropathic Pain.

PubMed

Kobiela Ketz, Ann; Byrnes, Kimberly R; Grunberg, Neil E; Kasper, Christine E; Osborne, Lisa; Pryor, Brian; Tosini, Nicholas L; Wu, Xingjia; Anders, Juanita J

2017-05-01

Neuropathic pain is common and debilitating with limited effective treatments. Macrophage/microglial activation along ascending somatosensory pathways following peripheral nerve injury facilitates neuropathic pain. However, polarization of macrophages/microglia in neuropathic pain is not well understood. Photobiomodulation treatment has been used to decrease neuropathic pain, has anti-inflammatory effects in spinal injury and wound healing models, and modulates microglial polarization in vitro. Our aim was to characterize macrophage/microglia response after peripheral nerve injury and modulate the response with photobiomodulation. Adult male Sprague-Dawley rats were randomly assigned to sham (N = 13), spared nerve injury (N = 13), or injury + photobiomodulation treatment groups (N = 7). Mechanical hypersensitivity was assessed with electronic von Frey. Photobiomodulation (980 nm) was applied to affected hind paw (output power 1 W, 20 s, 41cm above skin, power density 43.25 mW/cm 2 , dose 20 J), dorsal root ganglia (output power 4.5W, 19s, in skin contact, power density 43.25 mW/cm 2 , dose 85.5 J), and spinal cord regions (output power 1.5 W, 19s, in skin contact, power density 43.25 mW/cm 2 , dose 28.5 J) every other day from day 7-30 post-operatively. Immunohistochemistry characterized macrophage/microglial activation. Injured groups demonstrated mechanical hypersensitivity 1-30 days post-operatively. Photobiomodulation-treated animals began to recover after two treatments; at day 26, mechanical sensitivity reached baseline. Peripheral nerve injury caused region-specific macrophages/microglia activation along spinothalamic and dorsal-column medial lemniscus pathways. A pro-inflammatory microglial marker was expressed in the spinal cord of injured rats compared to photobiomodulation-treated and sham group. Photobiomodulation-treated dorsal root ganglion macrophages expressed anti-inflammatory markers. Photobiomodulation effectively reduced

An abundant tissue macrophage population in the adult murine heart with a distinct alternatively-activated macrophage profile.

PubMed

Pinto, Alexander R; Paolicelli, Rosa; Salimova, Ekaterina; Gospocic, Janko; Slonimsky, Esfir; Bilbao-Cortes, Daniel; Godwin, James W; Rosenthal, Nadia A

2012-01-01

Cardiac tissue macrophages (cTMs) are a previously uncharacterised cell type that we have identified and characterise here as an abundant GFP(+) population within the adult Cx(3)cr1(GFP/+) knock-in mouse heart. They comprise the predominant myeloid cell population in the myocardium, and are found throughout myocardial interstitial spaces interacting directly with capillary endothelial cells and cardiomyocytes. Flow cytometry-based immunophenotyping shows that cTMs exhibit canonical macrophage markers. Gene expression analysis shows that cTMs (CD45(+)CD11b(+)GFP(+)) are distinct from mononuclear CD45(+)CD11b(+)GFP(+) cells sorted from the spleen and brain of adult Cx(3)cr1(GFP/+) mice. Gene expression profiling reveals that cTMs closely resemble alternatively-activated anti-inflammatory M2 macrophages, expressing a number of M2 markers, including Mrc1, CD163, and Lyve-1. While cTMs perform normal tissue macrophage homeostatic functions, they also exhibit a distinct phenotype, involving secretion of salutary factors (including IGF-1) and immune modulation. In summary, the characterisation of cTMs at the cellular and molecular level defines a potentially important role for these cells in cardiac homeostasis.

Effect of macrophages on in vitro corrosion behavior of magnesium alloy.

PubMed

Zhang, Jian; Hiromoto, Sachiko; Yamazaki, Tomohiko; Niu, Jialin; Huang, Hua; Jia, Gaozhi; Li, Haiyan; Ding, Wenjiang; Yuan, Guangyin

2016-10-01

The influence of cells on the corrosion behavior of biomedical magnesium alloy is an important but less studied topic, which is helpful for understanding the inconsistent corrosion rates between in vitro and in vivo experiments. In this work, macrophages were directly cultured on Mg-2.1Nd-0.2Zn-0.5Zr (wt %, abbreviated as JDBM) alloy surface for 72 or 168 hours. Macrophages retained good viability and the generation of reactive oxygen species (ROS) was greatly promoted on the alloy. Weight loss, Mg(2+) concentration, and cross-section observation results demonstrated that macrophages accelerated the in vitro corrosion of JDBM. The coverage of cell body did not affect the local thickness of corrosion product layer. The corrosion product layer had a porous inner Mg(OH)2 layer and a dense outer layer mainly composed of O, P, Mg, and Ca. The uniform acceleration of JDBM corrosion was attributed to the omnidirection diffusion of ROS from macrophages. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2476-2487, 2016. © 2016 Wiley Periodicals, Inc.

A secretomics analysis reveals major differences in the macrophage responses towards different types of carbon nanotubes.

PubMed

Palomäki, Jaana; Sund, Jukka; Vippola, Minnamari; Kinaret, Pia; Greco, Dario; Savolainen, Kai; Puustinen, Anne; Alenius, Harri

2015-01-01

Certain types of carbon nanotubes (CNT) can evoke inflammation, fibrosis and mesothelioma in vivo, raising concerns about their potential health effects. It has been recently postulated that NLRP3 inflammasome activation is important in the CNT-induced toxicity. However, more comprehensive studies of the protein secretion induced by CNT can provide new information about their possible pathogenic mechanisms. Here, we studied protein secretion from human macrophages with a proteomic approach in an unbiased way. Human monocyte-derived macrophages (MDM) were exposed to tangled or rigid, long multi-walled CNT (MWCNT) or crocidolite asbestos for 6 h. The growth media was concentrated and secreted proteins were analyzed using 2D-DIGE and DeCyder software. Subsequently, significantly up- or down-regulated protein spots were in-gel digested and identified with an LC-MS/MS approach. Bioinformatics analysis was performed to reveal the different patterns of protein secretion induced by these materials. The results show that both long rigid MWCNT and asbestos elicited ample and highly similar protein secretion. In contrast, exposure to long tangled MWCNT induced weaker protein secretion with a more distinct profile. Secretion of lysosomal proteins followed the exposure to all materials, suggesting lysosomal damage. However, only long rigid MWCNT was associated with apoptosis. This analysis suggests that the CNT toxicity in human MDM is mediated via vigorous secretion of inflammation-related proteins and apoptosis. This study provides new insights into the mechanisms of toxicity of high aspect ratio nanomaterials and indicates that not all types of CNT are as hazardous as asbestos fibers.

Niacin and its metabolites as master regulators of macrophage activation.

PubMed

Montserrat-de la Paz, Sergio; Naranjo, M Carmen; Lopez, Sergio; Abia, Rocio; Muriana, Francisco J Garcia; Bermudez, Beatriz

2017-01-01

Niacin is a broad-spectrum lipid-regulating drug used for clinical therapy of chronic high-grade inflammatory diseases. However, the mechanisms by which either niacin or the byproducts of its catabolism ameliorate these inflammatory diseases are not clear yet. Human circulating monocytes and mature macrophages were used to analyze the effects of niacin and its metabolites (NAM, NUA and 2-Pyr) on oxidative stress, plasticity and inflammatory response by using biochemical, flow cytometry, quantitative real-time PCR and Western blot technologies. Niacin, NAM and 2-Pyr significantly decreased ROS, NO and NOS2 expression in LPS-treated human mature macrophages. Niacin and NAM skewed macrophage polarization toward antiinflammatory M2 macrophage whereas a trend toward proinflammatory M1 macrophage was noted following treatment with NUA. Niacin and NAM also reduced the inflammatory competence of LPS-treated human mature macrophages and promoted bias toward antiinflammatory CD14 + CD16 ++ nonclassical human primary monocytes. This study reveals for the first time that niacin and its metabolites possess antioxidant, reprogramming and antiinflammatory properties on human primary monocytes and monocyte-derived macrophages. Our findings imply a new understanding of the mechanisms by which niacin and its metabolites favor a continuous and gradual plasticity process in the human monocyte/macrophage system. Copyright © 2016 Elsevier Inc. All rights reserved.

Transcriptional Profiling in Experimental Visceral Leishmaniasis Reveals a Broad Splenic Inflammatory Environment that Conditions Macrophages toward a Disease-Promoting Phenotype

PubMed Central

Spratt, Heidi; Travi, Bruno L.; Luxon, Bruce A.

2017-01-01

Visceral Leishmaniasis (VL), caused by the intracellular protozoan Leishmania donovani, is characterized by relentlessly increasing visceral parasite replication, cachexia, massive splenomegaly, pancytopenia and ultimately death. Progressive disease is considered to be due to impaired effector T cell function and/or failure of macrophages to be activated to kill the intracellular parasite. In previous studies, we used the Syrian hamster (Mesocricetus auratus) as a model because it mimics the progressive nature of active human VL. We demonstrated previously that mixed expression of macrophage-activating (IFN-γ) and regulatory (IL-4, IL-10, IL-21) cytokines, parasite-induced expression of macrophage arginase 1 (Arg1), and decreased production of nitric oxide are key immunopathologic factors. Here we examined global changes in gene expression to define the splenic environment and phenotype of splenic macrophages during progressive VL. We used RNA sequencing coupled with de novo transcriptome assembly, because the Syrian hamster does not have a fully sequenced and annotated reference genome. Differentially expressed transcripts identified a highly inflammatory spleen environment with abundant expression of type I and type II interferon response genes. However, high IFN-γ expression was ineffective in directing exclusive M1 macrophage polarization, suppressing M2-associated gene expression, and restraining parasite replication and disease. While many IFN-inducible transcripts were upregulated in the infected spleen, fewer were induced in splenic macrophages in VL. Paradoxically, IFN-γ enhanced parasite growth and induced the counter-regulatory molecules Arg1, Ido1 and Irg1 in splenic macrophages. This was mediated, at least in part, through IFN-γ-induced activation of STAT3 and expression of IL-10, which suggests that splenic macrophages in VL are conditioned to respond to macrophage activation signals with a counter-regulatory response that is ineffective and even

Targeted destruction of murine macrophage cells with bioconjugated gold nanorods

NASA Astrophysics Data System (ADS)

Pissuwan, Dakrong; Valenzuela, Stella M.; Killingsworth, Murray C.; Xu, Xiaoda; Cortie, Michael B.

2007-12-01

Gold nanorods manifest a readily tunable longitudinal plasmon resonance with light and consequently have potential for use in photothermal therapeutics. Recent work by others has shown how gold nanoshells and rods can be used to target cancer cells, which can then be destroyed using relatively high power laser radiation (˜1×105 to 1×1010 W/m2). Here we extend this concept to demonstrate how gold nanorods can be modified to bind to target macrophage cells, and show that high intensity laser radiation is not necessary, with even 5×102 W/m2 being sufficient, provided that a total fluence of ˜30 J/cm2 is delivered. We used the murine cell line RAW 264.7 and the monoclonal antibody CD11b, raised against murine macrophages, as our model system and a 5 mW solid state diode laser as our energy source. Exposure of the cells labeled with gold nanorods to a laser fluence of 30 J/cm2 resulted in 81% cell death compared to only 0.9% in the control, non-labeled cells.

Pirfenidone ameliorates lipopolysaccharide-induced pulmonary inflammation and fibrosis by blocking NLRP3 inflammasome activation.

PubMed

Li, Yi; Li, Haitao; Liu, Shuai; Pan, Pinhua; Su, Xiaoli; Tan, Hongyi; Wu, Dongdong; Zhang, Lemeng; Song, Chao; Dai, Minhui; Li, Qian; Mao, Zhi; Long, Yuan; Hu, Yongbin; Hu, Chengping

2018-05-18

Acute respiratory distress syndrome(ARDS)is a severe clinical disorder characterized by its acute onset, diffuse alveolar damage, intractable hypoxemia, and non-cardiogenic pulmonary edema. Acute lung injury(ALI) can trigger persistent lung inflammation and fibrosis through activation of the NLRP3 inflammasome and subsequent secretion of mature IL-1β, suggesting that the NLRP3 inflammasome is a potential therapeutic target for ALI, for which new therapeutic approaches are needed. Our present study aims to assess whether pirfenidone,with anti-fibrotic and anti-inflammatory properties, can improve LPS-induced inflammation and fibrosis by inhibiting NLRP3 inflammasome activation. Male C57BL/6 J mice were intratracheally injected with LPS to induce ALI. Mice were administered pirfenidone by oral gavage throughout the entire experimental course. The mouse macrophage cell line (J774 A.1) was incubated with LPS and ATP, with or without PFD pre-treatment. We demonstrated that PFD remarkably ameliorated LPS-induced pulmonary inflammation and fibrosis and reduced IL-1β and TGF-β1 levels in bronchoalveolar lavage fluid(BALF). Pirfenidone substantially reduced NLRP3 and ASC expression and inhibited caspase-1 activation and IL-1β maturation in lung tissues. In vitro, the experiments revealed that PFD significantly suppressed LPS/ATP-induced production of reactive oxygen species (ROS) and decreased caspase-1 activation and the level of IL-1β in J774 A.1 cells. Taken together, the administration of PFD reduced LPS-induced lung inflammation and fibrosis by blocking NLRP3 inflammasome activation and subsequent IL-1β secretion. These findings indicated that PFD can down-regulate NLRP3 inflammasome activation and that it may offer a promising therapeutic approach for ARDS patients. Copyright © 2018 Elsevier Ltd. All rights reserved.

Replication of Salmonella enterica Serovar Typhimurium in Human Monocyte-Derived Macrophages

PubMed Central

Lathrop, Stephanie K.; Binder, Kelsey A.; Starr, Tregei; Cooper, Kendal G.; Chong, Audrey; Carmody, Aaron B.

2015-01-01

Salmonella enterica serovar Typhimurium is a common cause of food-borne gastrointestinal illness, but additionally it causes potentially fatal bacteremia in some immunocompromised patients. In mice, systemic spread and replication of the bacteria depend upon infection of and replication within macrophages, but replication in human macrophages is not widely reported or well studied. In order to assess the ability of Salmonella Typhimurium to replicate in human macrophages, we infected primary monocyte-derived macrophages (MDM) that had been differentiated under conditions known to generate different phenotypes. We found that replication in MDM depends greatly upon the phenotype of the cells, as M1-skewed macrophages did not allow replication, while M2a macrophages and macrophages differentiated with macrophage colony-stimulating factor (M-CSF) alone (termed M0) did. We describe how additional conditions that alter the macrophage phenotype or the gene expression of the bacteria affect the outcome of infection. In M0 MDM, the temporal expression of representative genes from Salmonella pathogenicity islands 1 and 2 (SPI1 and SPI2) and the importance of the PhoP/Q two-component regulatory system are similar to what has been shown in mouse macrophages. However, in contrast to mouse macrophages, where replication is SPI2 dependent, we observed early SPI2-independent replication in addition to later SPI2-dependent replication in M0 macrophages. Only SPI2-dependent replication was associated with death of the host cell at later time points. Altogether, our results reveal a very nuanced interaction between Salmonella and human macrophages. PMID:25895967

Disruption of the Phospholipase D Gene Attenuates the Virulence of Aspergillus fumigatus

PubMed Central

Li, Xianping; Gao, Meihua; Han, Xuelin; Tao, Sha; Zheng, Dongyu; Cheng, Ying; Yu, Rentao; Han, Gaige; Schmidt, Martina

2012-01-01

Aspergillus fumigatus is the most prevalent airborne fungal pathogen that induces serious infections in immunocompromised patients. Phospholipases are key enzymes in pathogenic fungi that cleave host phospholipids, resulting in membrane destabilization and host cell penetration. However, knowledge of the impact of phospholipases on A. fumigatus virulence is rather limited. In this study, disruption of the pld gene encoding phospholipase D (PLD), an important member of the phospholipase protein family in A. fumigatus, was confirmed to significantly decrease both intracellular and extracellular PLD activity of A. fumigatus. The pld gene disruption did not alter conidial morphological characteristics, germination, growth, and biofilm formation but significantly suppressed the internalization of A. fumigatus into A549 epithelial cells without affecting conidial adhesion to epithelial cells. Importantly, the suppressed internalization was fully rescued in the presence of 100 μM phosphatidic acid, the PLD product. Indeed, complementation of pld restored the PLD activity and internalization capacity of A. fumigatus. Phagocytosis of A. fumigatus conidia by J774 macrophages was not affected by the absence of the pld gene. Pretreatment of conidia with 1-butanol and a specific PLD inhibitor decreased the internalization of A. fumigatus into A549 epithelial cells but had no effect on phagocytosis by J774 macrophages. Finally, loss of the pld gene attenuated the virulence of A. fumigatus in mice immunosuppressed with hydrocortisone acetate but not with cyclophosphamide. These data suggest that PLD of A. fumigatus regulates its internalization into lung epithelial cells and may represent an important virulence factor for A. fumigatus infection. PMID:22083709

Reciprocal interactions between endothelial cells and macrophages in angiogenic vascular niches

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

Baer, Caroline; Squadrito, Mario Leonardo; Iruela-Arispe, M. Luisa, E-mail: arispe@mcdb.ucla.edu

The ability of macrophages to promote vascular growth has been associated with the secretion and local delivery of classic proangiogenic factors (e.g., VEGF-A and proteases). More recently, a series of studies have also revealed that physical contact of macrophages with growing blood vessels coordinates vascular fusion of emerging sprouts. Interestingly, the interactions between macrophages and vascular endothelial cells (ECs) appear to be bidirectional, such that activated ECs also support the expansion and differentiation of proangiogenic macrophages from myeloid progenitors. Here, we discuss recent findings suggesting that dynamic angiogenic vascular niches might also exist in vivo, e.g. in tumors, where sproutingmore » blood vessels and immature myeloid cells like monocytes engage in heterotypic interactions that are required for angiogenesis. Finally, we provide an account of emerging mechanisms of cell-to-cell communication that rely on secreted microvesicles, such as exosomes, which can offer a vehicle for the rapid exchange of molecules and genetic information between macrophages and ECs engaged in angiogenesis. -- Highlights: • Macrophages promote angiogenesis by secreting proangiogenic factors. • Macrophages modulate angiogenesis via cell-to-cell contacts with endothelial cells. • Endothelial cells promote the differentiation of proangiogenic macrophages. • Macrophages and endothelial cells may cooperate to form angiogenic vascular niches.« less

Interaction of murine macrophage-membrane proteins with components of the pathogenic fungus Histoplasma capsulatum

PubMed Central

Taylor, M L; Duarte-Escalante, E; Reyes-Montes, M R; Elizondo, N; Maldonado, G; Zenteno, E

1998-01-01

The interaction of macrophage-membrane proteins and histoplasmin, a crude antigen of the pathogenic fungus Histoplasma capsulatum, was studied using murine peritoneal macrophages. Membrane proteins were purified via membrane attachment to polycationic beads and solubilized in Tris–HCl/SDS/DTT/glycerol for protein extraction; afterwards they were adsorbed or not with H. capsulatum yeast or lectin binding-enriched by affinity chromatography. Membrane proteins and histoplasmin interactions were detected by ELISA and immunoblotting assays using anti-H. capsulatum human or mouse serum and biotinylated goat anti-human or anti-mouse IgG/streptavidin-peroxidase system to reveal the interaction. Results indicate that macrophage-membrane proteins and histoplasmin components interact in a dose-dependent reaction, and adsorption of macrophage-membrane proteins by yeast cells induces a critical decrease in the interaction. Macrophage-membrane glycoproteins with terminal d-galactosyl residues, purified by chromatography with Abrus precatorius lectin, bound to histoplasmin; and two bands of 68 kD and 180 kD of transferred membrane protein samples interacted with histoplasmin components, as revealed by immunoblot assays. Specificity for β-galactoside residues on the macrophage-membrane was confirmed by galactose inhibition of the interaction between macrophage-membrane proteins and histoplasmin components, in competitive ELISA using sugars, as well as by enzymatic cleavage of the galactoside residues. PMID:9737672

Association of β-catenin with P-Smad3 but not LEF-1 dissociates in vitro profibrotic from anti-inflammatory effects of TGF-β1.

