Innate Immune Responses In Macrophages Research Articles

Genetic analysis of long-lived families reveals novel variants influencing high density-lipoprotein cholesterol.

The plasma levels of high-density lipoprotein cholesterol (HDL) have an inverse relationship to the risks of atherosclerosis and cardiovascular disease (CVD), and have also been associated with longevity. We sought to identify novel loci for HDL that could potentially provide new insights into biological regulation of HDL metabolism in healthy-longevous subjects. We performed a genome-wide association (GWA) scan on HDL using a mixed model approach to account for family structure using kinship coefficients. A total of 4114 subjects of European descent (480 families) were genotyped at ~2.3 million SNPs and ~38 million SNPs were imputed using the 1000 Genome Cosmopolitan reference panel in MACH. We identified novel variants near-NLRP1 (17p13) associated with an increase of HDL levels at genome-wide significant level (p < 5.0E-08). Additionally, several CETP (16q21) and ZNF259-APOA5-A4-C3-A1 (11q23.3) variants associated with HDL were found, replicating those previously reported in the literature. A possible regulatory variant upstream of NLRP1 that is associated with HDL in these elderly Long Life Family Study (LLFS) subjects may also contribute to their longevity and health. Our NLRP1 intergenic SNPs show a potential regulatory function in Encyclopedia of DNA Elements (ENCODE); however, it is not clear whether they regulate NLRP1 or other more remote gene. NLRP1 plays an important role in the induction of apoptosis, and its inflammasome is critical for mediating innate immune responses. Nlrp1a (a mouse ortholog of human NLRP1) interacts with SREBP-1a (17p11) which has a fundamental role in lipid concentration and composition, and is involved in innate immune response in macrophages. The NLRP1 region is conserved in mammals, but also has evolved adaptively showing signals of positive selection in European populations that might confer an advantage. NLRP1 intergenic SNPs have also been associated with immunity/inflammasome disorders which highlights the biological importance of this chromosomal region.

Pro-inflammatory response and defective bactericidal activity by Mycobacterium tuberculosis/HIV-1 co-infected human macrophages (INC7P.421)

Abstract Mycobacterium tuberculosis (M.tb), the etiological agent of tuberculosis (TB), is the leading cause of death in people living with Human Immunodeficiency Virus (HIV). In contrast to most other opportunistic infections, susceptibility to M.tb occurs prior to significant loss of peripheral CD4+T cells in people with HIV. The molecular basis for pathogen synergy that leads to an aggressive course of TB in those with HIV-1 infection is poorly defined. The primary objective of this study is to determine how HIV-1 alters the host macrophage innate immune response to M.tb infection. Primary human monocyte-derived macrophages, alveolar macrophages from human donor bronchoalveolar lavage, and our recently developed humanized mouse model of M.tb/HIV co-infection were used for in vitro and in vivo studies. Preliminary results demonstrate that HIV-1 (JR-CSF)/M.tb (H37Rv) co-infection results in greater cell death and increased expression of pro-inflammatory cytokines and chemokines (e.g. IL-1β, IL-6, MCP) compared to mono-infection with either HIV-1 or M.tb. A corresponding increase in leukocyte recruitment into the lungs of M.tb-infected humanized mice was observed in animals with HIV-1 infection and coincided with greater mycobacterial dissemination. These studies suggest that inappropriate inflammatory responses by host macrophages may contribute to dysfunctional host immunity to M.tb in those with concurrent HIV-1 infection.

