ABSTRACT: VEGFB effect in resting and active macrophage. The goal of this study was to investigate the effects of Vascular Endothelial Growth Factor B (VEGFB) on gene expression in resting and active macrophages. Resting macrophages were obtained from peripheral blood of healthy volunteers. A part of macropahges were activated by Lipopolysaccharides (LPS). Both types of cells were treated by VEGFB. Total RNA was extracted using conventional Trizol extraction, quantified by Nanodrop and analyzed by Bioanalyzer. 1microg of RNA was amplified using AminoAllyl MessageAmp kit. 5µg amino allyl-coupled RNA was labeled with Cy3 or Cy5 dyes. Dye coupling yield >5% was a prerequisite for further analysis. 750ng of labeled RNA was hybridized on 25K gene microarrays for 17 hours at 60°C. 4 arrays per sample were hybridized and scanned with the Genepix 4000B Scanner (Molecular Devices). Microarray data quantification and pre-processing was performed with the MAIA software . The SAM tool was applied to identify differentially expressed genes. Overall design: Macrophages from 2 different volunteers were treated by vehicle, or VEGFB, or LPS, or LPS and VEGFB
Project description:VEGFB effect in resting and active macrophage. The goal of this study was to investigate the effects of Vascular Endothelial Growth Factor B (VEGFB) on gene expression in resting and active macrophages. Resting macrophages were obtained from peripheral blood of healthy volunteers. A part of macropahges were activated by Lipopolysaccharides (LPS). Both types of cells were treated by VEGFB. Total RNA was extracted using conventional Trizol extraction, quantified by Nanodrop and analyzed by Bioanalyzer. 1microg of RNA was amplified using AminoAllyl MessageAmp kit. 5µg amino allyl-coupled RNA was labeled with Cy3 or Cy5 dyes. Dye coupling yield >5% was a prerequisite for further analysis. 750ng of labeled RNA was hybridized on 25K gene microarrays for 17 hours at 60°C. 4 arrays per sample were hybridized and scanned with the Genepix 4000B Scanner (Molecular Devices). Microarray data quantification and pre-processing was performed with the MAIA software . The SAM tool was applied to identify differentially expressed genes. Macrophages from 2 different volunteers were treated by vehicle, or VEGFB, or LPS, or LPS and VEGFB
Project description:Sepsis is a consequence of systemic bacterial infections leading to hyper activation of immune cells by bacterial products resulting in enhanced release of mediators of inflammation. Endotoxin (LPS) is a major component of the outer membrane of Gram negative bacteria and a critical factor in pathogenesis of sepsis. Development of antagonists that inhibit the storm of inflammatory molecules by blocking Toll like receptors (TLR) has been the main stay of research efforts. We report here that a filarial glycoprotein binds to murine macrophages and human monocytes through TLR4 and activates them through alternate pathway and in the process inhibits LPS mediated classical activation which leads to inflammation associated with endotoxemia. The active component of the nematode glycoprotein mediating alternate activation of macrophages was found to be a carbohydrate residue, Chitohexaose. Murine macrophages and human monocytes up regulated Arginase-1 and released high levels of IL-10 when incubated with chitohexaose. Macrophages of C3H/HeJ mice (non-responsive to LPS) failed to get activated by chitohexaose suggesting that a functional TLR4 is critical for alternate activation of macrophages also. Chitohexaose inhibited LPS induced production of inflammatory molecules TNF-?, IL-1? and IL-6 by macropahges in vitro and in vivo in mice. Intraperitoneal injection of chitohexaose completely protected mice against endotoxemia when challenged with a lethal dose of LPS. Furthermore, Chitohexaose was found to reverse LPS induced endotoxemia in mice even 6/24/48 hrs after its onset. Monocytes of subjects with active filarial infection displayed characteristic alternate activation markers and were refractory to LPS mediated inflammatory activation suggesting an interesting possibility of subjects with filarial infections being less prone to develop of endotoxemia. These observations that innate activation of alternate pathway of macrophages by chtx through TLR4 has offered novel opportunities to cell biologists to study two mutually exclusive activation pathways of macrophages being mediated through a single receptor.
Project description:Maintenance of macrophages in their basal state and their rapid activation in response to pathogen detection are central to the innate immune system, acting to limit nonspecific oxidative damage and promote pathogen killing following infection. To identify possible age-related alterations in macrophage function, we have assayed the function of peritoneal macrophages from young (3-4 months) and aged (14-15 months) Balb/c mice. In agreement with prior suggestions, we observe age-dependent increases in the extent of recruitment of macrophages into the peritoneum, as well as ex vivo functional changes involving enhanced nitric oxide production under resting conditions that contribute to a reduction in the time needed for full activation of senescent macrophages following exposure to lipopolysaccharides (LPS). Further, we observe enhanced bactericidal activity following Salmonella uptake by macrophages isolated from aged Balb/c mice in comparison with those isolated from young animals. Pathways responsible for observed phenotypic changes were interrogated using tandem mass spectrometry, which identified age-dependent increases in levels of proteins linked to immune cell pathways under basal conditions and following LPS activation. Immune pathways upregulated in macrophages isolated from aged mice include proteins critical to the formation of the immunoproteasome. Detection of these latter proteins is dramatically enhanced following LPS exposure for macrophages isolated from aged animals; in comparison, the identification of immunoproteasome subunits is insensitive to LPS exposure for macrophages isolated from young animals. Consistent with observed global changes in the proteome, quantitative proteomic measurements indicate that there are age-dependent abundance changes involving specific proteins linked to immune cell function under basal conditions. LPS exposure selectively increases the levels of many proteins involved in immune cell function in aged Balb/c mice. Collectively, these results indicate that macrophages isolated from old mice are in a preactivated state that enhances their sensitivities to LPS exposure. The hyper-responsive activation of macrophages in aged animals may act to minimize infection by general bacterial threats that arise due to age-dependent declines in adaptive immunity. However, this hypersensitivity and the associated increase in the level of formation of reactive oxygen species are likely to contribute to observed age-dependent increases in the level of oxidative damage that underlie many diseases of the elderly.