PubMed

Tian, Xinrui; Zhang, Jianlin; Tan, Thian Kui; Lyons, J Guy; Zhao, Hong; Niu, Bo; Lee, So Ra; Tsatralis, Tania; Zhao, Ye; Wang, Ya; Cao, Qi; Wang, Changqi; Wang, Yiping; Lee, Vincent W S; Kahn, Michael; Zheng, Guoping; Harris, David C H

2013-01-01

Transforming growth factor β1 (TGF-β1) is known to be both anti-inflammatory and profibrotic. Cross-talk between TGF-β/Smad and Wnt/β-catenin pathways in epithelial-mesenchymal transition (EMT) suggests a specific role for β-catenin in profibrotic effects of TGF-β1. However, no such mechanistic role has been demonstrated for β-catenin in the anti-inflammatory effects of TGF-β1. In the present study, we explored the role of β-catenin in the profibrotic and anti-inflammatory effects of TGF-β1 by using a cytosolic, but not membrane, β-catenin knockdown chimera (F-TrCP-Ecad) and the β-catenin/CBP inhibitor ICG-001. TGF-β1 induced nuclear Smad3/β-catenin complex, but not β-catenin/LEF-1 complex or TOP-flash activity, during EMT of C1.1 (renal tubular epithelial) cells. F-TrCP-Ecad selectively degraded TGF-β1-induced cytoplasmic β-catenin and blocked EMT of C1.1 cells. Both F-TrCP-Ecad and ICG-001 blocked TGF-β1-induced Smad3/β-catenin and Smad reporter activity in C1.1 cells, suggesting that TGF-β1-induced EMT depends on β-catenin binding to Smad3, but not LEF-1 downstream of Smad3, through canonical Wnt. In contrast, in J774 macrophages, the β-catenin level was low and was not changed by interferon-γ (IFN-γ) or lipopolysaccharide (LPS) with or without TGF-β1. TGF-β1 inhibition of LPS-induced TNF-α and IFN-γ-stimulated inducible NO synthase (iNOS) expression was not affected by F-TrCP-Ecad, ICG-001 or by overexpression of wild-type β-catenin in J774 cells. Inhibition of β-catenin by either F-TrCP-Ecad or ICG-001 abolished LiCl-induced TOP-flash, but not TGF-β1-induced Smad reporter, activity in J774 cells. These results demonstrate for the first time that β-catenin is required as a co-factor of Smad in TGF-β1-induced EMT of C1.1 epithelial cells, but not in TGF-β1 inhibition of macrophage activation. Targeting β-catenin may dissociate the TGF-β1 profibrotic and anti-inflammatory effects.

Adenosine signalling mediates the anti-inflammatory effects of the COX-2 inhibitor nimesulide.

PubMed

Caiazzo, Elisabetta; Maione, Francesco; Morello, Silvana; Lapucci, Andrea; Paccosi, Sara; Steckel, Bodo; Lavecchia, Antonio; Parenti, Astrid; Iuvone, Teresa; Schrader, Jürgen; Ialenti, Armando; Cicala, Carla

2016-07-15

Extracellular adenosine formation from ATP is controlled by ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase/CD39) and ecto-5'-nucleotidase (e-5NT/CD73); the latter converts AMP to adenosine and inorganic phosphate, representing the rate limiting step controlling the ratio between extracellular ATP and adenosine. Evidence that cellular expression and activity of CD39 and CD73 may be subject to changes under pathophysiological conditions has identified this pathway as an endogenous modulator in several diseases and was shown to be involved in the molecular mechanism of drugs, such as methotrexate, salicylates , interferon-β. We evaluated whether CD73/adenosine/A2A signalling pathway is involved in nimesulide anti-inflammatory effect, in vivo and in vitro. We found that the adenosine A2A agonist, 4-[2-[[6-amino-9-(N-ethyl-β-d-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid hydrochloride (CGS21680, 2mg/kg ip.), inhibited carrageenan-induced rat paw oedema and the effect was reversed by co-administration of the A2A antagonist -(2-[7-amino-2-[2-furyl][1,2,4]triazolo[2,3-a][1,3,5]triazin-5-yl-amino]ethyl)phenol (ZM241385; 3mg/kg i.p.). Nimesulide (5mg/kg i.p.) anti-inflammatory effect was inhibited by pre-treatment with ZM241385 (3mg/kg i.p.) and by local administration of the CD73 inhibitor, adenosine 5'-(α,β-methylene)diphosphate (APCP; 400μg/paw). Furthermore, we found increased activity of 5'-nucleotidase/CD73 in paws and plasma of nimesulide treated rats, 4h following oedema induction. In vitro, the inhibitory effect of nimesulide on nitrite and prostaglandin E2 production by lipopolysaccharide-activated J774 cell line was reversed by ZM241385 and APCP. Furthermore, nimesulide increased CD73 activity in J774 macrophages while it did not inhibit nitrite accumulation by lipopolysaccharide-activated SiRNA CD73 silenced J774 macrophages. Our data demonstrate that the anti-inflammatory effect of nimesulide in part is mediated by CD73

EphA2 promotes cell adhesion and spreading of monocyte and monocyte/macrophage cell lines on integrin ligand-coated surfaces.

PubMed

Saeki, Noritaka; Nishino, Shingo; Shimizu, Tomohiro; Ogawa, Kazushige

2015-01-01

Eph signaling, which arises following stimulation by ephrins, is known to induce opposite cell behaviors such as promoting and inhibiting cell adhesion as well as promoting cell-cell adhesion and repulsion by altering the organization of the actin cytoskeleton and influencing the adhesion activities of integrins. However, crosstalk between Eph/ephrin with integrin signaling has not been fully elucidated in leukocytes, including monocytes and their related cells. Using a cell attachment stripe assay, we have shown that, following stimulation with ephrin-A1, kinase-independent EphA2 promoted cell spreading/elongation as well as adhesion to integrin ligand-coated surfaces in cultured U937 (monocyte) and J774.1 (monocyte/macrophage) cells as well as sublines of these cells expressing dominant negative EphA2 that lacks most of the intracellular region. Moreover, a pull-down assay showed that dominant negative EphA2 is recruited to the β2 integrin/ICAM1 and β2 integrin/VCAM1 molecular complexes in the subline cells following stimulation with ephrin-A1-Fc. Notably, this study is the first comprehensive analysis of the effects of EphA2 receptors on integrin-mediated cell adhesion in monocytic cells. Based on these findings we propose that EphA2 promotes cell adhesion by an unknown signaling pathway that largely depends on the extracellular region of EphA2 and the activation of outside-in integrin signaling.

Activation of murine macrophages by lipoprotein and lipooligosaccharide of Treponema denticola.

PubMed

Rosen, G; Sela, M N; Naor, R; Halabi, A; Barak, V; Shapira, L

1999-03-01

We have recently demonstrated that the periodontopathogenic oral spirochete Treponema denticola possesses membrane-associated lipoproteins in addition to lipooligosaccharide (LOS). The aim of the present study was to test the potential of these oral spirochetal components to induce the production of inflammatory mediators by human macrophages, which in turn may stimulate tissue breakdown as observed in periodontal diseases. An enriched lipoprotein fraction (dLPP) from T. denticola ATCC 35404 obtained upon extraction of the treponemes with Triton X-114 was found to stimulate the production of nitric oxide (NO), tumor necrosis factor alpha (TNF-alpha), and interleukin-1 (IL-1) by mouse macrophages in a dose-dependent manner. Induction of NO by dLPP was at 25% of the levels obtained by Salmonella typhosa lipopolysaccharide (LPS) at similar concentrations, while IL-1 was produced at similar levels by both inducers. dLPP-mediated macrophage activation was unaffected by amounts of polymyxin B that neutralized the induction produced by S. typhosa LPS. dLPP also induced NO and TNF-alpha secretion from macrophages isolated from endotoxin-unresponsive C3H/HeJ mice to an extent similar to the stimulation produced in endotoxin-responsive mice. Purified T. denticola LOS also produced a concentration-dependent activation of NO and TNF-alpha in LPS-responsive and -nonresponsive mouse macrophages. However, macrophage activation by LOS was inhibited by polymyxin B. These results suggest that T. denticola lipoproteins and LOS may play a role in the inflammatory processes that characterize periodontal diseases.

Gpr132 sensing of lactate mediates tumor–macrophage interplay to promote breast cancer metastasis

PubMed Central

Chen, Peiwen; Zuo, Hao; Xiong, Hu; Kolar, Matthew J.; Chu, Qian; Saghatelian, Alan; Siegwart, Daniel J.; Wan, Yihong

2017-01-01

Macrophages are prominent immune cells in the tumor microenvironment that exert potent effects on cancer metastasis. However, the signals and receivers for the tumor–macrophage communication remain enigmatic. Here, we show that G protein-coupled receptor 132 (Gpr132) functions as a key macrophage sensor of the rising lactate in the acidic tumor milieu to mediate the reciprocal interaction between cancer cells and macrophages during breast cancer metastasis. Lactate activates macrophage Gpr132 to promote the alternatively activated macrophage (M2)-like phenotype, which, in turn, facilitates cancer cell adhesion, migration, and invasion. Consequently, Gpr132 deletion reduces M2 macrophages and impedes breast cancer lung metastasis in mice. Clinically, Gpr132 expression positively correlates with M2 macrophages, metastasis, and poor prognosis in patients with breast cancer. These findings uncover the lactate-Gpr132 axis as a driver of breast cancer metastasis by stimulating tumor–macrophage interplay, and reveal potential new therapeutic targets for breast cancer treatment. PMID:28049847

PPARγ Expression Is Diminished in Macrophages of Recurrent Miscarriage Placentas.

PubMed

Kolben, Theresa Maria; Rogatsch, Elisabeth; Vattai, Aurelia; Hester, Anna; Kuhn, Christina; Schmoeckel, Elisa; Mahner, Sven; Jeschke, Udo; Kolben, Thomas

2018-06-26

PPARγ belongs to the group of nuclear receptors which is expressed in the trophoblast and together with other factors is responsible for the maintenance of pregnancy. Apart from that PPARγ is also a main factor for macrophage polarization. The aim of this study was to investigate the combined expression pattern and frequency of PPARγ under physiological circumstances and in spontaneous and recurrent miscarriages in the trophoblast and in maternal macrophages of the decidua. Human placental tissues of the first trimester (15 physiologic pregnancies, 15 spontaneous abortion and 16 recurrent miscarriage placentas) were analyzed for expression of the nuclear receptor PPARγ. Expression changes were evaluated by immunohistochemistry and real time PCR (RT-PCR) in trophoblast and in maternal macrophages of the decidua. Maternal macrophages were identified by double immunofluorescence using cluster of differentiation 68 (CD68) as marker for macrophages and further characterized regarding their M1/M2 polarization status. The intermediate villous trophoblast revealed a significantly lower PPARγ expression in spontaneous and recurrent abortion. Maternal macrophages express PPARγ. Their number is significantly enhanced in the decidua of spontaneous miscarriages whereas in recurrent miscarriages maternal macrophages seem to express PPARγ only in very few cases. PPARγ is associated with an M2 polarization state that is common for decidual macrophages. The lack of PPARγ in recurrent miscarriage decidual macrophages seems to be associated with a specific inflammatory response against the fetus.

Zoledronic acid inhibits macrophage/microglia-assisted breast cancer cell invasion

PubMed Central

Rietkötter, Eva; Menck, Kerstin; Bleckmann, Annalen; Farhat, Katja; Schaffrinski, Meike; Schulz, Matthias; Hanisch, Uwe-Karsten; Binder, Claudia; Pukrop, Tobias

2013-01-01

The bisphosphonate zoledronic acid (ZA) significantly reduces complications of bone metastasis by inhibiting resident macrophages, the osteoclasts. Recent clinical trials indicate additional anti-metastatic effects of ZA outside the bone. However, which step of metastasis is influenced and whether this is due to direct toxicity on cancer cells or inhibition of the tumor promoting microenvironment, is unknown. In particular, tumor-associated and resident macrophages support each step of organ metastasis and could be a crucial target of ZA. Thus, we comparatively investigate the ZA effects on: i) different types of macrophages, ii) on breast cancer cells but also iii) on macrophage-induced invasion. We demonstrate that ZA concentrations reflecting the plasma level affected viability of human macrophages, murine bone marrow-derived macrophages as well as their resident brain equivalents, the microglia, while it did not influence the tested cancer cells. However, the effects on the macrophages subsequently reduced the macrophage/microglia-induced invasiveness of the cancer cells. In line with this, manipulation of microglia by ZA in organotypic brain slice cocultures reduced the tissue invasion by carcinoma cells. The characterization of human macrophages after ZA treatment revealed a phenotype/response shift, in particular after external stimulation. In conclusion, we show that therapeutic concentrations of ZA affect all types of macrophages but not the cancer cells. Thus, anti-metastatic effects of ZA are predominantly caused by modulating the microenvironment. Most importantly, our findings demonstrate that ZA reduced microglia-assisted invasion of cancer cells to the brain tissue, indicating a potential therapeutic role in the prevention of cerebral metastasis. PMID:24036536

An iso-α-acid-rich extract from hops (Humulus lupulus) attenuates acute alcohol-induced liver steatosis in mice.

PubMed

Hege, Marianne; Jung, Finn; Sellmann, Cathrin; Jin, Chengjun; Ziegenhardt, Doreen; Hellerbrand, Claus; Bergheim, Ina

2018-01-01

Results of in vitro and in vivo studies suggest that consumption of beer is less harmful for the liver than consumption of spirits. It also has been suggested that secondary plant compounds derived from hops such as xanthohumol or iso-α-acids may have beneficial effects on the development of liver diseases of various etiologies. The aim of this study was to determine whether iso-α-acids consumed in doses achieved by "normal" beer consumption have beneficial effects on health. Female C57 Bl/6 J mice, pretreated for 4 d with an iso-α-acid-rich extract (∼30% iso-α-acids from hops, 0.75 mg/kg body weight), were fed one bolus of ethanol (6 g/kg body weight intragastric) or an iso-caloric maltodextrin solution. Markers of liver damage, toll-like receptor-4 signaling, and lipid peroxidation were determined. Furthermore, the effect of isohumulone on the lipopolysaccharide-dependent activation of J774 A.1 macrophages, used as a model of Kupffer cells, was determined. In the liver, acute ethanol administration led to a significant accumulation of fat (∼10-fold), which was accompanied by significantly higher inducible nitric oxide synthase protein level, elevated nitric oxide production, and increased plasminogen activator inhibitor 1 protein concentration when compared to controls. In mice pretreated with iso-α-acids, these effects of alcohol were markedly attenuated. Pretreatment of J774 A.1 macrophages with isohumulone significantly attenuated lipopolysaccharide-induced mRNA expression of inducible nitric oxide synthase and interleukin-6 as well as the release of nitric oxide. Taken together, iso-α-acids markedly attenuated the development of acute alcohol-induced damage in mice. Copyright © 2017 Elsevier Inc. All rights reserved.

Salmonella employs multiple mechanisms to subvert the TLR-inducible zinc-mediated antimicrobial response of human macrophages.