Chemokine Receptor CCR8 Is Required for Lipopolysaccharide-Triggered Cytokine Production in Mouse Peritoneal Macrophages

Chemokine (C-C motif) receptor 8 (CCR8), the chemokine receptor for chemokine (C-C motif) ligand 1 (CCL1), is expressed in T-helper type-2 lymphocytes and peritoneal macrophages (PMφ) and is involved in various pathological conditions, including peritoneal adhesions. However, the role of CCR8 in inflammatory responses is not fully elucidated. To investigate the function of CCR8 in macrophages, we compared cytokine secretion from mouse PMφ or bone marrow-derived macrophages (BMMφ) stimulated with various Toll-like receptor (TLR) ligands in CCR8 deficient (CCR8- /-) and wild-type (WT) mice. We found that CCR8-/- PMφ demonstrated attenuated secretion of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-10 when stimulated with lipopolysaccharide (LPS). In particular, LPS-induced IL-10 production absolutely required CCR8. CCR8-dependent cytokine secretion was characteristic of PMφ but not BMMφ. To further investigate this result, we selected the small molecule compound R243 from a library of compounds with CCR8-antagonistic effects on CCL1-induced Ca2+ flux and CCL1-driven PMφ aggregation. Similar to CCR8-/- PMφ, R243 attenuated secretion of TNF-α, IL-6, and most strikingly IL-10 from WT PMφ, but not BMMφ. CCR8-/- PMφ and R243-treated WT PMφ both showed suppressed c-jun N-terminal kinase activity and nuclear factor-κB signaling after LPS treatment when compared with WT PMφ. A c-Jun signaling pathway inhibitor also produced an inhibitory effect on LPS-induced cytokine secretion that was similar to that of CCR8 deficiency or R243 treatment. As seen in CCR8-/- mice, administration of R243 attenuated peritoneal adhesions in vivo. R243 also prevented hapten-induced colitis. These results are indicative of cross talk between signaling pathways downstream of CCR8 and TLR-4 that induces cytokine production by PMφ. Through use of CCR8-/- mice and the new CCR8 inhibitor, R243, we identified a novel macrophage innate immune response pathway that involves a chemokine receptor.

Stimulation of inflammasome‐dependent innate response by group A streptococcus ADP‐ribosyltransferase protects host from bacterial infections (539.9)

Streptococcus pyogenes (GAS) is a causative agent of severe invasive infections including necrotizing fasciitis, bacteremia and toxic shock syndrome. SpyA (S. pyogenes ADP‐ribosyltransferase) is one of a subset of virulence factors significantly up‐regulated in the hyper‐invasive strain of a globally disseminated M1T1 GAS. Surprisingly, mice systematically challenged with GAS ΔspyA suffered significantly higher mortality and displayed reduced bacterial clearance. We recovered higher levels of ΔspyA GAS compared to the wild‐type (WT) strain from bone marrow‐derived macrophage (BMDM) killing assays. Complementation of the GAS ΔspyA mutant restored the WT BMDM survival phenotype, heterologous expression of SpyA in Staphylococcus aureus or the macrophage itself led to a significant increase in bacterial killing. Increased bacterial survival of the ΔspyA mutant was associated with severe attenuation of caspase‐1 dependent inflammasome activation and impaired IL‐1β production. BMDMs deficient in caspase‐1, NLRP3, ASC or pro‐ IL‐1β reversed the effect of SpyA expression on macrophage survival and abolished protection seen in WT infected mice. In conclusion, SpyA triggers caspase‐1 dependent inflammatory responses, or “pyroptosis” in macrophages, highlighting the importance of this macrophage innate immune response pathway in defense against invasive bacterial pathogens.Grant Funding Source: Canadian Institutes of Health Research (CIHR)

A novel immunomodulatory function of PHLPP1: inhibition of iNOS via attenuation of STAT1 ser727 phosphorylation in mouse macrophages