Project description:When macrophages are activated by sensing bacterial lipopolysaccharides (LPS), they greatly increase their motility, mRNA synthesis and protein production. Most of the ATP needed for these responses is derived from the uptake and catabolism of glucose, a relatively inefficient ATP source. Although the stimulated cells also increase their uptake of free fatty acids, they store a large fraction as triglycerides (TAG). We report here that both Toll-like receptor 4 (TLR4) and TLR2 agonists stimulate prolonged TAG retention by primary murine and human macrophages. Agonist-induced TAG storage lasted at least 72-96 hrs in vitro and was associated with increases in fatty acid (FA) uptake, FA esterification, and FA incorporation into TAG; FA oxidation decreased. The results of expression and inhibitor studies support a prominent role for increases in long chain acyl CoA synthase 1 (ACSL1) and diacylglycerol acyltransferase-2 (DGAT2) during the sustained response to TLR2/4 activation; decreases in adipose triglyceride lipase (ATGL, Pnpla2) and monoacylglycerol lipase (MgII) may also contribute. Stimulated murine macrophages that retained TAG carried out phagocytosis more effectively and were protected from saturated fatty acid-induced cell death (lipotoxicity). TLR agonist-induced TAG retention in macrophages is thus an active, sustained process that may have important adaptive functions. It may also contribute to the persistence of lipid-laden macrophages in infected tissues, host susceptibility to some microbial pathogens, and the pathogenesis of atherosclerosis. RNA from macrophage loaded with Fatty Acids, stimulated with bacterial lipopolysaccharides (LPS), or both compared to untreated controls (FA, LPS, FA+LPS, untreated). Replicates from 4 independent experiments.
Project description:Identification of diffuse signals from the chromatin immunoprecipitation and high-throughput massively parallel sequencing (ChIP-Seq) technology poses significant computational challenges, and there are few methods currently available. We present a novel global clustering approach to enrich diffuse CHIP-Seq signals of RNA polymerase II and histone 3 lysine 4 trimethylation (H3K4Me3) and apply it to identify putative long intergenic non-coding RNAs (lincRNAs) in macrophage cells. Our global clustering method compares favorably to the local clustering method SICER that was also designed to identify diffuse CHIP-Seq signals. The validity of the algorithm is confirmed at several levels. First, 8 out of a total of 11 selected putative lincRNA regions in primary macrophages respond to lipopolysaccharides (LPS) treatment as predicted by our computational method. Second, the genes nearest to lincRNAs are enriched with biological functions related to metabolic processes under resting conditions but with developmental and immune-related functions under LPS treatment. Third, the putative lincRNAs have conserved promoters, modestly conserved exons, and expected secondary structures by prediction. Last, they are enriched with motifs of transcription factors such as PU.1 and AP.1, previously shown to be important lineage determining factors in macrophages, and 83% of them overlap with distal enhancers markers. In summary, GCLS based on RNA polymerase II and H3K4Me3 CHIP-Seq method can effectively detect putative lincRNAs that exhibit expected characteristics, as exemplified by macrophages in the study.
Project description:Upon stimulation with Th1 cytokines or bacterial lipopolysaccharides, resting macrophages shift their phenotype toward a pro-inflammatory state as part of the innate immune response. LPS-activated macrophages undergo profound metabolic changes to adapt to these new physiological requirements. One key step to mediate this metabolic adaptation is the stabilization of HIF1α, which leads to increased glycolysis and lactate release, as well as decreased oxygen consumption. HIF1 abundance can result in the induction of the gene encoding pyruvate dehydrogenase kinase 1 (PDK1), which inhibits pyruvate dehydrogenase (PDH) via phosphorylation. Therefore, it has been speculated that pyruvate oxidation through PDH is decreased in pro-inflammatory macrophages. However, to answer this open question, an in-depth analysis of this metabolic branching point was so far lacking. In this work, we applied stable isotope-assisted metabolomics techniques and demonstrate that pyruvate oxidation is maintained in mature pro-inflammatory macrophages. Glucose-derived pyruvate is oxidized via PDH to generate citrate in the mitochondria. Citrate is used for the synthesis of the antimicrobial metabolite itaconate and for lipogenesis. An increased demand for these metabolites decreases citrate oxidation through the tricarboxylic acid cycle, whereas increased glutamine uptake serves to replenish the TCA cycle. Furthermore, we found that the PDH flux is maintained by unchanged PDK1 abundance, despite the presence of HIF1. By pharmacological intervention, we demonstrate that the PDH flux is an important node for M(LPS) macrophage activation. Therefore, PDH represents a metabolic intervention point that might become a research target for translational medicine to treat chronic inflammatory diseases.