PubMed

Kapetanovic, Ronan; Bokil, Nilesh J; Achard, Maud E S; Ong, Cheryl-Lynn Y; Peters, Kate M; Stocks, Claudia J; Phan, Minh-Duy; Monteleone, Mercedes; Schroder, Kate; Irvine, Katharine M; Saunders, Bernadette M; Walker, Mark J; Stacey, Katryn J; McEwan, Alastair G; Schembri, Mark A; Sweet, Matthew J

2016-05-01

We aimed to characterize antimicrobial zinc trafficking within macrophages and to determine whether the professional intramacrophage pathogen Salmonella enterica serovar Typhimurium (S Typhimurium) subverts this pathway. Using both Escherichia coli and S Typhimurium, we show that TLR signaling promotes the accumulation of vesicular zinc within primary human macrophages. Vesicular zinc is delivered to E. coli to promote microbial clearance, whereas S. Typhimurium evades this response via Salmonella pathogenicity island (SPI)-1. Even in the absence of SPI-1 and the zinc exporter ZntA, S Typhimurium resists the innate immune zinc stress response, implying the existence of additional host subversion mechanisms. We also demonstrate the combinatorial antimicrobial effects of zinc and copper, a pathway that S. Typhimurium again evades. Our use of complementary tools and approaches, including confocal microscopy, direct assessment of intramacrophage bacterial zinc stress responses, specific E. coli and S Typhimurium mutants, and inductively coupled plasma mass spectroscopy, has enabled carefully controlled characterization of this novel innate immune antimicrobial pathway. In summary, our study provides new insights at the cellular level into the well-documented effects of zinc in promoting host defense against infectious disease, as well as the complex host subversion strategies employed by S Typhimurium to combat this pathway.-Kapetanovic, R., Bokil, N. J., Achard, M. E. S., Ong, C.-L. Y., Peters, K. M., Stocks, C. J., Phan, M.-D., Monteleone, M., Schroder, K., Irvine, K. M., Saunders, B. M., Walker, M. J., Stacey, K. J., McEwan, A. G., Schembri, M. A., Sweet, M. J. Salmonella employs multiple mechanisms to subvert the TLR-inducible zinc-mediated antimicrobial response of human macrophages. © FASEB.

The alveolar macrophage.

PubMed

Bowden, D H

1984-04-01

The pulmonary macrophagic system is critical to the defense of the lung, keeping the alveoli clean and sterile and responding on demand with an adaptive outpouring of new cells into the air sacs. Under basal conditions alveolar macrophages, in common with other mononuclear phagocytes, are derived from the bone marrow. A population of macrophage precursors within the pulmonary interstitium provides a reserve pool capable of proliferation and delivery of phagocytes in response to unusually heavy loads of inhaled particles. This reserve system also produces macrophages when monocytic precursors in the bone marrow are depleted by diseases such as leukemia. The alveolar macrophage is destined to ingest particulate matter and to be eliminated along the mucociliary pathway; clearance by lymphatics is of minor importance and macrophages probably do not recross the alveolar epithelium to reach the pulmonary interstitial compartment. Although the protective role of the macrophage is dominant, this cell may participate, directly or indirectly, in the genesis of two major groups of chronic pulmonary disease, interstitial fibrosis and emphysema. Such inappropriate responses involve interactions with fibroblastic cells and tissue injury initiated by proteases secreted by the macrophage.

SHP-1-dependent macrophage differentiation exacerbates virus-induced myositis.

PubMed

Watson, Neva B; Schneider, Karin M; Massa, Paul T

2015-03-15

Virus-induced myositis is an emerging global affliction that remains poorly characterized with few treatment options. Moreover, muscle-tropic viruses often spread to the CNS, causing dramatically increased morbidity. Therefore, there is an urgent need to explore genetic factors involved in this class of human disease. This report investigates critical innate immune pathways affecting murine virus-induced myositis. Of particular importance, the key immune regulator src homology region 2 domain-containing phosphatase 1 (SHP-1), which normally suppresses macrophage-mediated inflammation, is a major factor in promoting clinical disease in muscle. We show that Theiler's murine encephalomyelitis virus (TMEV) infection of skeletal myofibers induces inflammation and subsequent dystrophic calcification, with loss of ambulation in wild-type (WT) mice. Surprisingly, although similar extensive myofiber infection and inflammation are observed in SHP-1(-/-) mice, these mice neither accumulate dead calcified myofibers nor lose ambulation. Macrophages were the predominant effector cells infiltrating WT and SHP-1(-/-) muscle, and an increased infiltration of immature monocytes/macrophages correlated with an absence of clinical disease in SHP-1(-/-) mice, whereas mature M1-like macrophages corresponded with increased myofiber degeneration in WT mice. Furthermore, blocking SHP-1 activation in WT macrophages blocked virus-induced myofiber degeneration, and pharmacologic ablation of macrophages inhibited muscle calcification in TMEV-infected WT animals. These data suggest that, following TMEV infection of muscle, SHP-1 promotes M1 differentiation of infiltrating macrophages, and these inflammatory macrophages are likely involved in damaging muscle fibers. These findings reveal a pathological role for SHP-1 in promoting inflammatory macrophage differentiation and myofiber damage in virus-infected skeletal muscle, thus identifying SHP-1 and M1 macrophages as essential mediators of virus

Replication of Salmonella enterica Serovar Typhimurium in Human Monocyte-Derived Macrophages.

PubMed

Lathrop, Stephanie K; Binder, Kelsey A; Starr, Tregei; Cooper, Kendal G; Chong, Audrey; Carmody, Aaron B; Steele-Mortimer, Olivia

2015-07-01

Salmonella enterica serovar Typhimurium is a common cause of food-borne gastrointestinal illness, but additionally it causes potentially fatal bacteremia in some immunocompromised patients. In mice, systemic spread and replication of the bacteria depend upon infection of and replication within macrophages, but replication in human macrophages is not widely reported or well studied. In order to assess the ability of Salmonella Typhimurium to replicate in human macrophages, we infected primary monocyte-derived macrophages (MDM) that had been differentiated under conditions known to generate different phenotypes. We found that replication in MDM depends greatly upon the phenotype of the cells, as M1-skewed macrophages did not allow replication, while M2a macrophages and macrophages differentiated with macrophage colony-stimulating factor (M-CSF) alone (termed M0) did. We describe how additional conditions that alter the macrophage phenotype or the gene expression of the bacteria affect the outcome of infection. In M0 MDM, the temporal expression of representative genes from Salmonella pathogenicity islands 1 and 2 (SPI1 and SPI2) and the importance of the PhoP/Q two-component regulatory system are similar to what has been shown in mouse macrophages. However, in contrast to mouse macrophages, where replication is SPI2 dependent, we observed early SPI2-independent replication in addition to later SPI2-dependent replication in M0 macrophages. Only SPI2-dependent replication was associated with death of the host cell at later time points. Altogether, our results reveal a very nuanced interaction between Salmonella and human macrophages. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Specific Macrophage Subtypes Influence the Progression of Rhabdomyolysis-Induced Kidney Injury

PubMed Central

Belliere, Julie; Casemayou, Audrey; Ducasse, Laure; Zakaroff-Girard, Alexia; Martins, Frédéric; Iacovoni, Jason S.; Guilbeau-Frugier, Céline; Buffin-Meyer, Bénédicte; Pipy, Bernard; Chauveau, Dominique

2015-01-01

Rhabdomyolysis can be life threatening if complicated by AKI. Macrophage infiltration has been observed in rat kidneys after glycerol-induced rhabdomyolysis, but the role of macrophages in rhabdomyolysis-induced AKI remains unknown. Here, in a patient diagnosed with rhabdomyolysis, we detected substantial macrophage infiltration in the kidney. In a mouse model of rhabdomyolysis-induced AKI, diverse renal macrophage phenotypes were observed depending on the stage of the disease. Two days after rhabdomyolysis, F4/80lowCD11bhighLy6bhighCD206low kidney macrophages were dominant, whereas by day 8, F4/80highCD11b+Ly6blowCD206high cells became the most abundant. Single-cell gene expression analyses of FACS-sorted macrophages revealed that these subpopulations were heterogeneous and that individual cells simultaneously expressed both M1 and M2 markers. Liposomal clodronate-mediated macrophage depletion significantly reduced the early infiltration of F4/80lowCD11bhighLy6bhighCD206low macrophages. Furthermore, transcriptionally regulated targets potentially involved in disease progression, including fibronectin, collagen III, and chemoattractants that were identified via single-cell analysis, were verified as macrophage-dependent in situ. In vitro, myoglobin treatment induced proximal tubular cells to secrete chemoattractants and macrophages to express proinflammatory markers. At day 30, liposomal clodronate-mediated macrophage depletion reduced fibrosis and improved both kidney repair and mouse survival. Seven months after rhabdomyolysis, histologic lesions were still present but were substantially reduced with prior depletion of macrophages. These results suggest an important role for macrophages in rhabdomyolysis-induced AKI progression and advocate the utility of long-term follow-up for patients with this disease. PMID:25270069

Macrophages redirect phagocytosis by non-professional phagocytes and influence inflammation.

PubMed

Han, Claudia Z; Juncadella, Ignacio J; Kinchen, Jason M; Buckley, Monica W; Klibanov, Alexander L; Dryden, Kelly; Onengut-Gumuscu, Suna; Erdbrügger, Uta; Turner, Stephen D; Shim, Yun M; Tung, Kenneth S; Ravichandran, Kodi S

2016-11-24

Professional phagocytes (such as macrophages) and non-professional phagocytes (such as epithelial cells) clear billions of apoptotic cells and particles on a daily basis. Although professional and non-professional macrophages reside in proximity in most tissues, whether they communicate with each other during cell clearance, and how this might affect inflammation, is not known. Here we show that macrophages, through the release of a soluble growth factor and microvesicles, alter the type of particles engulfed by non-professional phagocytes and influence their inflammatory response. During phagocytosis of apoptotic cells or in response to inflammation-associated cytokines, macrophages released insulin-like growth factor 1 (IGF-1). The binding of IGF-1 to its receptor on non-professional phagocytes redirected their phagocytosis, such that uptake of larger apoptotic cells was reduced whereas engulfment of microvesicles was increased. IGF-1 did not alter engulfment by macrophages. Macrophages also released microvesicles, whose uptake by epithelial cells was enhanced by IGF-1 and led to decreased inflammatory responses by epithelial cells. Consistent with these observations, deletion of IGF-1 receptor in airway epithelial cells led to exacerbated lung inflammation after allergen exposure. These genetic and functional studies reveal that IGF-1- and microvesicle-dependent communication between macrophages and epithelial cells can critically influence the magnitude of tissue inflammation in vivo.

Huaier extract suppresses breast cancer via regulating tumor-associated macrophages

PubMed Central

Li, Yaming; Qi, Wenwen; Song, Xiaojin; Lv, Shangge; Zhang, Hanwen; Yang, Qifeng

2016-01-01

Macrophages in tumor microenvironment are mostly M2-polarized - and have been reported to promote tumorigenesis, which are also defined as tumor-associated macrophages (TAMs). Here, we examined the regulatory effects of Huaier extract on TAMs using RAW264.7 murine macrophage cell line. Our data demonstrated that Huaier extract could inhibit the infiltration of macrophages into tumor microenvironment in a dose-dependent manner. By performing RT-PCR, immunofluorescence and phagocytosis assay, we were able to find that Huaier extract could regulate the polarization of macrophages, with decreased M2-polarization and increased phagocytosis of RAW264.7 cells. Moreover, we identified that Huaier extract could suppress macrophages-induced angiogenesis by using HUVEC migration assay, tube formation and chorioallantoic membrane assay. Additionally, western blotting showed decreased expression of MMP2, MMP9 and VEGF with the use of Huaier extract. Finally, we found that Huaier extract could inhibit M2-macrophages infiltration and angiogenesis through treating 4T1 tumor bearing mice with Huaier extract. Our study revealed a novel mechanism of the anti-tumor effect of Huaier extract which inhibited angiogenesis by targeting TAMs. These findings provided that Huaier was a promising drug for clinical treatment of breast cancer. PMID:26831282

Dual transcriptome sequencing reveals resistance of TLR4 ligand-activated bone marrow-derived macrophages to inflammation mediated by the BET inhibitor JQ1

PubMed Central

Das, Amitabh; Chai, Jin Choul; Yang, Chul-su; Lee, Young Seek; Das, Nando Dulal; Jung, Kyoung Hwa; Chai, Young Gyu

2015-01-01

Persistent macrophage activation is associated with the expression of various pro-inflammatory genes, cytokines and chemokines, which may initiate or amplify inflammatory disorders. A novel synthetic BET inhibitor, JQ1, was proven to exert immunosuppressive activities in macrophages. However, a genome-wide search for JQ1 molecular targets has not been undertaken. The present study aimed at evaluating the anti-inflammatory function and underlying genes that are targeted by JQ1 in LPS-stimulated primary bone marrow-derived macrophages (BMDMs) using global transcriptomic RNA sequencing and quantitative real-time PCR. Among the annotated genes, transcriptional sequencing of BMDMs that were treated with JQ1 revealed a selective effect on LPS-induced gene expression in which the induction of cytokines/chemokines, interferon-stimulated genes, and prominent (transcription factors) TFs was suppressed. Additionally, we found that JQ1 reduced the expression of previously unidentified genes that are important in inflammation. Importantly, these inflammatory genes were not affected by JQ1 treatment alone. Furthermore, we confirmed that JQ1 reduced cytokines/chemokines in the supernatants of LPS treated BMDMs. Moreover, the biological pathways and gene ontology of the differentially expressed genes were determined in the JQ1 treatment of BMDMs. These unprecedented results suggest that the BET inhibitor JQ1 is a candidate for the prevention or therapeutic treatment of inflammatory disorders. PMID:26582142

Replication of Norovirus in Cell Culture Reveals a Tropism for Dendritic Cells and Macrophages

PubMed Central

Wobus, Christiane E; Karst, Stephanie M; Thackray, Larissa B; Chang, Kyeong-Ok; Sosnovtsev, Stanislav V; Belliot, Gaël; Krug, Anne; Mackenzie, Jason M; Green, Kim Y

2004-01-01

Noroviruses are understudied because these important enteric pathogens have not been cultured to date. We found that the norovirus murine norovirus 1 (MNV-1) infects macrophage-like cells in vivo and replicates in cultured primary dendritic cells and macrophages. MNV-1 growth was inhibited by the interferon-αβ receptor and STAT-1, and was associated with extensive rearrangements of intracellular membranes. An amino acid substitution in the capsid protein of serially passaged MNV-1 was associated with virulence attenuation in vivo. This is the first report of replication of a norovirus in cell culture. The capacity of MNV-1 to replicate in a STAT-1-regulated fashion and the unexpected tropism of a norovirus for cells of the hematopoietic lineage provide important insights into norovirus biology. PMID:15562321

Brucella infection inhibits macrophages apoptosis via Nedd4-dependent degradation of calpain2.

PubMed

Cui, Guimei; Wei, Pan; Zhao, Yuxi; Guan, Zhenhong; Yang, Li; Sun, Wanchun; Wang, Shuangxi; Peng, Qisheng

2014-11-07

The calcium-dependent protease calpain2 is involved in macrophages apoptosis. Brucella infection-induced up-regulation of intracellular calcium level is an essential factor for the intracellular survival of Brucella within macrophages. Here, we hypothesize that calcium-dependent E3 ubiquitin ligase Nedd4 ubiquitinates calpain2 and inhibits Brucella infection-induced macrophage apoptosis via degradation of calpain2.Our results reveal that Brucella infection induces increases in Nedd4 activity in an intracellular calcium dependent manner. Furthermore, Brucella infection-induced degradation of calpain2 is mediated by Nedd4 ubiquitination of calpain2. Brucella infection-induced calpain2 degradation inhibited macrophages apoptosis. Treatment of Brucella infected macrophages with calcium chelator BAPTA or Nedd4 knock-down decreased Nedd4 activity, prevented calpain2 degradation, and resulted in macrophages apoptosis. Copyright © 2014 Elsevier B.V. All rights reserved.

Antiorthostatic suspension stimulates profiles of macrophage activation in mice

NASA Technical Reports Server (NTRS)

Miller, E. S.; Bates, R. A.; Koebel, D. A.; Sonnenfeld, G.

1999-01-01

The antiorthostatic suspension model simulates certain physiological effects of spaceflight. We have previously reported BDF1 mice suspended by the tail in the antiorthostatic orientation for 4 days express high levels of resistance to virulent Listeria monocytogenesinfection. In the present study, we examined whether the increased resistance to this organism correlates with profiles of macrophage activation, given the role of the macrophage in killing this pathogen in vivo. We infected BDF1 mice with a lethal dose of virulent L. monocytogenes on day 4 of antiorthostatic suspension and 24 h later constructed profiles of macrophage activation. Viable listeria could not be detected in mice suspended in the antiorthostatic orientation 24 h after infection. Flow cytometric analysis revealed the numbers of granulocytes and mononuclear phagocytes in the spleen of infected mice were not significantly altered as a result of antiorthostatic suspension. Splenocytes from antiorthostatically suspended infected mice produced increased titers of IL-1. Serum levels of neopterin, a nucleotide metabolite secreted by activated macrophages, were enhanced in mice infected during antiorthostatic suspension, but not in antiorthostatically suspended naive mice. Splenic macrophages from mice infected on day 4 of suspension produced enhanced levels of lysozyme. In contrast to the results from antiorthostatically suspended infected mice, macrophages from antiorthostatically suspended uninfected mice did not express enhanced bactericidal activities. The collective results indicate that antiorthostatic suspension can stimulate profiles of macrophage activation which correlate with increased resistance to infection by certain classes of pathogenic bacteria.

Dexamethasone targeted directly to macrophages induces macrophage niches that promote erythroid expansion.

PubMed

Falchi, Mario; Varricchio, Lilian; Martelli, Fabrizio; Masiello, Francesca; Federici, Giulia; Zingariello, Maria; Girelli, Gabriella; Whitsett, Carolyn; Petricoin, Emanuel F; Moestrup, Søren Kragh; Zeuner, Ann; Migliaccio, Anna Rita

2015-02-01

Cultures of human CD34(pos) cells stimulated with erythroid growth factors plus dexamethasone, a model for stress erythropoiesis, generate numerous erythroid cells plus a few macrophages (approx. 3%; 3:1 positive and negative for CD169). Interactions occurring between erythroblasts and macrophages in these cultures and the biological effects associated with these interactions were documented by live phase-contrast videomicroscopy. Macrophages expressed high motility interacting with hundreds/thousands of erythroblasts per hour. CD169(pos) macrophages established multiple rapid 'loose' interactions with proerythroblasts leading to formation of transient erythroblastic island-like structures. By contrast, CD169(neg) macrophages established 'tight' interactions with mature erythroblasts and phagocytosed these cells. 'Loose' interactions of CD169(pos) macrophages were associated with proerythroblast cytokinesis (the M phase of the cell cycle) suggesting that these interactions may promote proerythroblast duplication. This hypothesis was tested by experiments that showed that as few as 103 macrophages significantly increased levels of 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide incorporation frequency in S/G2/M and cytokinesis expressed by proerythroblasts over 24 h of culture. These effects were observed also when macrophages were co-cultured with dexamethasone directly conjugated to a macrophage-specific CD163 antibody. In conclusion, in addition to promoting proerythroblast proliferation directly, dexamethasone stimulates expansion of these cells indirectly by stimulating maturation and cytokinesis supporting activity of macrophages. Copyright© Ferrata Storti Foundation.