PHLPP1 is a novel tumor suppressor, but its role in the regulation of innate immune responses, which are frequently dysregulated in cancer, is unexplored. Here, we report that LPS attenuated PHLPP1 expression at mRNA and protein levels in immune cells, suggesting its involvement in immune responses. To test this, we overexpressed PHLPP1 in RAW 264.7 macrophages and observed a dramatic reduction in LPS/IFN-γ-induced iNOS expression. Conversely, silencing of PHLPP1 by siRNA or by shRNA robustly augmented LPS/IFN-γ-induced iNOS expression. qPCR and iNOS promoter reporter experiments showed that PHLPP1 inhibited iNOS transcription. Mechanistic analysis revealed that PHLPP1 suppressed LPS/IFN-γ-induced phosphorylation of ser727 STAT1; however, the underlying mechanisms differed. PHLPP1 reduced IFN-γ-stimulated but not LPS-induced ERK1/2 phosphorylation, and inhibition of ERK1/2 abolished IFN-γ-induced ser727 STAT1 phosphorylation and iNOS expression. In contrast, PHLPP1 knockdown augmented LPS-induced but not IFN-γ-elicited p38 phosphorylation. Blockade of p38 abolished LPS-stimulated phosphorylation of ser727 STAT1 and iNOS expression. Furthermore, PHLPP1 suppressed LPS-induced phosphorylation of tyr701 STAT1 by dampening p38-dependent IFN-β feedback. Collectively, our data demonstrate for the first time that PHLPP1 plays a vital role in restricting innate immune responses of macrophages, and further studies may show it to be a potential therapeutic target within the context of dysregulated macrophage activity.

Is RXRα Crucially Involved in Intestinal Inflammation?

Retinoid X receptors (RXRs) are nuclear receptors that control several physiological processes [1]. Three closely related RXR isotypes are encoded on chromosome 9: RXRα (activated by 9-cis-retinoic acid), RXRβ, and RXRγ. RXRs form heterodimers, either spontaneously or in a ligand-dependent manner, with other nuclear receptors including retinoic acid receptors (RAR), peroxisome proliferator-activated receptors (PPAR), estrogen receptors (ER), vitamin D receptors (VDR), thyroid hormone receptors (TR), farnesoid X receptors (FXR), liver X receptors (LXR), constitutive androstane receptors (CAR), and pregnane X receptors (PXR) [2, 3]. Dimerization of RXR with other nuclear receptors transcriptionally controls a variety of cellular processes by binding to specific DNA response elements within regions of target gene promoters [4]. Nuclear receptor binding to RXR can be functionally divided into “permissive” and “non-permissive” groups. Heterodimers that form when binding to a “permissive” receptor, for example PPAR, can be activated by agonists to either heterodimeric partner. In contrast, “non-permissive” RXR nuclear receptor heterodimers, for example VDR and THR, can be activated only by agonists binding to the partner of RXR [5]. 1,25(OH)2D3 binds to its receptor, VDR, which requires RXR to bind to vitamin D response elements for transcriptional regulation. Several pieces of evidence are indicative of loss of vitamin D-VDR signals in the pathogenesis of inflammatory bowel disease. First, VDR polymorphisms are associated with IBD risk in some populations [6]. In a murine model, VDR/IL-10 double knockout mice develop more severe colitis and mortality is higher [7]. In IL-10(−/−) mice, vitamin D deficiency exacerbates the symptoms of colitis, whereas vitamin D supplementation ameliorates symptoms and reduces inflammation [8]. Another mechanism whereby vitamin D protects against IBD development is by maintaining or restoring the integrity of the intestinal mucosal barrier by preventing intestinal epithelial apoptosis [9]. In this regard, 1,25(OH)2D3 has several effects on the immune system, including suppression of T cell activation and modulation of antigen presentation [10]. Furthermore, VDR signaling is also involved in regulation of the innate immune response in macrophages [11]. In this issue of Digestive Diseases and Sciences, Knack-stedt et al. report the potential involvement of RXRα in mucosal inflammation [12]. Although the study did not demonstrate differences in colitis between RXRα heterozygotes and wild-type mice in a murine model of early colitis, there were significant increases in Snail1 and Snail2 in the RXR+/− mice. Snail 1 and Snail 2 are transcription factors linked to the epithelial–mesenchymal transition that mediate cell migration during inflammation [13]. These transcription factors are up-regulated in ulcerative colitis and can silence VDR expression, which is often down-regulated in active mucosal inflammation and colon cancer [9, 14]. Through this mechanism, RXRα is likely to be important in modulating inflammation and carcinogenesis, because, for example, RXR and VDR polymorphisms alter the risk of sporadic colon cancer [15, 16]. It is difficult to understand, however, why mucosal inflammation was no different despite up-regulation of Snail 1 and Snail 2 in this study. Although the RXRα heterozygous model used in this work did not result in significant down-regulation of RXRα protein expression, a model with conditional RXRα homozygous deletion might reveal a greater effect on mucosal inflammation. Furthermore, extending the time of investigation from a model of early colitis to one of chronic colitis might reveal different effects of RXRα hemizygous deletion in colonic inflammation. Nevertheless, further mechanistic evaluation of the effect of RXRα in mucosal inflammation is needed, because it is unknown whether the RXR–VDR heterodimer, as suggested by the authors, or alternative nuclear receptors or other pathways regulate the inflammatory cascade or mediate carcinogenesis, as supported by the observation that RXR and PPARγ agonists reduced inflammation in a TNBS-induced colitis murine model [17]. There is increasing evidence that low serum vitamin D concentrations are a risk factor for the development of inflammatory bowel disease [18]. Although evidence for humans is lacking, in murine models, treatment with vitamin D does improve symptoms of colitis [8]. Potentially, therapy targeting RXRα, in conjunction with vitamin D, could also be used to treat bowel inflammation. Because VDR forms a non-permissive heterodimer with RXR, RXR agonists are unlikely to be beneficial in regulating transcription at VDREs. Furthermore, because RXRα seems to be excess in most cells, therapeutic intervention to increase RXR expression is unlikely to enhance RXR–VDR signaling [19]. For this reason, the greatest potential for therapeutic intervention involving this pathway is likely to involve increasing the interaction of RXR with its heterodimeric partners.