Project description:Inflammatory response plays an essential role in the resolution of infections. However, inflammation can be detrimental to an organism and cause irreparable damage. For example, during sepsis, a cytokine storm can lead to multiple organ failures and often results in death. One of the strongest triggers of the inflammatory response is bacterial lipopolysaccharides (LPS), acting mostly through Toll-like receptor 4 (TLR4). Paradoxically, while exposure to LPS triggers a robust inflammatory response, repeated or prolonged exposure to LPS can induce a state of endotoxin tolerance, a phenomenon where macrophages and monocytes do not respond to new endotoxin challenges, and it is often associated with secondary infections and negative outcomes. The cellular mechanisms regulating this phenomenon remain elusive. We used metabolic measurements to confirm differences in the cellular metabolism of naïve macrophages and that of macrophages responding to LPS stimulation or those in the LPS-tolerant state. In parallel, we performed an unbiased secretome survey using quantitative mass spectrometry during the induction of LPS tolerance, creating the first comprehensive secretome profile of endotoxin-tolerant cells. The secretome changes confirmed that LPS-tolerant macrophages have significantly decreased cellular metabolism and that the proteins secreted by LPS-tolerant macrophages have a strong association with cell survival, protein metabolism, and the metabolism of reactive oxygen species.
Project description:NK cells can be stimulated by bacterial lipopolysaccharides (LPS). Unlike macrophages, human NK cells do not express or express very low level of surface TLR4 receptor normally required for the LPS stimulation. This has led to the assumption that the mechanisms of stimulating action of LPS on macrophages and NK cells differs. In this work, we investigated the effects of different forms of E. coli LPS, including mutants lacking O-antigen structures, and deacylated LPS on IFN? production by purified human NK cells. The main findings were the following: (1) NK cells were more sensitive to the S-forms of LPS than the R-forms (LPS lacking O-antigen); (2) LPS triggered a significant increase in IFN? production by NK cells in concentrations about 1000 times higher than those that can induce cytokine production by macrophages; (3) the composition and structure of saccharide part of LPS have a strong influence on its observed effects on NK cells; and (4) LPS fully retained the ability to trigger cytokine production in NK cells in serum-free media. The acquired data demonstrated that the presence and structure of O-antigen affects the LPS-induced activation of human NK cells.
Project description:The goal of this study was to investigate the effects of the cardioprotective nucleoside adenosine on gene expression in early and late endothelial progenitor cells. Adenosine mod Overall design: Early outgrowth endothelial progenitor cells were obtained by adhesion of peripheral blood mononuclear cells of healthy volunteers. Late endothelial progenitor cells were obtained by purification of CD34+ peripheral blood cells and were cultured and amplified in endothelial-specific medium containing growth factors. Both cell types were treated by adenosine (10micromol/L) for 6 hours. Total RNA was extracted using conventional Trizol extraction, quantified by Nanodrop and analyzed by Bioanalyzer. 1microg of RNA was amplified using AminoAllyl MessageAmp kit. 5µg amino allyl-coupled RNA was labeled with Cy3 or Cy5 dyes. Dye coupling yield >5% was a prerequisite for further analysis. 750ng of labeled RNA was hybridized on 25,000 gene microarrays for 17 hours at 60°C. 4 arrays per sample were hybridized and scanned with the Genepix 4000B Scanner (Molecular Devices). Six independent experiments were performed. Microarray data quantification and pre-processing was performed with the MAIA software and intensity values were log-transformed. Gene expression values were standardized across experiments with mean = 0 and standard deviation = 1. The SAM tool was applied to identify differentially expressed genes.
Project description:In mammalian cells, inflammatory caspases detect Gram-negative bacterial invasion by binding lipopolysaccharides (LPS). Murine caspase-11 binds cytosolic LPS, stimulates pyroptotic cell death, and drives sepsis pathogenesis. Extracellular priming factors enhance caspase-11-dependent pyroptosis. Herein we compare priming agents and demonstrate that IFN? priming elicits the most rapid and amplified macrophage response to cytosolic LPS. Previous studies indicate that IFN-induced expression of caspase-11 and guanylate binding proteins (GBPs) are causal events explaining the effects of priming on cytosolic LPS sensing. We demonstrate that these events cannot fully account for the increased response triggered by IFN? treatment. Indeed, IFN? priming elicits higher pyroptosis levels in response to cytosolic LPS when macrophages stably express caspase-11. In macrophages lacking GBPs encoded on chromosome 3, IFN? priming enhanced pyroptosis in response to cytosolic LPS as compared with other priming agents. These results suggest an unknown regulator of caspase-11-dependent pyroptosis exists, whose activity is upregulated by IFN?.