Purinergic signaling during macrophage differentiation results in M2 alternative activated macrophages.

PubMed

Barberà-Cremades, Maria; Baroja-Mazo, Alberto; Pelegrín, Pablo

2016-02-01

Macrophages represent a highly heterogenic cell population of the innate immune system, with important roles in the initiation and resolution of the inflammatory response. Purinergic signaling regulates both M1 and M2 macrophage function at different levels by controlling the secretion of cytokines, phagocytosis, and the production of reactive oxygen species. We found that extracellular nucleotides arrest macrophage differentiation from bone marrow precursors via adenosine and P2 receptors. This results in a mature macrophage with increased expression of M2, but not M1, genes. Similar to adenosine and ATP, macrophage growth arrested with LPS treatment resulted in an increase of the M2-related marker Ym1. Recombinant Ym1 was able to affect macrophage proliferation and could, potentially, be involved in the arrest of macrophage growth during hematopoiesis. © Society for Leukocyte Biology.

Specific macrophage subtypes influence the progression of rhabdomyolysis-induced kidney injury.

PubMed

Belliere, Julie; Casemayou, Audrey; Ducasse, Laure; Zakaroff-Girard, Alexia; Martins, Frédéric; Iacovoni, Jason S; Guilbeau-Frugier, Céline; Buffin-Meyer, Bénédicte; Pipy, Bernard; Chauveau, Dominique; Schanstra, Joost P; Bascands, Jean-Loup

2015-06-01

Rhabdomyolysis can be life threatening if complicated by AKI. Macrophage infiltration has been observed in rat kidneys after glycerol-induced rhabdomyolysis, but the role of macrophages in rhabdomyolysis-induced AKI remains unknown. Here, in a patient diagnosed with rhabdomyolysis, we detected substantial macrophage infiltration in the kidney. In a mouse model of rhabdomyolysis-induced AKI, diverse renal macrophage phenotypes were observed depending on the stage of the disease. Two days after rhabdomyolysis, F4/80(low)CD11b(high)Ly6b(high)CD206(low) kidney macrophages were dominant, whereas by day 8, F4/80(high)CD11b(+)Ly6b(low)CD206(high) cells became the most abundant. Single-cell gene expression analyses of FACS-sorted macrophages revealed that these subpopulations were heterogeneous and that individual cells simultaneously expressed both M1 and M2 markers. Liposomal clodronate-mediated macrophage depletion significantly reduced the early infiltration of F4/80(low)CD11b(high)Ly6b(high)CD206(low) macrophages. Furthermore, transcriptionally regulated targets potentially involved in disease progression, including fibronectin, collagen III, and chemoattractants that were identified via single-cell analysis, were verified as macrophage-dependent in situ. In vitro, myoglobin treatment induced proximal tubular cells to secrete chemoattractants and macrophages to express proinflammatory markers. At day 30, liposomal clodronate-mediated macrophage depletion reduced fibrosis and improved both kidney repair and mouse survival. Seven months after rhabdomyolysis, histologic lesions were still present but were substantially reduced with prior depletion of macrophages. These results suggest an important role for macrophages in rhabdomyolysis-induced AKI progression and advocate the utility of long-term follow-up for patients with this disease. Copyright © 2015 by the American Society of Nephrology.

Ultrastructural analysis of the pigment dispersion syndrome in DBA/2J mice.

PubMed

Schraermeyer, Mareike; Schnichels, Sven; Julien, Sylvie; Heiduschka, Peter; Bartz-Schmidt, Karl-Ulrich; Schraermeyer, Ulrich

2009-11-01

To characterise ocular pigment abnormalities associated with iris atrophy in DBA/2J mice as a model for human pigment dispersion syndrome. Immunohistochemistry, electron and light microscopy were performed to examine the eyes of DBA/2J mice ranging in age from 2.5 to 18 months old. The focus of our study was the description of the ultrastructural modifications in the irides of DBA/2J mice. The DBA/2J mice presented modifications in the melanosomes in all the pigmented parts of the eye, including the retinal pigment epithelial cells and choroidal melanocytes of the ciliary pigment epithelium. The extracellular matrix of the iris stroma disappeared with ageing. Pigmented cells detached from the iris and migrated into the trabecular meshwork exclusively on the anterior iris surface. These cells were identified as macrophages by immunohistochemistry and electron microscopy. There was no evidence that melanocytes or iris pigment epithelial cells migrated into the trabecular meshwork, but they became more and more depigmented. The aqueous outflow was blocked by pigment-laden cells, but not by cellular debris or melanosomes. No substantial amount of extracellular melanosomes was observed. The morphology of melanosomes is aberrant in all pigment cells in the eyes of DBA/2J mice. We conclude that the disease process begins with the transfer of both immature melanosomes from the iris pigment epithelium (IPE) and melanocytes to macrophages, which subsequently migrate into the trabecular meshwork. Accumulating macrophages cause a blockade of the chamber angle. As the disease progresses, the IPE, melanocytes and iris stroma, including blood vessels, disappear, leading to iris atrophy. It is speculated that the loss of these pigment cells is partly caused by reduction of the iris stroma.

Macrophage Depletion Ameliorates Peripheral Neuropathy in Aging Mice.

PubMed

Yuan, Xidi; Klein, Dennis; Kerscher, Susanne; West, Brian L; Weis, Joachim; Katona, Istvan; Martini, Rudolf

2018-05-09

Aging is known as a major risk factor for the structure and function of the nervous system. There is urgent need to overcome such deleterious effects of age-related neurodegeneration. Here we show that peripheral nerves of 24-month-old aging C57BL/6 mice of either sex show similar pathological alterations as nerves from aging human individuals, whereas 12-month-old adult mice lack such alterations. Specifically, nerve fibers showed demyelination, remyelination and axonal lesion. Moreover, in the aging mice, neuromuscular junctions showed features typical for dying-back neuropathies, as revealed by a decline of presynaptic markers, associated with α-bungarotoxin-positive postsynapses. In line with these observations were reduced muscle strengths. These alterations were accompanied by elevated numbers of endoneurial macrophages, partially comprising the features of phagocytosing macrophages. Comparable profiles of macrophages could be identified in peripheral nerve biopsies of aging persons. To determine the pathological impact of macrophages in aging mice, we selectively targeted the cells by applying an orally administered CSF-1R specific kinase (c-FMS) inhibitor. The 6-month-lasting treatment started before development of degenerative changes at 18 months and reduced macrophage numbers in mice by ∼70%, without side effects. Strikingly, nerve structure was ameliorated and muscle strength preserved. We show, for the first time, that age-related degenerative changes in peripheral nerves are driven by macrophages. These findings may pave the way for treating degeneration in the aging peripheral nervous system by targeting macrophages, leading to reduced weakness, improved mobility, and eventually increased quality of life in the elderly. SIGNIFICANCE STATEMENT Aging is a major risk factor for the structure and function of the nervous system. Here we show that peripheral nerves of 24-month-old aging mice show similar degenerative alterations as nerves from aging

Intravital live cell triggered imaging system reveals monocyte patrolling and macrophage migration in atherosclerotic arteries

NASA Astrophysics Data System (ADS)

McArdle, Sara; Chodaczek, Grzegorz; Ray, Nilanjan; Ley, Klaus

2015-02-01

Intravital multiphoton imaging of arteries is technically challenging because the artery expands with every heartbeat, causing severe motion artifacts. To study leukocyte activity in atherosclerosis, we developed the intravital live cell triggered imaging system (ILTIS). This system implements cardiac triggered acquisition as well as frame selection and image registration algorithms to produce stable movies of myeloid cell movement in atherosclerotic arteries in live mice. To minimize tissue damage, no mechanical stabilization is used and the artery is allowed to expand freely. ILTIS performs multicolor high frame-rate two-dimensional imaging and full-thickness three-dimensional imaging of beating arteries in live mice. The external carotid artery and its branches (superior thyroid and ascending pharyngeal arteries) were developed as a surgically accessible and reliable model of atherosclerosis. We use ILTIS to demonstrate Cx3cr1GFP monocytes patrolling the lumen of atherosclerotic arteries. Additionally, we developed a new reporter mouse (Apoe-/-Cx3cr1GFP/+Cd11cYFP) to image GFP+ and GFP+YFP+ macrophages "dancing on the spot" and YFP+ macrophages migrating within intimal plaque. ILTIS will be helpful to answer pertinent open questions in the field, including monocyte recruitment and transmigration, macrophage and dendritic cell activity, and motion of other immune cells.

Activation of Murine Macrophages by Lipoprotein and Lipooligosaccharide of Treponema denticola

PubMed Central

Rosen, Graciela; Sela, Michael N.; Naor, Ronit; Halabi, Amal; Barak, Vivian; Shapira, Lior

1999-01-01

We have recently demonstrated that the periodontopathogenic oral spirochete Treponema denticola possesses membrane-associated lipoproteins in addition to lipooligosaccharide (LOS). The aim of the present study was to test the potential of these oral spirochetal components to induce the production of inflammatory mediators by human macrophages, which in turn may stimulate tissue breakdown as observed in periodontal diseases. An enriched lipoprotein fraction (dLPP) from T. denticola ATCC 35404 obtained upon extraction of the treponemes with Triton X-114 was found to stimulate the production of nitric oxide (NO), tumor necrosis factor alpha (TNF-α), and interleukin-1 (IL-1) by mouse macrophages in a dose-dependent manner. Induction of NO by dLPP was at 25% of the levels obtained by Salmonella typhosa lipopolysaccharide (LPS) at similar concentrations, while IL-1 was produced at similar levels by both inducers. dLPP-mediated macrophage activation was unaffected by amounts of polymyxin B that neutralized the induction produced by S. typhosa LPS. dLPP also induced NO and TNF-α secretion from macrophages isolated from endotoxin-unresponsive C3H/HeJ mice to an extent similar to the stimulation produced in endotoxin-responsive mice. Purified T. denticola LOS also produced a concentration-dependent activation of NO and TNF-α in LPS-responsive and -nonresponsive mouse macrophages. However, macrophage activation by LOS was inhibited by polymyxin B. These results suggest that T. denticola lipoproteins and LOS may play a role in the inflammatory processes that characterize periodontal diseases. PMID:10024558

Relationship between complement activation, cellular uptake and surface physicochemical aspects of novel PEG-modified nanocapsules.

PubMed

Mosqueira, V C; Legrand, P; Gulik, A; Bourdon, O; Gref, R; Labarre, D; Barratt, G

2001-11-01

The aim of our work was to examine the relationship between modifications of the surface of nanocapsules (NC) by adsorption or covalent grafting of poly(ethylene oxide) (PEG), and changes in their phospholipid (PL) content on complement activation (C3 cleavage) and on uptake by macrophages. The physicochemical characterization of the NC included an investigation of their properties, such as surface charge, size, hydrophilicity, morphology and homogeneity. This is the first time that such properties have been correlated with biological interactions for NC, a novel carrier system with a structure more complex than nanospheres. C3 crossed immunoelectrophoresis revealed the reduced activation for NC with longer PEG chain and higher density, although all formulations induced C3 cleavage to a lesser or greater extent. NC bearing PEG covalently bound to the surface were weaker activators of complement than plain PLA [poly(D,L-lactide)] NC or nanospheres (NS). Furthermore, the fluorescent/confocal microscopy of J774A1 cells in contact with NC reveal a dramatically reduced interaction with PEG-bearing NC. However, the way in which PEG was attached (covalent or adsorbed) seemed to affect the mechanism of uptake. Taken together, these results suggest that the low level of protein binding to NC covered with a high density of 20kDa PEG chains is likely to be due to the steric barriers surrounding these particles, which prevents protein adsorption and reduces their interaction with macrophages.

Macrophage-selective toxicity as a mechanism of hydroquinone-induced myelotoxicity

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

Thomas, D.J.

1989-01-01

This research has focused upon the role of the bone marrow stroma in the etiology of benzene hematotoxicity. Treatment with the metabolite hydroquinone results in a reduced capacity of the stroma to support myelopoiesis. The goal of this research was to examine stromal cell selective toxicity following hydroquinone treatment. Populations of macrophages and a fibroblastoid cell line (LTF) or primary fibroblasts were developed from mouse bone marrow. Following treatment of with hydroquinone, treated or control fibroblastoid cells were reconstituted with control or treated macrophages, respectively, and the cultures were assayed for their ability to support myelopoiesis. To examine mechanisms ofmore » selective toxicity, macrophage and LTF cultures were incubated with 14C-hydroquinone and bioactivation was examined. After 24 hours, macrophages had 16-fold higher levels of bound {sup 14}C than LTF cells. Peroxide-dependent bioactivation in cell homogenates revealed that peroxide could support formation of covalent-binding species in macrophage homogenates but not in LTF homogenates. It was determined that macrophages, but not LTF cells, contained detectable levels of peroxidase activity which was consistent with the postulate that increased binding was due to peroxidase-mediated bioactivation of hydroquinone. Accordingly, purified myeloperoxidase incubated with {sup 14}C-hydroquinone, resulted in bioactivation to a covalent-binding species. This study provided evidence supporting selective bioactivation as a mechanism of selective toxicity of hydroquinone to bone marrow stromal macrophages.« less

Macrophages under pressure: the role of macrophage polarization in hypertension.

PubMed

Harwani, Sailesh C

2018-01-01

Hypertension is a multifactorial disease involving the nervous, renal, and cardiovascular systems. Macrophages are the most abundant and ubiquitous immune cells, placing them in a unique position to serve as key mediators between these components. The polarization of macrophages confers vast phenotypic and functional plasticity, allowing them to act as proinflammatory, homeostatic, and anti-inflammatory agents. Key differences between the M1 and M2 phenotypes, the 2 subsets at the extremes of this polarization spectrum, place macrophages at a juncture to mediate many mechanisms involved in the pathogenesis of hypertension. Neuronal and non-neuronal regulation of the immune system, that is, the "neuroimmuno" axis, plays an integral role in the polarization of macrophages. In hypertension, the neuroimmuno axis results in synchronization of macrophage mobilization from immune cell reservoirs and their chemotaxis, via increased expression of chemoattractants, to end organs critical in the development of hypertension. This complicated system is largely coordinated by the dichotomous actions of the autonomic neuronal and non-neuronal activation of cholinergic, adrenergic, and neurohormonal receptors on macrophages, leading to their ability to "switch" between phenotypes at sites of active inflammation. Data from experimental models and human studies are in concordance with each other and support a central role for macrophage polarization in the pathogenesis of hypertension. Copyright © 2017 Elsevier Inc. All rights reserved.

Effects of resveratrol-related hydroxystilbenes on the nitric oxide production in macrophage cells: structural requirements and mechanism of action.

PubMed

Cho, Dong-Im; Koo, Na-Youn; Chung, Woon Jae; Kim, Tae-Sung; Ryu, Shi Yong; Im, Suhn Young; Kim, Kyeong-Man

2002-09-13

NF-kappaB that plays an important role in iNOS expression is one of the targets of various potential anti-inflammatory agents including resveratrol. Resveratrol contains a structural similarity with estrogen, and there has been speculation about resveratrol as estrogen agonist. In this study, the mechanism and structural requirements of resveratrol and related hydroxystilbenes for the inhibition of LPS-induced nitric oxide production were studied in macrophage cells (RAW 264.7 and J774) by comparing its effect on LPS-induced NF-kappaB translocation and nitric oxide production, and by considering the possibility of involvement of an estrogen receptor. LPS-induced nitric oxide production was inhibited only when cells were treated with resveratrol prior to stimulation with LPS, suggesting that resveratrol does not affect the enzyme itself. A higher concentration of resveratrol than needed for the inhibition of nitric oxide production was required for the inhibition of NF-kappaB mobilization or iNOS expression. Estrogen and diethylstilbesterol, an estrogen agonist, caused only weak inhibition of nitric oxide production, and the effects of resveratrol were not noticeably blocked by ICI-182780, an estrogen antagonist. Structure-activity analysis of resveratrol and nine hydroxystilbenes suggests that the structural balance between oxygen functional groups on the benzene rings is important for their activity. Our results suggest that resveratrol might act on other cellular targets as well as NF-kappaB at the initial stage of gene expression. Unique structural features of hydroxystilbenes are needed for suppression of nitric oxide production and it is unlikely that estrogen receptor is involved in it.

Different responses to oxidized low-density lipoproteins in human polarized macrophages

PubMed Central

2011-01-01

Background Oxidized low-density lipoprotein (oxLDL) uptake by macrophages plays an important role in foam cell formation. It has been suggested the presence of heterogeneous subsets of macrophage, such as M1 and M2, in human atherosclerotic lesions. To evaluate which types of macrophages contribute to atherogenesis, we performed cDNA microarray analysis to determine oxLDL-induced transcriptional alterations of each subset of macrophages. Results Human monocyte-derived macrophages were polarized toward the M1 or M2 subset, followed by treatment with oxLDL. Then gene expression levels during oxLDL treatment in each subset of macrophages were evaluated by cDNA microarray analysis and quantitative real-time RT-PCR. In terms of high-ranking upregulated genes and functional ontologies, the alterations during oxLDL treatment in M2 macrophages were similar to those in nonpolarized macrophages (M0). Molecular network analysis showed that most of the molecules in the oxLDL-induced highest scoring molecular network of M1 macrophages were directly or indirectly related to transforming growth factor (TGF)-β1. Hierarchical cluster analysis revealed commonly upregulated genes in all subset of macrophages, some of which contained antioxidant response elements (ARE) in their promoter regions. A cluster of genes that were specifically upregulated in M1 macrophages included those encoding molecules related to nuclear factor of kappa light polypeptide gene enhancer in B-cells (NF-κB) signaling pathway. Quantitative real-time RT-PCR showed that the gene expression of interleukin (IL)-8 after oxLDL treatment in M2 macrophages was markedly lower than those in M0 and M1 cells. HMOX1 gene expression levels were almost the same in all 3 subsets of macrophages even after oxLDL treatment. Conclusions The present study demonstrated transcriptional alterations in polarized macrophages during oxLDL treatment. The data suggested that oxLDL uptake may affect TGF-β1- and NF

Dexamethasone palmitate ameliorates macrophages-rich graft-versus-host disease by inhibiting macrophage functions.