Autophagy in macrophages: impacting inflammation and bacterial infection.

Macrophages are on the front line of host defense. They possess an array of germline-encoded pattern recognition receptors/sensors (PRRs) that recognize pathogen-associated molecular patterns (PAMPs) and which activate downstream effectors/pathways to help mediate innate immune responses and host defense. Innate immune responses include the rapid induction of transcriptional networks that trigger the production of cytokines, chemokines, and cytotoxic molecules; the mobilization of cells including neutrophils and other leukocytes; the engulfment of pathogens by phagocytosis and their delivery to lysosome for degradation; and the induction of autophagy. Autophagy is a catabolic process that normally maintains cellular homeostasis in a lysosome-dependent manner, but it also functions as a cytoprotective response that intersects with a variety of general stress-response pathways. This review focuses on the intimately linked molecular mechanisms that help govern the autophagic pathway and macrophage innate immune responses.

Resolvin D1 primes the resolution process initiated by calorie restriction in obesity‐induced steatohepatitis

Insulin resistance and nonalcoholic steatohepatitis (NASH), characterized by hepatic steatosis combined with inflammation, are major sequelae of obesity. Currently, lifestyle modification (i.e., weight loss) is the first-line therapy for NASH. However, weight loss resolves steatosis but not inflammation. In this study, we tested the ability of resolvin D1 (RvD1), an anti-inflammatory and proresolving molecule, to promote the resolution initiated by calorie restriction in obese mice with NASH. Calorie restriction reduced adipose and liver weight (-56 and -13%, respectively; P<0.001), serum leptin and resistin levels, hepatic steatosis, and insulin resistance. In addition to these, mice receiving RvD1 during the dietary intervention showed increased adiponectin expression at both the mRNA and protein levels and reduced liver macrophage infiltration (-15%, P<0.01). Moreover, RvD1 skewed macrophages from an M1- to an M2-like anti-inflammatory phenotype, induced a specific hepatic miRNA signature (i.e., miR-219-5p and miR-199a-5p), and reduced inflammatory adipokine mRNA and protein expression and macrophage innate immune response. In precision-cut liver slices (PCLSs), which override the influence of circulating factors, RvD1 attenuated hypoxia-induced mRNA and protein expression of COX-2, IL-1β, IL-6, and CCR7. Of note, RvD1 anti-inflammatory actions were absent in macrophage-depleted PCLSs. In summary, RvD1 acts as a facilitator of the hepatic resolution process by reducing the inflammatory component of obesity-induced NASH.