PubMed

Nishiwaki, Satoshi; Nakayama, Takayuki; Murata, Makoto; Nishida, Tetsuya; Terakura, Seitaro; Saito, Shigeki; Kato, Tomonori; Mizuno, Hiroki; Imahashi, Nobuhiko; Seto, Aika; Ozawa, Yukiyasu; Miyamura, Koichi; Ito, Masafumi; Takeshita, Kyosuke; Kato, Hidefumi; Toyokuni, Shinya; Nagao, Keisuke; Ueda, Ryuzo; Naoe, Tomoki

2014-01-01

Macrophage infiltration of skin GVHD lesions correlates directly with disease severity, but the mechanisms underlying this relationship remain unclear and GVHD with many macrophages is a therapeutic challenge. Here, we characterize the macrophages involved in GVHD and report that dexamethasone palmitate (DP), a liposteroid, can ameliorate such GVHD by inhibiting macrophage functions. We found that host-derived macrophages could exacerbate GVHD in a mouse model through expression of higher levels of pro-inflammatory TNF-α and IFN-γ, and lower levels of anti-inflammatory IL-10 than resident macrophages in mice without GVHD. DP significantly decreased the viability and migration capacity of primary mouse macrophages compared to conventional dexamethasone in vitro. DP treatment on day 7 and day 14 decreased macrophage number, and attenuated GVHD score and subsequent mortality in a murine model. This is the first study to provide evidence that therapy for GVHD should be changed on the basis of infiltrating cell type.

Jellyfish collagen stimulates production of TNF-α and IL-6 by J774.1 cells through activation of NF-κB and JNK via TLR4 signaling pathway.

PubMed

Putra, Agus Budiawan Naro; Nishi, Kosuke; Shiraishi, Ryusuke; Doi, Mikiharu; Sugahara, Takuya

2014-03-01

We previously reported that jellyfish collagen stimulates both the acquired and innate immune responses. In the acquired immune response, jellyfish collagen enhanced immunoglobulin production by lymphocytes in vitro and in vivo. Meanwhile, in the innate immune response jellyfish collagen promoted cytokine production and phagocytotic activity of macrophages. The facts that jellyfish collagen plays several potential roles in stimulating cytokine production by macrophages have further attracted us to uncover its mechanisms. We herein describe that the cytokine production-stimulating activity of jellyfish collagen was canceled by a Toll-like receptor 4 (TLR4) inhibitor. Moreover, jellyfish collagen stimulated phosphorylation of inhibitor of κBα (IκBα), promoted the translocation of nucleus factor-κB (NF-κB), and activated c-Jun N-terminal kinase (JNK). A JNK inhibitor also abrogated the cytokine production-stimulating activity of jellyfish collagen. These results suggest that jellyfish collagen may facilitate cytokine production by macrophages through activation of NF-κB and JNK via the TLR4 signaling pathways. Copyright © 2013 Elsevier Ltd. All rights reserved.

Rhinovirus infection induces distinct transcriptome profiles in polarized human macrophages.

PubMed

Rajput, Charu; Walsh, Megan P; Eder, Breanna N; Metitiri, Ediri E; Popova, Antonia P; Hershenson, Marc B

2018-05-01

Infections with rhinovirus (RV) cause asthma exacerbations. Recent studies suggest that macrophages play a role in asthmatic airway inflammation and the innate immune response to RV infection. Macrophages exhibit phenotypes based on surface markers and gene expression. We hypothesized that macrophage polarization state alters gene expression in response to RV infection. Cells were derived from human peripheral blood derived monocytes. M1 and M2 polarization was carried out by using IFN-γ and IL-4, respectively, and RNA was extracted for Affymetrix Human Gene ST2.1 exon arrays. Selected genes were validated by quantitative (q)PCR. Treatment of nonactivated (M0) macrophages with IFN-γ and IL-4 induced the expression of 252 and 153 distinct genes, respectively, including previously-identified M1 and M2 markers. RV infection of M0 macrophages induced upregulation of 232 genes; pathway analysis showed significant overrepresentation of genes involved in IFN-α/β signaling and cytokine signaling in the immune system. RV infection induced differential expression of 195 distinct genes in M1-like macrophages but only seven distinct genes in M2-like-polarized cells. In a secondary analysis, comparison between M0-, RV-infected, and M1-like-polarized, RV-infected macrophages revealed differential expression of 227 genes including those associated with asthma and its exacerbation. qPCR demonstrated increased expression of CCL8, CXCL10, TNFSF10, TNFSF18, IL6, NOD2, and GSDMD and reduced expression of VNN1, AGO1, and AGO2. Together, these data show that, in contrast to M2-like-polarized macrophages, gene expression of M1-like macrophages is highly regulated by RV.

Heme induces programmed necrosis on macrophages through autocrine TNF and ROS production

PubMed Central

Fortes, Guilherme B.; Alves, Leticia S.; de Oliveira, Rosane; Dutra, Fabianno F.; Rodrigues, Danielle; Fernandez, Patricia L.; Souto-Padron, Thais; De Rosa, María José; Kelliher, Michelle; Golenbock, Douglas; Chan, Francis K. M.

2012-01-01

Diseases that cause hemolysis or myonecrosis lead to the leakage of large amounts of heme proteins. Free heme has proinflammatory and cytotoxic effects. Heme induces TLR4-dependent production of tumor necrosis factor (TNF), whereas heme cytotoxicity has been attributed to its ability to intercalate into cell membranes and cause oxidative stress. We show that heme caused early macrophage death characterized by the loss of plasma membrane integrity and morphologic features resembling necrosis. Heme-induced cell death required TNFR1 and TLR4/MyD88-dependent TNF production. Addition of TNF to Tlr4−/− or to Myd88−/− macrophages restored heme-induced cell death. The use of necrostatin-1, a selective inhibitor of receptor-interacting protein 1 (RIP1, also known as RIPK1), or cells deficient in Rip1 or Rip3 revealed a critical role for RIP proteins in heme-induced cell death. Serum, antioxidants, iron chelation, or inhibition of c-Jun N-terminal kinase (JNK) ameliorated heme-induced oxidative burst and blocked macrophage cell death. Macrophages from heme oxygenase-1 deficient mice (Hmox1−/−) had increased oxidative stress and were more sensitive to heme. Taken together, these results revealed that heme induces macrophage necrosis through 2 synergistic mechanisms: TLR4/Myd88-dependent expression of TNF and TLR4-independent generation of ROS. PMID:22262768

Macrophage responses to 316L stainless steel and cobalt chromium alloys with different surface topographies.

PubMed

Anderson, Jordan A; Lamichhane, Sujan; Mani, Gopinath

2016-11-01

The surface topography of a biomaterial plays a vital role in determining macrophage interactions and influencing immune response. In this study, we investigated the effect of smooth and microrough topographies of commonly used metallic biomaterials such as 316 L stainless steel (SS) and cobalt-chromium (CoCr) alloys on macrophage interactions. The macrophage adhesion was greater on CoCr compared to SS, irrespective of their topographies. The macrophage activation and the secretion of most pro-inflammatory cytokines (TNF-α, IL-6, and IP-10) were greater on microrough surfaces than on smooth surfaces by day-1. However, by day-2, the macrophage activation on smooth surfaces was also significantly increased up to the same level as observed on the microrough surfaces, with more amount of cytokines secreted. The secretion of anti-inflammatory cytokine (IL-10) was significantly increased from day-1 to day-2 on all the alloy surfaces with the effect most prominently observed on microrough surfaces. The production of nitric oxide by the macrophages did not show any major substrate-dependent effect. The foreign body giant cells formed by macrophages were least observed on the microrough surfaces of CoCr. Thus, this study demonstrated that the nature of material (SS or CoCr) and their surface topographies (smooth or microrough) strongly influence the macrophage responses. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2658-2672, 2016. © 2016 Wiley Periodicals, Inc.

Granulocyte-Macrophage Colony-Stimulating Factor: More Than a Hemopoietin

DTIC Science & Technology

1990-01-01

Sullivan, R., Elias, A., Antman , K.. Schnipper, L.. and Griffin, D., Granulocyte-macrophage colony-stimulating factor induces the expression of the CDI lb...surface adhesion molecule on human granulocytes in vivo. Blood 72, 691--697, 1988. 38. Socinski, M. A., Cannistra, S., Elias, A., Antman , K. H...1989. 82. Antman , K.. Griffin, J., Elias, A., Socinski. M., Ryan, L., Cannistra, S., Gette, D., Whitly, M., Frei, E., and Schnipper, L., Effect of

Cathepsins limit macrophage necroptosis through cleavage of Rip1 kinase.

PubMed

McComb, Scott; Shutinoski, Bojan; Thurston, Susan; Cessford, Erin; Kumar, Kriti; Sad, Subash

2014-06-15

It has recently been shown that programmed necrosis, necroptosis, may play a key role in the development of inflammation. Deciphering the regulation of this pathway within immune cells may therefore have implications in pathology associated with inflammatory diseases. We show that treatment of macrophages with the pan caspase inhibitor (zVAD-FMK) results in both increased phosphorylation and decreased cleavage of receptor interacting protein kinase-1 (Rip1), leading to necroptosis that is dependent on autocrine TNF signaling. Stimulation of cells with TLR agonists such as LPS in the presence of zVAD-FMK also induced Rip1-phosphorylation via a TNFR-independent mechanism. Further examination of Rip1 expression under these stimulatory conditions revealed a regulatory cleavage of Rip1 in macrophages that is not apparently attributable to caspase-8. Instead, we provide novel evidence that cysteine family cathepsins, which are highly abundant in myeloid cells, can also cleave Rip1 kinase. Using small interfering RNA knockdown, specific cathepsin inhibitors, and cell-free cleavage assays, we demonstrate that cysteine cathepsins B and S can directly cleave Rip1. Finally, we demonstrate that only through combined inhibition of cathepsins and caspase-8 could a potent induction of macrophage necroptosis be achieved. These data reveal a novel mechanism of regulation of necroptosis by cathepsins within macrophage cells. Copyright © 2014 by The American Association of Immunologists, Inc.

Novel phosphate-activated macrophages prevent ectopic calcification by increasing extracellular ATP and pyrophosphate

PubMed Central

Villa-Bellosta, Ricardo; Hamczyk, Magda R.; Andrés, Vicente

2017-01-01

Purpose Phosphorus is an essential nutrient involved in many pathobiological processes. Less than 1% of phosphorus is found in extracellular fluids as inorganic phosphate ion (Pi) in solution. High serum Pi level promotes ectopic calcification in many tissues, including blood vessels. Here, we studied the effect of elevated Pi concentration on macrophage polarization and calcification. Macrophages, present in virtually all tissues, play key roles in health and disease and display remarkable plasticity, being able to change their physiology in response to environmental cues. Methods and results High-throughput transcriptomic analysis and functional studies demonstrated that Pi induces unpolarized macrophages to adopt a phenotype closely resembling that of alternatively-activated M2 macrophages, as revealed by arginine hydrolysis and energetic and antioxidant profiles. Pi-induced macrophages showed an anti-calcifying action mediated by increased availability of extracellular ATP and pyrophosphate. Conclusion We conclude that the ability of Pi-activated macrophages to prevent calcium-phosphate deposition is a compensatory mechanism protecting tissues from hyperphosphatemia-induced pathologic calcification. PMID:28362852

Estimation of the membrane potential of cultured macrophages from the fast potential transient upon microelectrode entry

PubMed Central

Ince, C; Ypey, DL; Van Furth, R; Verveen, AA

1983-01-01

Analysis of membrane potential recordings upon microelectrode impalement of four types of macrophages (cell lines P388D1 and PU5-1.8, cultured mouse peritoneal macrophages, and cultured human monocytes) reveals that these cells have membrane potentials at least two times more negative than sustained potential values (E(s)) frequently reported. Upon microelectrode entry into the cell (P388D1), the recorded potential drops to a peak value (E(p)) (mean -37 mV for 50 cells, range -15 to -70 mV) within 2 ms, after which it decays to a depolarized potential (E(n)) (mean -12 mV) in about 20 ms. Thereafter, the membrane develops one or a series of slow hyperpolarizations before a final sustained membrane potential (E(s)) (mean -14 mV, range -5 to -40) is established. The mean value of the peak of the first hyperpolarization (E(h)) is -30 mV (range -10 to -55 mV). The initial fast peak transient, measured upon microelectrode entry, was first described and analyzed by Lassen et al. (Lassen, U.V., A.M. T. Nielson, L. Pape, and L. O. Simonsen, 1971, J. Membr. Biol. 6:269-288 for other change in the membrane potential from its real value before impalement to a sustained depolarized value. This was shown to be true for macrophages by two-electrode impalements of single cells. Values of E(p), E(n), E(h), E(s), and membrane resistance (R(m)) measured for the other macrophages were similar to those of P388D1. From these results we conclude that E(p) is a better estimate of the true membrane potential of macrophages than E(s), and that the slow hyperpolarizations upon impalement should be regarded as transient repolarizations back to the original membrane potentials. Thus, analysis of the initial fast impalement transient can be a valuable aid in the estimation of the membrane potential of various sorts of small isolated cells by microelectrodes. PMID:6833384

PDT-treated apoptotic cells induce macrophage synthesis NO

NASA Astrophysics Data System (ADS)

Song, S.; Xing, D.; Zhou, F. F.; Chen, W. R.

2009-11-01

Nitric oxide (NO) is a biologically active molecule which has multi-functional in different species. As a second messenger and neurotransmitter, NO is not only an important regulatory factor between cells' information transmission, but also an important messenger in cell-mediated immunity and cytotoxicity. On the other side, NO is involving in some diseases' pathological process. In pathological conditions, the macrophages are activated to produce a large quantity of nitric oxide synthase (iNOS), which can use L-arginine to produce an excessive amount of NO, thereby killing bacteria, viruses, parasites, fungi, tumor cells, as well as in other series of the immune process. In this paper, photofrin-based photodynamic therapy (PDT) was used to treat EMT6 mammary tumors in vitro to induce apoptotic cells, and then co-incubation both apoptotic cells and macrophages, which could activate macrophage to induce a series of cytotoxic factors, especially NO. This, in turn, utilizes macrophages to activate a cytotoxic response towards neighboring tumor cells. These results provided a new idea for us to further study the immunological mechanism involved in damaging effects of PDT, also revealed the important function of the immune effect of apoptotic cells in PDT.

Efficacies of gentamicin-loaded magnetite block ionomer complexes against chronic Brucella melitensis infection

NASA Astrophysics Data System (ADS)

Jain-Gupta, Neeta; Pothayee, Nipon; Pothayee, Nikorn; Tyler, Ronald; Caudell, David L.; Balasubramaniam, Sharavanan; Hu, Nan; Davis, Richey M.; Riffle, Judy S.; Sriranganathan, Nammalwar

2013-11-01

Anionic copolymers can enable intracellular delivery of cationic drugs which otherwise cannot cross cell membrane barriers. We tested the efficacy of gentamicin-loaded magnetite block ionomer complexes (MBICs) against intracellular Brucella melitensis. Anionic block copolymers were used to coat nanomagnetite through adsorption of a portion of anions on the particle surfaces, then the remaining anions were complexed with 30-32 weight percentage of gentamicin. The zeta potential changed from -39 to -13 mV after encapsulation of the drug with complementary charge. The gentamicin-loaded MBICs had intensity average hydrodynamic diameters of 62 nm, while the polymer-coated nanomagnetite particles without drug were 34 nm in size. No toxicity as measured by a MTS assay was observed upon incubation of the MBICs with J774A.1 murine macrophage-like cells. Confocal microscopic images showed that the MBICs were taken up by the macrophages and distributed in the cell cytoplasm and endosomal/lysosomal compartments. Upon treatment with gentamicin-loaded MBICs (3.5 Log10), B. melitensis-infected macrophages showed significantly higher clearance of Brucella compared to the treatment with free g (0.9 Log10). Compared to doxycycline alone, a combination of doxycycline and gentamicin (either free or encapsulated in MBICs) showed significantly higher clearance of B. melitensis from chronically infected mice. Histopathological examination of kidneys from the MBICs-treated mice revealed multifocal infiltration of macrophages containing intracytoplasmic iron (MBICs) in peri-renal adipose. Although MBICs showed similar efficacy as free gentamicin against Brucella in mice, our strategy presents an effective way to deliver higher loads of drugs intracellularly and ability to study the bio-distribution of drug carriers.

Macrophage cell lines derived from major histocompatibility complex II-negative mice

NASA Technical Reports Server (NTRS)

Beharka, A. A.; Armstrong, J. W.; Chapes, S. K.; Spooner, B. S. (Principal Investigator)

1998-01-01

Two bone-marrow-derived macrophage cell lines, C2D and C2Dt, were isolated from major histocompatibility class II negative knock-out mice. The C2D cell line was stabilized by continuous culture in colony-stimulating factor-1 and the C2Dt cell line was transformed with SV40 virus large T antigen. These cells exhibited phenotypic properties of macrophages including morphology and expression of Mac 1 and Mac 2 cell surface molecules. These cells also had comparable growth to the bone-marrow-derived macrophage cell line B6MP102. These new cell lines were not spontaneously cytotoxic and were only capable of modest killing of F5b tumor cells when stimulated with LPS and interferon-gamma, but not when stimulated with LPS alone or with staphylococcal exotoxin. C2D and C2Dt cells phagocytosed labeled Staphylococcus aureus similarly to B6MP102 cells but less well than C2D peritoneal macrophages. These cell lines secreted interleukin-6, but not tumor necrosis factor or nitric oxide in response to LPS or staphlococcal enterotoxins A or B C2D(t) cells were tumorigenic in C2D and C57BL/6J mice but C2D cells were not. These data suggest that macrophage cell lines can be established from bone marrow cells of major histocompatibility complex II-negative mice.

Severe Acinetobacter baumannii Sepsis Is Associated with Elevation of Pentraxin 3

PubMed Central

Ketter, Patrick M.; Guentzel, M. Neal; Schaffer, Beverly; Herzig, Maryanne; Wu, Xiaowu; Montgomery, Robbie K.; Parida, Bijaya K.; Fedyk, Chriselda G.; Yu, Jieh-Juen; Jorgensen, James; Chambers, James P.; Cap, Andrew P.

2014-01-01

Multidrug-resistant Acinetobacter baumannii is among the most prevalent bacterial pathogens associated with trauma-related wound and bloodstream infections. Although septic shock and disseminated intravascular coagulation have been reported following fulminant A. baumannii sepsis, little is known about the protective host immune response to this pathogen. In this study, we examined the role of PTX3, a soluble pattern recognition receptor with reported antimicrobial properties and stored within neutrophil granules. PTX3 production by murine J774a.1 macrophages was assessed following challenge with A. baumannii strains ATCC 19606 and clinical isolates (CI) 77, 78, 79, 80, and 86. Interestingly, only CI strains 79, 80, and 86 induced PTX3 synthesis in murine J774a.1 macrophages, with greatest production observed following CI 79 and 86 challenge. Subsequently, C57BL/6 mice were challenged intraperitoneally with CI 77 and 79 to assess the role of PTX3 in vivo. A. baumannii strain CI 79 exhibited significantly (P < 0.0005) increased mortality, with an approximate 50% lethal dose (LD50) of 105 CFU, while an equivalent dose of CI 77 exhibited no mortality. Plasma leukocyte chemokines (KC, MCP-1, and RANTES) and myeloperoxidase activity were also significantly elevated following challenge with CI 79, indicating neutrophil recruitment/activation associated with significant elevation in serum PTX3 levels. Furthermore, 10-fold-greater PTX3 levels were observed in mouse serum 12 h postchallenge, comparing CI 79 to CI 77 (1,561 ng/ml versus 145 ng/ml), with concomitant severe pathology (liver and spleen) and coagulopathy. Together, these results suggest that elevation of PTX3 is associated with fulminant disease during A. baumannii sepsis. PMID:25001601

Live Candida albicans suppresses production of reactive oxygen species in phagocytes.

PubMed

Wellington, Melanie; Dolan, Kristy; Krysan, Damian J

2009-01-01

Production of reactive oxygen species (ROS) is an important aspect of phagocyte-mediated host responses. Since phagocytes play a crucial role in the host response to Candida albicans, we examined the ability of Candida to modulate phagocyte ROS production. ROS production was measured in the murine macrophage cell line J774 and in primary phagocytes using luminol-enhanced chemiluminescence. J774 cells, murine polymorphonuclear leukocytes (PMN), human monocytes, and human PMN treated with live C. albicans produced significantly less ROS than phagocytes treated with heat-killed C. albicans. Live C. albicans also suppressed ROS production in murine bone marrow-derived macrophages from C57BL/6 mice, but not from BALB/c mice. Live C. albicans also suppressed ROS in response to external stimuli. C. albicans and Candida glabrata suppressed ROS production by phagocytes, whereas Saccharomyces cerevisiae stimulated ROS production. The cell wall is the initial point of contact between Candida and phagocytes, but isolated cell walls from both heat-killed and live C. albicans stimulated ROS production. Heat-killed C. albicans has increased surface exposure of 1,3-beta-glucan, a cell wall component that can stimulate phagocytes. To determine whether surface 1,3-beta-glucan exposure accounted for the difference in ROS production, live C. albicans cells were treated with a sublethal dose of caspofungin to increase surface 1,3-beta-glucan exposure. Caspofungin-treated C. albicans was fully able to suppress ROS production, indicating that suppression of ROS overrides stimulatory signals from 1,3-beta-glucan. These studies indicate that live C. albicans actively suppresses ROS production in phagocytes in vitro, which may represent an important immune evasion mechanism.

Live Candida albicans Suppresses Production of Reactive Oxygen Species in Phagocytes▿ †

PubMed Central

Wellington, Melanie; Dolan, Kristy; Krysan, Damian J.

2009-01-01

Production of reactive oxygen species (ROS) is an important aspect of phagocyte-mediated host responses. Since phagocytes play a crucial role in the host response to Candida albicans, we examined the ability of Candida to modulate phagocyte ROS production. ROS production was measured in the murine macrophage cell line J774 and in primary phagocytes using luminol-enhanced chemiluminescence. J774 cells, murine polymorphonuclear leukocytes (PMN), human monocytes, and human PMN treated with live C. albicans produced significantly less ROS than phagocytes treated with heat-killed C. albicans. Live C. albicans also suppressed ROS production in murine bone marrow-derived macrophages from C57BL/6 mice, but not from BALB/c mice. Live C. albicans also suppressed ROS in response to external stimuli. C. albicans and Candida glabrata suppressed ROS production by phagocytes, whereas Saccharomyces cerevisiae stimulated ROS production. The cell wall is the initial point of contact between Candida and phagocytes, but isolated cell walls from both heat-killed and live C. albicans stimulated ROS production. Heat-killed C. albicans has increased surface exposure of 1,3-β-glucan, a cell wall component that can stimulate phagocytes. To determine whether surface 1,3-β-glucan exposure accounted for the difference in ROS production, live C. albicans cells were treated with a sublethal dose of caspofungin to increase surface 1,3-β-glucan exposure. Caspofungin-treated C. albicans was fully able to suppress ROS production, indicating that suppression of ROS overrides stimulatory signals from 1,3-β-glucan. These studies indicate that live C. albicans actively suppresses ROS production in phagocytes in vitro, which may represent an important immune evasion mechanism. PMID:18981256

Monocyte/Macrophage-derived IGF-1 Orchestrates Murine Skeletal Muscle Regeneration and Modulates Autocrine Polarization

PubMed Central

Tonkin, Joanne; Temmerman, Lieve; Sampson, Robert D; Gallego-Colon, Enrique; Barberi, Laura; Bilbao, Daniel; Schneider, Michael D; Musarò, Antonio; Rosenthal, Nadia

2015-01-01

Insulin-like growth factor 1 (IGF-1) is a potent enhancer of tissue regeneration, and its overexpression in muscle injury leads to hastened resolution of the inflammatory phase. Here, we show that monocytes/macrophages constitute an important initial source of IGF-1 in muscle injury, as conditional deletion of the IGF-1 gene specifically in mouse myeloid cells (ϕIGF-1 CKO) blocked the normal surge of local IGF-1 in damaged muscle and significantly compromised regeneration. In injured muscle, Ly6C+ monocytes/macrophages and CD206+ macrophages expressed equivalent IGF-1 levels, which were transiently upregulated during transition from the inflammation to repair. In injured ϕIGF-1 CKO mouse muscle, accumulation of CD206+ macrophages was impaired, while an increase in Ly6C+ monocytes/macrophages was favored. Transcriptional profiling uncovered inflammatory skewing in ϕIGF-1 CKO macrophages, which failed to fully induce a reparative gene program in vitro or in vivo, revealing a novel autocrine role for IGF-1 in modulating murine macrophage phenotypes. These data establish local macrophage-derived IGF-1 as a key factor in inflammation resolution and macrophage polarization during muscle regeneration. PMID:25896247

[Macrophage activation in atherosclerosis. Message 1: Activation of macrophages normally and in atherosclerotic lesions].

PubMed

Nikiforov, N G; Kornienko, V Y; Karagodin, V P; Orekhov, A N

2015-01-01

Macrophages play important role in initiation and progression of inflammation in atherosclerosis. Plaque macrophages were shown to exhibit a phenotypic range that is intermediate between two extremes, M1 (proinflammatory) and M2 (anti-inflammatory). Indeed, in atherosclerosis, macrophages demonstrate phenotypic plasticity to rapidly adjust to changing microenvironmental conditions. In plaque macrophages demonstrate different phenotypes, and besides macrophage phenotypes could be changed. Phenotypes M1, M2, M4, Mhem, HA-mac, M(Hb) u Mox are described in the article. Ability of macrophages change their phenotype also considered.

Melanocortin 1 Receptor Signaling Regulates Cholesterol Transport in Macrophages.

PubMed

Rinne, Petteri; Rami, Martina; Nuutinen, Salla; Santovito, Donato; van der Vorst, Emiel P C; Guillamat-Prats, Raquel; Lyytikäinen, Leo-Pekka; Raitoharju, Emma; Oksala, Niku; Ring, Larisa; Cai, Minying; Hruby, Victor J; Lehtimäki, Terho; Weber, Christian; Steffens, Sabine

2017-07-04

The melanocortin 1 receptor (MC1-R) is expressed by monocytes and macrophages, where it exerts anti-inflammatory actions on stimulation with its natural ligand α-melanocyte-stimulating hormone. The present study was designed to investigate the specific role of MC1-R in the context of atherosclerosis and possible regulatory pathways of MC1-R beyond anti-inflammation. Human and mouse atherosclerotic samples and primary mouse macrophages were used to study the regulatory functions of MC1-R. The impact of pharmacological MC1-R activation on atherosclerosis was assessed in apolipoprotein E-deficient mice. Characterization of human and mouse atherosclerotic plaques revealed that MC1-R expression localizes in lesional macrophages and is significantly associated with the ATP-binding cassette transporters ABCA1 and ABCG1, which are responsible for initiating reverse cholesterol transport. Using bone marrow-derived macrophages, we observed that α-melanocyte-stimulating hormone and selective MC1-R agonists similarly promoted cholesterol efflux, which is a counterregulatory mechanism against foam cell formation. Mechanistically, MC1-R activation upregulated the levels of ABCA1 and ABCG1. These effects were accompanied by a reduction in cell surface CD36 expression and in cholesterol uptake, further protecting macrophages from excessive lipid accumulation. Conversely, macrophages deficient in functional MC1-R displayed a phenotype with impaired efflux and enhanced uptake of cholesterol. Pharmacological targeting of MC1-R in atherosclerotic apolipoprotein E-deficient mice reduced plasma cholesterol levels and aortic CD36 expression and increased plaque ABCG1 expression and signs of plaque stability. Our findings identify a novel role for MC1-R in macrophage cholesterol transport. Activation of MC1-R confers protection against macrophage foam cell formation through a dual mechanism: It prevents cholesterol uptake while concomitantly promoting ABCA1- and ABCG1-mediated reverse

Akt, mTOR and NF-κB pathway activation in Treponema pallidum stimulates M1 macrophages.

PubMed

Lin, Li-Rong; Gao, Zheng-Xiang; Lin, Yong; Zhu, Xiao-Zhen; Liu, Wei; Liu, Dan; Gao, Kun; Tong, Man-Li; Zhang, Hui-Lin; Liu, Li-Li; Xiao, Yao; Niu, Jian-Jun; Liu, Fan; Yang, Tian-Ci

2018-06-01

The polarization of macrophages and the molecular mechanism involved during the early process of syphilis infection remain unknown. This study was conducted to explore the influence of Treponema pallidum (T. pallidum) treatment on macrophage polarization and the Akt-mTOR-NFκB signaling pathway mechanism involved in this process. M0 macrophages derived from the phorbol-12-myristate-13-acetate-induced human acute monocytic leukemia cell line THP-1 were cultured with T. pallidum. T. pallidum induced inflammatory cytokine (IL-1β and TNF-α) expression in a dose- and time-dependent manner. However IL-10 cytokine expression decreased at the mRNA and protein levels. Additionally, the expression of the M1 surface marker iNOS was up-regulated with incubation time, and the expression of the M2 surface marker CD206 was low (vs. PBS treated macrophages, P < 0.001) and did not fluctuate over 12 h. Further studies revealed that Akt-mTOR-NFκB pathway proteins, including p-Akt, p-mTOR, p-S6, p-p65, and p-IκBα, were significantly higher in the T. pallidum-treated macrophages than in the PBS-treated macrophages (P < 0.05). In addition, inflammatory cytokine expression was suppressed in T. pallidum-induced M1 macrophages pretreated with LY294002 (an Akt-specific inhibitor) or PDTC (an NF-κB inhibitor), while inflammatory cytokine levels increased in T. pallidum-induced M1 macrophages pretreated with rapamycin (an mTOR inhibitor). These findings revealed that T. pallidum promotes the macrophage transition to pro-inflammatory M1 macrophages in vitro. The present study also provides evidence that Akt, mTOR and NF-κB pathway activation in T. pallidum stimulates M1 macrophages. This study provides novel insights into the innate immune response to T. pallidum infection. Copyright © 2018. Published by Elsevier B.V.

Granulocyte-Macrophage Colony Stimulatory Factor Enhances the Pro-Inflammatory Response of Interferon-γ-Treated Macrophages to Pseudomonas aeruginosa Infection

PubMed Central

Singh, Sonali; Barr, Helen; Liu, Yi-Chia; Robins, Adrian; Heeb, Stephan; Williams, Paul; Fogarty, Andrew; Cámara, Miguel; Martínez-Pomares, Luisa

2015-01-01

Pseudomonas aeruginosa is an opportunistic pathogen that can cause severe infections at compromised epithelial surfaces, such those found in burns, wounds, and in lungs damaged by mechanical ventilation or recurrent infections, particularly in cystic fibrosis (CF) patients. CF patients have been proposed to have a Th2 and Th17-biased immune response suggesting that the lack of Th1 and/or over exuberant Th17 responses could contribute to the establishment of chronic P. aeruginosa infection and deterioration of lung function. Accordingly, we have observed that interferon (IFN)-γ production by peripheral blood mononuclear cells from CF patients positively correlated with lung function, particularly in patients chronically infected with P. aeruginosa. In contrast, IL-17A levels tended to correlate negatively with lung function with this trend becoming significant in patients chronically infected with P. aeruginosa. These results are in agreement with IFN-γ and IL-17A playing protective and detrimental roles, respectively, in CF. In order to explore the protective effect of IFN-γ in CF, the effect of IFN-γ alone or in combination with granulocyte-macrophage colony-stimulating factor (GM-CSF), on the ability of human macrophages to control P. aeruginosa growth, resist the cytotoxicity induced by this bacterium or promote inflammation was investigated. Treatment of macrophages with IFN-γ, in the presence and absence of GM-CSF, failed to alter bacterial growth or macrophage survival upon P. aeruginosa infection, but changed the inflammatory potential of macrophages. IFN-γ caused up-regulation of monocyte chemoattractant protein-1 (MCP-1) and TNF-α and down-regulation of IL-10 expression by infected macrophages. GM-CSF in combination with IFN-γ promoted IL-6 production and further reduction of IL-10 synthesis. Comparison of TNF-α vs. IL-10 and IL-6 vs. IL-10 ratios revealed the following hierarchy in regard to the pro-inflammatory potential of human macrophages

Granulocyte-macrophage colony stimulatory factor enhances the pro-inflammatory response of interferon-γ-treated macrophages to Pseudomonas aeruginosa infection.

PubMed

Singh, Sonali; Barr, Helen; Liu, Yi-Chia; Robins, Adrian; Heeb, Stephan; Williams, Paul; Fogarty, Andrew; Cámara, Miguel; Martínez-Pomares, Luisa

2015-01-01

Pseudomonas aeruginosa is an opportunistic pathogen that can cause severe infections at compromised epithelial surfaces, such those found in burns, wounds, and in lungs damaged by mechanical ventilation or recurrent infections, particularly in cystic fibrosis (CF) patients. CF patients have been proposed to have a Th2 and Th17-biased immune response suggesting that the lack of Th1 and/or over exuberant Th17 responses could contribute to the establishment of chronic P. aeruginosa infection and deterioration of lung function. Accordingly, we have observed that interferon (IFN)-γ production by peripheral blood mononuclear cells from CF patients positively correlated with lung function, particularly in patients chronically infected with P. aeruginosa. In contrast, IL-17A levels tended to correlate negatively with lung function with this trend becoming significant in patients chronically infected with P. aeruginosa. These results are in agreement with IFN-γ and IL-17A playing protective and detrimental roles, respectively, in CF. In order to explore the protective effect of IFN-γ in CF, the effect of IFN-γ alone or in combination with granulocyte-macrophage colony-stimulating factor (GM-CSF), on the ability of human macrophages to control P. aeruginosa growth, resist the cytotoxicity induced by this bacterium or promote inflammation was investigated. Treatment of macrophages with IFN-γ, in the presence and absence of GM-CSF, failed to alter bacterial growth or macrophage survival upon P. aeruginosa infection, but changed the inflammatory potential of macrophages. IFN-γ caused up-regulation of monocyte chemoattractant protein-1 (MCP-1) and TNF-α and down-regulation of IL-10 expression by infected macrophages. GM-CSF in combination with IFN-γ promoted IL-6 production and further reduction of IL-10 synthesis. Comparison of TNF-α vs. IL-10 and IL-6 vs. IL-10 ratios revealed the following hierarchy in regard to the pro-inflammatory potential of human macrophages

The lethal effects of cytokine-induced nitric oxide on cardiac myocytes are blocked by nitric oxide synthase antagonism or transforming growth factor beta.

PubMed Central

Pinsky, D J; Cai, B; Yang, X; Rodriguez, C; Sciacca, R R; Cannon, P J

1995-01-01

Inducible nitric oxide (NO) produced by macrophages is cytotoxic to invading organisms and has an important role in host defense. Recent studies have demonstrated inducible NO production within the heart, and that cytokine-induced NO mediates alterations in cardiac contractility, but the cytotoxic potential of nitric oxide with respect to the heart has not been defined. To evaluate the role of inducible nitric oxide synthase (iNOS) on cardiac myocyte cytotoxicity, we exposed adult rat cardiac myocytes to either cytokines alone or to activated J774 macrophages in coculture. Increased expression of both iNOS message and protein was seen in J774 macrophages treated with IFN gamma and LPS and cardiac myocytes treated with TNF-alpha, IL-1 beta, and IFN gamma. Increased NO synthesis was confirmed in both the coculture and isolated myocyte preparations by increased nitrite production. Increased NO synthesis was associated with a parallel increase in myocyte death as measured by CPK release into the culture medium as well as by loss of membrane integrity, visualized by trypan blue staining. Addition of the competitive NO synthase inhibitor L-NMMA to the culture medium prevented both the increased nitrite production and the cytotoxicity observed after cytokine treatment in both the isolated myocyte and the coculture experiments. Because transforming growth-factor beta modulates iNOS expression in other cell types, we evaluated its effects on cardiac myocyte iNOS expression and NO-mediated myocyte cytotoxicity. TGF-beta reduced expression of cardiac myocyte iNOS message and protein, reduced nitrite production, and reduced NO-mediated cytotoxicity in parallel. Taken together, these experiments show the cytotoxic potential of endogenous NO production within the heart, and suggest a role for TGF-beta or NO synthase antagonists to mute these lethal effects. These findings may help explain the cardiac response to sepsis or allograft rejection, as well as the progression of

Nanonization increases the antileishmanial efficacy of amphotericin B: an ex vivo approach.

PubMed

Manandhar, Krishna Das; Yadav, Thakur Prasad; Prajapati, Vijay Kumar; Basukala, Om; Aganja, Ram Prasad; Dude, Anuradha; Shrivastav, Onkar Nath; Sundar, Shyam

2014-01-01

With widespread resistance to pentavalent antimonial in the endemic eastern terai belt of Nepal and Bihar, India, Amphotericin B deoxycholate is now the first-line antileishmanial drug for the treatment of visceral leishmaniasis (VL). However, universal occurrence of infusion-related fever and rigors with amphotericin B (AmB), occasional serious life-threatening toxicities like cardiotoxicity, anaphylaxis, hypokalemia, and nephrotoxicity are major barriers to its use in areas with limited medical facilities. Liposomal amphotericins, however, are devoid of adverse effects, high cost makes it unaffordable. We had formulated nanoparticles (10-20 nm) from amphotericin B deoxycholate (1-2 μm) applying high pressure (150 atm) milling homogenization in argon atmosphere and tested its ex vivo efficacy in Leishmania infected J774A cell line and peritoneal macrophage. The ex vivo ED50 for intracellular amastigotes in peritoneal macrophage by nano-amphotericin was 0.0027 ± 0.001 μg/mL which was significantly less (p = 0.0029) than the required dose of amphotericin B (0.0426 ± 0.003 μg/mL). Similarly, in J774A cell line, 50 % of intracellular amastigotes were cleared by 0.0038 ± 0.001 μg/mL of nano-amphotericin while the dose was a bit more for AmB (0.0196 ± 0.001 μg/mL) illustrating the significant difference (p value, 0.0122). The nanoformulation has also shown high efficacy (ED50, 0.0028-0.0035 μg/mL) in inhibition of infected macrophage count. The new formulation accumulated to spleen, the targeted organ, 7 days after inoculation of drug to the infected hamster as traced in vivo by TEM convincing it as potential drug. Given a favorable safety profile and very low cost of production contemplated, it may prove to be a feasible alternative for conventional amphotericin B.

[Macrophages in human semen].

PubMed

Bouvet, Beatriz Reina; Brufman, Adriana Silvia; Paparella, Cecilia Vicenta; Feldman, Rodolfo Nestor; Gatti, Vanda Nora; Solis, Edita Amalia

2003-11-01

To investigate the presence of macrophages in human semen samples and the function they carry out in the seminal fluid. Their presence was studied in relation to spermatic morphology, percentage of spermatozoids with native DNA, and presence of antispermatic antibodies. The work was performed with semen samples from 31 unfertile males from 63 couples in which the "female factor" was ruled out as the cause of infertility. Sperm study according to WHO (1992) was carried out in all samples, in addition to: DNA study with acridine orange as fluorocrom, macrophage concentration by neutral red in a Neubauer camera, and detection of antispermatic antibodies with a mixed agglutination test (TAC II) (validated with Mar Screen-Fertility technologies). Sperm morphology was evaluated by Papanicolaou test. 19/31 selected sperm samples (61.3%) showed increased concentration of macrophages, 13 of them (41.9%) with denaturalized DNA, and 8 (25.8%) abnormal morphology. Six samples showed increased macrophage concentration and predominance of native DNA, whereas 11 samples showed increased macrophages and abnormal morphology. Among 18 (58.1%) samples showing antispermatic antibodies 14 (77.7%) had an increased concentration of macrophages. Statistical analysis resulted in a high correlation between macrophage concentration and increased percentage of spermatozoids with denaturalized DNA (p < 0.05). An increased concentration of macrophages is associated with the presence of antispermatic antibodies (p < 0.05). There was not evidence of significant association between concentration of macrophages and percentage of morphologically normal spermatozoids (p < 0.05). We can conclude that macrophages are present in human semen and participate in immunovigilance contributing to improve the seminal quality.

Nanoparticles Containing Curcumin Useful for Suppressing Macrophages In Vivo in Mice

PubMed Central

Amano, Chie; Minematsu, Hideki; Fujita, Kazuyo; Iwashita, Shinki; Adachi, Masaki; Igarashi, Koichi; Hinuma, Shuji

2015-01-01

To explore a novel method using liposomes to suppress macrophages, we screened food constituents through cell culture assays. Curcumin was one of the strongest compounds exhibiting suppressive effects on macrophages. We subsequently tried various methods to prepare liposomal curcumin, and eventually succeeded in preparing liposomes with sufficient amounts of curcumin to suppress macrophages by incorporating a complex of curcumin and bovine serum albumin. The diameter of the resultant nanoparticles, the liposomes containing curcumin, ranged from 60 to 100 nm. Flow cytometric analyses revealed that after intraperitoneal administration of the liposomes containing curcumin into mice, these were incorporated mainly by macrophages positive for F4/80, CD36, and CD11b antigens. Peritoneal cells prepared from mice injected in vivo with the liposomes containing curcumin apparently decreased interleukin-6-producing activities. Major changes in body weight and survival rates in the mice were not observed after administrating the liposomes containing curcumin. These results indicate that the liposomes containing curcumin are safe and useful for the selective suppression of macrophages in vivo in mice. PMID:26361331

Changing pattern of the subcellular distribution of erythroblast macrophage protein (Emp) during macrophage differentiation.

PubMed

Soni, Shivani; Bala, Shashi; Kumar, Ajay; Hanspal, Manjit

2007-01-01

Erythroblast macrophage protein (Emp) mediates the attachment of erythroid cells to macrophages and is required for normal differentiation of both cell lineages. In erythroid cells, Emp is believed to be involved in nuclear extrusion, however, its role in macrophage differentiation is unknown. Information on the changes in the expression level and subcellular distribution of Emp in differentiating macrophages is essential for understanding the function of Emp. Macrophages of varying maturity were examined by immunofluorescence microscopy and biochemical methods. Our data show that Emp is expressed in all stages of maturation, but its localization pattern changes dramatically during maturation: in immature macrophages, a substantial fraction of Emp is associated with the nuclear matrix, whereas in more mature cells, Emp is expressed largely at cell surface. Pulse-chase experiments show that nascent Emp migrates intracellularly from the cytoplasm to the plasma membrane more efficiently in mature macrophages than in immature cells. Incubation of erythroid cells with macrophages in culture shows that erythroid cells attach to mature macrophages but not to immature macrophage precursors. Together, our data show that the temporal and spatial expression of Emp correlates with its role in erythroblastic island formation and suggest that Emp may be involved in multiple cellular functions.

*CHANGING PATTERN OF THE SUBCELLULAR DISTRIBUTION OF ERYTHROBLAST MACROPHAGE PROTEIN (EMP) DURING MACROPHAGE DIFFERENTIATION

PubMed Central

Soni, Shivani; Bala, Shashi; Kumar, Ajay; Hanspal, Manjit

2007-01-01

Erythroblast macrophage protein (Emp), mediates the attachment of erythroid cells to macrophages, and is required for normal differentiation of both cell lineages. In erythroid cells Emp is believed to be involved in nuclear extrusion however, its role in macrophage differentiation is unknown. Information on the changes in the expression level and subcellular distribution of Emp in differentiating macrophages is essential for understanding the function of Emp. Macrophages of varying maturity were examined by immunofluorescence microscopy and biochemical methods. Our data shows that Emp is expressed in all stages of maturation, but its localization pattern changes dramatically during maturation: in immature macrophages, a substantial fraction of Emp is associated with the nuclear matrix, whereas in more mature cells, Emp is expressed largely at cell surface. Pulse-chase experiments show that nascent Emp migrates intracellularly from the cytoplasm to the plasma membrane more efficiently in mature macrophages than in immature cells. Incubation of erythroid cells with macrophages in culture show that erythroid cells attach to mature macrophages but not to immature macrophage precursors. Together, our data shows that the temporal and spatial expression of Emp correlates with its role in erythroblastic island formation, and suggests that Emp may be involved in multiple cellular functions. PMID:17071116

In vitro evaluation of newly synthesised [1,2,4]triazolo[1,5a]pyrimidine derivatives against Trypanosoma cruzi, Leishmania donovani and Phytomonas staheli.

PubMed

Luque, F; Fernández-Ramos, C; Entrala, E; Rosales, M J; Navarro, J A; Romero, M A; Salas, J M; Sánchez-Moreno, M

2000-05-01

The antiprotozoal activity of newly synthesised compounds, all [1,2,4]triazolo [1,5a]pyrimidine derivatives, was tested against the protozoan parasites Trypanosoma cruzi, Leishmania donovani and Phytotmonas staheli. Six of these compounds significantly inhibited in vitro cell growth of the epimastigote forms of T. cruzi, and the promastigote forms of L. donovani and P. staheli. Some of the compounds reached complete growth inhibition at 1 microg/ml for 48 h of parasite/drug interaction. None of the compounds tested showed significant toxicity against cells of Aedes albopictus, mouse macrophages J-774A.1 and Lycopersicum esculentum at dosages five times greater than used against parasites.

Macrophages Exhibit a Large Repertoire of Activation States via Multiple Mechanisms of Macrophage-activating Factors.

PubMed

Sumiya, Y U; Inoue, Takahiro; Ishikawa, Mami; Inui, Toshio; Kuchiike, Daisuke; Kubo, Kentaro; Uto, Yoshihiro; Nishikata, Takahito

2016-07-01

Macrophages are important components of human defense systems and consequently key to antitumor immunity. Human-serum macrophage activation factor (serum MAF) can activate macrophages, making it a promising reagent for anticancer therapy. We established four different macrophage subtypes through introduction of different culture conditions to THP-1- and U937-derived macrophages. We assessed phagocytic activity to understand subtype responses to typical macrophage activation factors (MAFs) and the activation mechanisms of serum MAF. All four macrophage subtypes differed in their response to all MAFs. Moreover, serum MAF had two different activation mechanisms: N-acetylgalactosamine (GalNAc)-dependent and GalNAc-independent. Macrophage activation states and mechanisms are heterogeneous. Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

Cell Elasticity Determines Macrophage Function

PubMed Central

Patel, Naimish R.; Bole, Medhavi; Chen, Cheng; Hardin, Charles C.; Kho, Alvin T.; Mih, Justin; Deng, Linhong; Butler, James; Tschumperlin, Daniel; Fredberg, Jeffrey J.; Krishnan, Ramaswamy; Koziel, Henry

2012-01-01

Macrophages serve to maintain organ homeostasis in response to challenges from injury, inflammation, malignancy, particulate exposure, or infection. Until now, receptor ligation has been understood as being the central mechanism that regulates macrophage function. Using macrophages of different origins and species, we report that macrophage elasticity is a major determinant of innate macrophage function. Macrophage elasticity is modulated not only by classical biologic activators such as LPS and IFN-γ, but to an equal extent by substrate rigidity and substrate stretch. Macrophage elasticity is dependent upon actin polymerization and small rhoGTPase activation, but functional effects of elasticity are not predicted by examination of gene expression profiles alone. Taken together, these data demonstrate an unanticipated role for cell elasticity as a common pathway by which mechanical and biologic factors determine macrophage function. PMID:23028423

Targeting Androgen Receptor to Suppress Macrophage-induced EMT and Benign Prostatic Hyperplasia (BPH) Development

PubMed Central

Lu, Tianjing; Lin, Wen-Jye; Izumi, Kouji; Wang, Xiaohai; Xu, Defeng; Fang, Lei-Ya; Li, Lei; Jiang, Qi

2012-01-01

Early studies suggested macrophages might play roles in inflammation-associated benign prostatic hyperplasia (BPH) development, yet the underlying mechanisms remain unclear. Here we first showed that CD68+ macrophages were identified in both epithelium and the stromal area of human BPH tissues. We then established an in vitro co-culture model with prostate epithelial and macrophage cell lines to study the potential impacts of infiltrating macrophages in the BPH development and found that co-culturing prostate epithelial cells with macrophages promoted migration of macrophages. In a three-dimensional culture system, the sphere diameter of BPH-1 prostate cells was significantly increased during coculture with THP-1 macrophage cells. Mechanism dissection suggested that expression levels of epithelial-mesenchymal transition (EMT) markers, such as N-cadherin, Snail, and TGF-β2, were increased, and administration of anti-TGF-β2 neutralizing antibody during co-culture suppressed the EMT and THP-1-mediated growth of BPH-1 cells, suggesting THP-1 might go through EMT to influence the BPH development and progression. Importantly, we found that modulation of androgen receptor (AR) in BPH-1 and mPrE cells significantly increased THP-1 and RAW264.7 cell migration, respectively, and enhanced expression levels of EMT markers, suggesting that AR in prostate epithelial cells might play a role in promoting macrophage-mediated EMT in prostate epithelial cells. Silencing AR function via an AR degradation enhancer, ASC-J9, decreased the macrophage migration to BPH-1 cells and suppressed EMT marker expression. Together, these results provide the first evidence to demonstrate that prostate epithelial AR function is important for macrophage-mediated EMT and proliferation of prostate epithelial cells, which represents a previously unrecognized role of AR in the cross-talk between macrophages and prostate epithelial cells. These results may provide new insights for a new therapeutic

Targeting androgen receptor to suppress macrophage-induced EMT and benign prostatic hyperplasia (BPH) development.

PubMed

Lu, Tianjing; Lin, Wen-Jye; Izumi, Kouji; Wang, Xiaohai; Xu, Defeng; Fang, Lei-Ya; Li, Lei; Jiang, Qi; Jin, Jie; Chang, Chawnshang

2012-10-01

Early studies suggested macrophages might play roles in inflammation-associated benign prostatic hyperplasia (BPH) development, yet the underlying mechanisms remain unclear. Here we first showed that CD68(+) macrophages were identified in both epithelium and the stromal area of human BPH tissues. We then established an in vitro co-culture model with prostate epithelial and macrophage cell lines to study the potential impacts of infiltrating macrophages in the BPH development and found that co-culturing prostate epithelial cells with macrophages promoted migration of macrophages. In a three-dimensional culture system, the sphere diameter of BPH-1 prostate cells was significantly increased during coculture with THP-1 macrophage cells. Mechanism dissection suggested that expression levels of epithelial-mesenchymal transition (EMT) markers, such as N-cadherin, Snail, and TGF-β2, were increased, and administration of anti-TGF-β2 neutralizing antibody during co-culture suppressed the EMT and THP-1-mediated growth of BPH-1 cells, suggesting THP-1 might go through EMT to influence the BPH development and progression. Importantly, we found that modulation of androgen receptor (AR) in BPH-1 and mPrE cells significantly increased THP-1 and RAW264.7 cell migration, respectively, and enhanced expression levels of EMT markers, suggesting that AR in prostate epithelial cells might play a role in promoting macrophage-mediated EMT in prostate epithelial cells. Silencing AR function via an AR degradation enhancer, ASC-J9, decreased the macrophage migration to BPH-1 cells and suppressed EMT marker expression. Together, these results provide the first evidence to demonstrate that prostate epithelial AR function is important for macrophage-mediated EMT and proliferation of prostate epithelial cells, which represents a previously unrecognized role of AR in the cross-talk between macrophages and prostate epithelial cells. These results may provide new insights for a new therapeutic

Impact of a 1-year lifestyle modification program on cholesterol efflux capacities in men with abdominal obesity and dyslipidemia.

PubMed

Boyer, Marjorie; Mitchell, Patricia L; Poirier, Dr Paul; Alméras, Natalie; Tremblay, Angelo; Bergeron, Jean; Despres, Jean-Pierre; Arsenault, Benoit J

2018-06-05

Cholesterol efflux capacities (CECs) are negatively associated with cardiovascular disease risk, irrespective of plasma high-density lipoprotein (HDL) cholesterol levels. Whether interventions targeting lifestyle improve HDL-CECs is unknown. Our objective was to determine whether improving dietary quality and increasing physical activity levels improves HDL-CECs in abdominally obese men with dyslipidemia. Our study sample included men (488.5 years) with an elevated waist circumference ({greater than or equal to}90 cm) associated with dyslipidemia (triglycerides {greater than or equal to}1.69 and/or HDL cholesterol <1.03 mmol/l); 113 men completed a 1-year intervention, consisting of a healthy eating and physical activity/exercise program and 32 were included in a control group. An oral lipid tolerance test (OLTT) was performed in a subsample of 28 men who completed the intervention and blood was collected every 2 hours during 8 hours. HDL-CECs were measured using 3 H cholesterol labeled J774 macrophages and HepG2 hepatocytes. The lifestyle modification program led to an overall improvement in the cardiometabolic risk profile, increases in J774-HDL-CEC by 14.1% (+0.881.09%, p<0.0001), HepG2-HDL-CEC by 3.4% (+0.170.75%, p=0.01), HDL-C and apolipoprotein A-1 levels (13.5%, p<0.0001 and 14.9%, p<0.0001, respectively). J774-HDL-CECs and HepG2-HDL-CECs did not change in the control group. The best predictor for changes in HDL-CEC was Apo A1 level. The lifestyle modification program also improved HDL-CECs response in postprandial lipemia during an OLTT. HDL-CEC did not change during the OLTT. Our results suggest that increasing physical activity levels and improving diet quality can have a positive impact on both HDL quantity and quality in abdominally obese men with dyslipidemia.

Localization of azithromycin in Toxoplasma gondii-infected cells.

PubMed Central

Schwab, J C; Cao, Y; Slowik, M R; Joiner, K A

1994-01-01

Agents effective against intracellular pathogens must enter infected cells, crossing vacuolar membranes surrounding the organisms and then penetrating into the microbe and localizing to the microbial target site. We have characterized these parameters for azithromycin entry into Toxoplasma gondii-infected Chinese hamster ovary cells and murine macrophage-like J774 cells. Azithromycin uptake into infected host cells was concentrative and was dependent upon proton gradients. Subcellular fractionation of azithromycin-loaded infected CHO cells demonstrated > 95% intracellular drug in host cell lysosomes and cytosol, with < 5% associated with the parasite. Uptake of azithromycin into the T. gondii vacuole increased if parasites were coated with antibody prior to internalization by murine J774 cells, conditions which result in the formation of acidified phagolysosomes. No redistribution or retention of azithromycin in the parasite was observed when drug efflux from antibiotic-loaded infected CHO cells was monitored. Azithromycin entry into extracellular T. gondii was concentrative, was temperature and pH dependent, and was not different when azithromycin-sensitive and -resistant parasites were compared. These results demonstrate that azithromycin concentrates primarily in acidified compartments in parasites and host cells. The high concentration of azithromycin within these compartments may not be biologically relevant to inhibition of intracellular parasite growth by this agent. PMID:7979295

Antileishmanial pharmacomodulation in 8-nitroquinolin-2(1H)-one series.

PubMed

Kieffer, Charline; Cohen, Anita; Verhaeghe, Pierre; Paloque, Lucie; Hutter, Sébastien; Castera-Ducros, Caroline; Laget, Michèle; Rault, Sylvain; Valentin, Alexis; Rathelot, Pascal; Azas, Nadine; Vanelle, Patrice

2015-05-15

An antileishmanial pharmacomodulation at position 4 of 8-nitroquinolin-2(1H)-one was conducted by using the Sonogashira and Suzuki-Miyaura coupling reactions. A series of 25 derivatives was tested in vitro on the promastigote stage of Leishmania donovani along with an in vitro cytotoxicity evaluation on the human HepG2 cell line. Only the derivatives bearing a phenyl moiety at position 4 of the quinoline ring displayed interesting biologic profile, when the phenyl moiety was substituted at the para position by a Br or Cl atom, or by a CF3 group. Among them, molecules 17 and 19 were the most selective and were then tested in vitro on the intracellular amastigote stage of both L. donovani and Leishmania infantum, in parallel with complementary in vitro cytotoxicity assays on the macrophage cell lines THP-1 and J774A.1. Molecule 19 showed no activity on the amastigote stages of the parasites and some cytotoxicity on the J774A.1 cell line while molecule 17, less cytotoxic than 19, showed anti-amastigote activity in L. infantum, being 3 times less active than miltefosine but more active and selective than pentamidine. Nevertheless, hit-molecule 17 did not appear as selective as the parent compound. Copyright © 2015 Elsevier Ltd. All rights reserved.

Identification of a Novel Pathway of Transforming Growth Factor-β1 Regulation by Extracellular NAD+ in Mouse Macrophages

PubMed Central

Zamora, Ruben; Azhar, Nabil; Namas, Rajaie; Metukuri, Mallikarjuna R.; Clermont, Thierry; Gladstone, Chase; Namas, Rami A.; Hermus, Linda; Megas, Cristina; Constantine, Gregory; Billiar, Timothy R.; Fink, Mitchell P.; Vodovotz, Yoram

2012-01-01

Extracellular β-nicotinamide adenine dinucleotide (NAD+) is anti-inflammatory. We hypothesized that NAD+ would modulate the anti-inflammatory cytokine Transforming Growth Factor (TGF)-β1. Indeed, NAD+ led to increases in both active and latent cell-associated TGF-β1 in RAW 264.7 mouse macrophages as well as in primary peritoneal macrophages isolated from both C3H/HeJ (TLR4-mutant) and C3H/HeOuJ (wild-type controls for C3H/HeJ) mice. NAD+ acts partially via cyclic ADP-ribose (cADPR) and subsequent release of Ca2+. Treatment of macrophages with the cADPR analog 3-deaza-cADPR or Ca2+ ionophores recapitulated the effects of NAD+ on TGF-β1, whereas the cADPR antagonist 8-Br-cADPR, Ca2+ chelation, and antagonism of L-type Ca2+ channels suppressed these effects. The time and dose effects of NAD+ on TGF-β1 were complex and could be modeled both statistically and mathematically. Model-predicted levels of TGF-β1 protein and mRNA were largely confirmed experimentally but also suggested the presence of other mechanisms of regulation of TGF-β1 by NAD+. Thus, in vitro and in silico evidence points to NAD+ as a novel modulator of TGF-β1. PMID:22829588

Global Secretome Characterization of Herpes Simplex Virus 1-Infected Human Primary Macrophages

PubMed Central

Miettinen, Juho J.; Matikainen, Sampsa

2012-01-01

Herpes simplex virus 1 (HSV-1) is a common pathogen infecting the majority of people worldwide at some stage in their lives. The early host response to viral infection is initiated by the cells of the innate immune response, including macrophages. Here, we have characterized the secretome of HSV-1-infected human primary macrophages using high-throughput quantitative proteomics. We identified and quantified 516 distinct human proteins with high confidence from the macrophage secretome upon HSV-1 infection, and the secretion of 411 proteins was >2-fold increased upon beta interferon (IFN-β) priming and/or HSV-1 infection. Bioinformatics analysis of the secretome data revealed that most of the secreted proteins were intracellular, and almost 80% of the proteins whose secretion increased more than 2-fold were known exosomal proteins. This strongly suggests that nonclassical, vesicle-mediated protein secretion is activated in IFN-β-primed and HSV-1-infected macrophages. Proteins related to immune and inflammatory responses, interferon-induced proteins, and endogenous danger signal proteins were efficiently secreted upon IFN-β priming and HSV-1 infection. The secreted IFN-induced proteins include interferon-induced tetratricopeptide protein 2 (IFIT2), IFIT3, signal transducer and activator of transcription 1 (STAT1), and myxovirus resistance protein A (MxA), implicating that these proteins also have important extracellular antiviral functions. Proinflammatory cytokine interleukin-1β was not released by HSV-1-infected macrophages, demonstrating that HSV-1 can antagonize inflammasome function. In conclusion, our results provide a global view of the secretome of HSV-1-infected macrophages, revealing host factors possibly having a role in antiviral defense. PMID:22973042

Proprotein convertase 1/3 inhibited macrophages: A novel therapeutic based on drone macrophages.

PubMed

Duhamel, Marie; Rodet, Franck; Murgoci, Adriana; Wisztorski, Maxence; Day, Robert; Fournier, Isabelle; Salzet, Michel

2016-06-01

We demonstrated here thanks to proteomic, that proprotein convertase 1/3 knockdown macrophages present all the characteristic of activated pro-inflammatory macrophages. TLR4 and TLR9 signaling pathways can be enhanced leading to the secretion of pro-inflammatory factors and antitumor factors. We can control their activation by controlling one enzyme, PC1/3. In a tumor context, PC1/3 inhibition in macrophages may reactivate them and lead to a cytokine storm after stimulation "at distance" with a TLR ligand. Therefore, we name these proprotein convertase inhibited macrophages the "drone macrophages". They constitute an innovative cell therapy to treat efficiently tumors.

Susceptibility of bone marrow-derived macrophages to influenza virus infection is dependent on macrophage phenotype.

PubMed

Campbell, Gillian M; Nicol, Marlynne Q; Dransfield, Ian; Shaw, Darren J; Nash, Anthony A; Dutia, Bernadette M

2015-10-01

The role of the macrophage in influenza virus infection is complex. Macrophages are critical for resolution of influenza virus infections but implicated in morbidity and mortality in severe infections. They can be infected with influenza virus and consequently macrophage infection is likely to have an impact on the host immune response. Macrophages display a range of functional phenotypes, from the prototypical pro-inflammatory classically activated cell to alternatively activated anti-inflammatory macrophages involved in immune regulation and wound healing. We were interested in how macrophages of different phenotype respond to influenza virus infection and therefore studied the infection of bone marrow-derived macrophages (BMDMs) of classical and alternative phenotype in vitro. Our results show that alternatively activated macrophages are more readily infected and killed by the virus than classically activated. Classically activated BMDMs express the pro-inflammatory markers inducible nitric oxide synthase (iNOS) and TNF-α, and TNF-α expression was further upregulated following infection. Alternatively activated macrophages express Arginase-1 and CD206; however, following infection, expression of these markers was downregulated whilst expression of iNOS and TNF-α was upregulated. Thus, infection can override the anti-inflammatory state of alternatively activated macrophages. Importantly, however, this results in lower levels of pro-inflammatory markers than those produced by classically activated cells. Our results showed that macrophage phenotype affects the inflammatory macrophage response following infection, and indicated that modulating the macrophage phenotype may provide a route to develop novel strategies to prevent and treat influenza virus infection.

Lung tumor production and tissue metal distribution after exposure to manual metal ARC-stainless steel welding fume in A/J and C57BL/6J mice.

PubMed

Zeidler-Erdely, Patti C; Battelli, Lori A; Salmen-Muniz, Rebecca; Li, Zheng; Erdely, Aaron; Kashon, Michael L; Simeonova, Petia P; Antonini, James M

2011-01-01

Stainless steel welding produces fumes that contain carcinogenic metals. Therefore, welders may be at risk for the development of lung cancer, but animal data are inadequate in this regard. Our main objective was to examine lung tumor production and histopathological alterations in lung-tumor-susceptible (A/J) and -resistant C57BL/6J (B6) mice exposed to manual metal arc-stainless steel (MMA-SS) welding fume. Male mice were exposed to vehicle or MMA-SS welding fume (20 mg/kg) by pharyngeal aspiration once per month for 4 mo. At 78 wk postexposure, gross tumor counts and histopathological changes were assessed and metal analysis was done on extrapulmonary tissue (aorta, heart, kidney, and liver). At 78 wk postexposure, gross lung tumor multiplicity and incidence were unremarkable in mice exposed to MMA-SS welding fume. Histopathology revealed that only the exposed A/J mice contained minimal amounts of MMA-SS welding fume in the lung and statistically increased lymphoid infiltrates and alveolar macrophages. A significant increase in tumor multiplicity in the A/J strain was observed at 78 wk. Metal analysis of extrapulmonary tissue showed that only the MMA-SS-exposed A/J mice had elevated levels of Cr, Cu, Mn, and Zn in kidney and Cr in liver. In conclusion, this study further supports that MMA-SS welding fume does not produce a significant tumorigenic response in an animal model, but may induce a chronic lung immune response. In addition, long-term extrapulmonary tissue alterations in metals in the susceptible A/J mouse suggest that the adverse effects of this fume might be cumulative.

TraJ-dependent Escherichia coli K1 interactions with professional phagocytes are important for early systemic dissemination of infection in the neonatal rat.

PubMed

Hill, Val T; Townsend, Stacy M; Arias, Robyn S; Jenabi, Jasmine M; Gomez-Gonzalez, Ignacio; Shimada, Hiroyuki; Badger, Julie L

2004-01-01

Escherichia coli is a major cause of neonatal bacterial sepsis and meningitis. We recently identified a gene, traJ, which contributes to the ability of E. coli K1 to penetrate the blood-brain barrier in the neonatal rat. Because very little is known regarding the most critical step in disease progression, translocation to the gut and dissemination to the lymphoid tissues after a natural route of infection, we assessed the ability of a traJ mutant to cause systemic disease in the neonatal rat. Our studies determined that the traJ mutant is significantly less virulent than the wild type in the neonatal rat due to a decreased ability to disseminate from the mesenteric lymph nodes to the deeper tissues of the liver and spleen and to the blood during the early stages of systemic disease. Histopathologic studies determined that although significantly less or no mutant bacteria were recovered from the spleen and livers of infected neonatal rats, the inflammatory response was considerably greater than that in wild-type-colonized tissues. In vitro studies revealed that macrophages internalize the traJ mutant less frequently than they do the wild type and by a morphologically distinct process. Furthermore, we determined that tissue macrophages and dendritic cells within the liver and spleen are the major cellular targets of E. coli K1 and that TraJ significantly contributes to the predominantly intracellular nature of E. coli K1 within these professional phagocytes exclusively during the early stages of systemic disease. These data indicate that, contrary to earlier indications, E. coli K1 resides within professional phagocytes, and this is essential for the efficient progression of systemic disease.

Blocking fatty acid-fueled mROS production within macrophages alleviates acute gouty inflammation.

PubMed

Hall, Christopher J; Sanderson, Leslie E; Lawrence, Lisa M; Pool, Bregina; van der Kroef, Maarten; Ashimbayeva, Elina; Britto, Denver; Harper, Jacquie L; Lieschke, Graham J; Astin, Jonathan W; Crosier, Kathryn E; Dalbeth, Nicola; Crosier, Philip S

2018-05-01

Gout is the most common inflammatory arthritis affecting men. Acute gouty inflammation is triggered by monosodium urate (MSU) crystal deposition in and around joints that activates macrophages into a proinflammatory state, resulting in neutrophil recruitment. A complete understanding of how MSU crystals activate macrophages in vivo has been difficult because of limitations of live imaging this process in traditional animal models. By live imaging the macrophage and neutrophil response to MSU crystals within an intact host (larval zebrafish), we reveal that macrophage activation requires mitochondrial ROS (mROS) generated through fatty acid oxidation. This mitochondrial source of ROS contributes to NF-κB-driven production of IL-1β and TNF-α, which promote neutrophil recruitment. We demonstrate the therapeutic utility of this discovery by showing that this mechanism is conserved in human macrophages and, via pharmacologic blockade, that it contributes to neutrophil recruitment in a mouse model of acute gouty inflammation. To our knowledge, this study is the first to uncover an immunometabolic mechanism of macrophage activation that operates during acute gouty inflammation. Targeting this pathway holds promise in the management of gout and, potentially, other macrophage-driven diseases.

Isolation and Differentiation of Murine Macrophages.

PubMed

Rios, Francisco J; Touyz, Rhian M; Montezano, Augusto C

2017-01-01

Macrophages play a major role in inflammation, wound healing, and tissue repair. Infiltrated monocytes differentiate into different macrophage subtypes with protective or pathogenic activities in vascular lesions. In the heart and vascular tissues, pathological activation promotes cardiovascular inflammation and remodeling and there is increasing evidence that macrophages play important mechanisms in this environment. Primary murine macrophages can be obtained from: bone marrow by different treatments (granulocyte-macrophage colony-stimulating factor-GM-CSF, macrophage colony-stimulating factor-M-CSF or supernatant of murine fibroblast L929), peritoneal cavity (resident or thioglycolate elicit macrophages), from the lung (alveolar macrophages) or from adipose tissue. In this chapter we describe some protocols to obtain primary murine macrophages and how to identify a pure macrophage population or activation phenotypes using different markers.

Bacteroides fragilis induce necrosis on mice peritoneal macrophages: In vitro and in vivo assays

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

Vieira, J.M.B.D., E-mail: jmanya@terra.com.br; Laboratorio de Biologia de Anaerobios, IMPPG, UFRJ, Rio de Janeiro; Seabra, S.H.

Bacteroides fragilis is an anaerobic bacteria component of human intestinal microbiota and agent of infections. In the host B. fragilis interacts with macrophages, which produces toxic radicals like NO. The interaction of activated mice peritoneal macrophages with four strains of B. fragilis was evaluated on this study. Previously was shown that such strains could cause metabolic and morphologic alterations related to macrophage death. In this work propidium iodide staining showed the strains inducing macrophage necrosis in that the labeling was evident. Besides nitroblue tetrazolium test showed that B. fragilis stimulates macrophage to produce oxygen radicals. In vivo assays performed inmore » BalbC mice have results similar to those for in vitro tests as well as scanning electron microscopy, which showed the same surface pore-like structures observed in vitro before. The results revealed that B. fragilis strains studied lead to macrophage death by a process similar to necrosis.« less

Increase of radiocarbon concentration in tree rings from Kujawy (SE Poland) around AD 774-775

NASA Astrophysics Data System (ADS)

Rakowski, Andrzej Z.; Krąpiec, Marek; Huels, Mathias; Pawlyta, Jacek; Dreves, Alexander; Meadows, John

2015-10-01

Evidence of a rapid increase in atmospheric radiocarbon (14C) content in AD 774-775 was presented by Miyake et al. (2012), who observed an increase of about 12‰ in the 14C content in annual tree rings from Japanese cedar. Usoskin et al. (2013) report a similar 14C spike in German oak, and attribute it to exceptional solar activity. If this phenomenon is global in character, such rapid changes in 14C concentration may affect the accuracy of calibrated dates, as the existing calibration curve is composed mainly of decadal samples. Single-year samples of dendro-chronologically dated tree rings of deciduous oak (Quercus robur) from Kujawy, a village near Krakow (SE Poland), spanning the years AD 765-796, were collected and their 14C content was measured using the AMS system in the Leibniz Laboratory. The results clearly show a rapid increase of 9.2 ± 2.1‰ in the 14C concentration in tree rings between AD 774 and AD 775, with maximum Δ14C = 4.1 ± 2.3‰ noted in AD 776.

Blood-brain barrier breakdown promotes macrophage infiltration and cognitive impairment in leptin receptor-deficient mice.