IRF3 helps control acute TMEV infection through IL-6 expression but contributes to acute hippocampus damage following TMEV infection

IRF3 is an innate anti-viral factor whose role in limiting Theiler's murine encephalomyelitis virus (TMEV) infection and preventing TMEV-induced disease is unclear. Acute disease and innate immune responses of macrophages were examined in IRF3 knockout mice compared with C57Bl/6 mice following in vitro or intracranial infection with either TMEV GDVII or DA. IRF3 deficiency augmented viral infection, as well as morbidity and mortality following intracranial infection with neurovirulent TMEV GDVII. In contrast, IRF3 deficiency prevented hippocampal injury following intracranial infection with persistent TMEV DA. The extent of TMEV infection in macrophages from C57Bl/6 mice was significantly less than that in IRF3 deficient macrophages, which was associated with poor IFN-β and IL-6 expression in response to TMEV. Reestablishing IRF3 expression in IRF3 deficient macrophages increased control of TMEV replication and increased expression of IFN-β and IL-6. In addition, IRF3 deficient macrophages failed to exhibit IL-6 antiviral effects, which was associated with inability to sustain IL-6-induced STAT1 activation compared with C57BL/6 macrophages. Altogether, IRF3 contributes to early control of TMEV replication through induction of IL-6 and IFN-β and support of IL-6 antiviral effects, but contributes to TMEV-induced hippocampal injury.

Deletion of the Vaccinia Virus Gene A46R, Encoding for an Inhibitor of TLR Signalling, Is an Effective Approach to Enhance the Immunogenicity in Mice of the HIV/AIDS Vaccine Candidate NYVAC-C

Viruses have developed strategies to counteract signalling through Toll-like receptors (TLRs) that are involved in the detection of viruses and induction of proinflammatory cytokines and IFNs. Vaccinia virus (VACV) encodes A46 protein which disrupts TLR signalling by interfering with TLR: adaptor interactions. Since the innate immune response to viruses is critical to induce protective immunity, we studied whether deletion of A46R gene in a NYVAC vector expressing HIV-1 Env, Gag, Pol and Nef antigens (NYVAC-C) improves immune responses against HIV-1 antigens. This question was examined in human macrophages and in mice infected with a single A46R deletion mutant of the vaccine candidate NYVAC-C (NYVAC-C-ΔA46R). The viral gene A46R is not required for virus replication in primary chicken embryo fibroblast (CEF) cells and its deletion in NYVAC-C markedly increases TNF, IL-6 and IL-8 secretion by human macrophages. Analysis of the immune responses elicited in BALB/c mice after DNA prime/NYVAC boost immunization shows that deletion of A46R improves the magnitude of the HIV-1-specific CD4 and CD8 T cell immune responses during adaptive and memory phases, maintains the functional profile observed with the parental NYVAC-C and enhances anti-gp120 humoral response during the memory phase. These findings establish the immunological role of VACV A46R on innate immune responses of macrophages in vitro and antigen-specific T and B cell immune responses in vivo and suggest that deletion of viral inhibitors of TLR signalling is a useful approach for the improvement of poxvirus-based vaccine candidates.

Endocytosis of Mycobacterium tuberculosis Heat Shock Protein 60 Is Required to Induce Interleukin-10 Production in Macrophages*

Understanding the signaling pathways involved in the regulation of anti-inflammatory and pro-inflammatory responses in tuberculosis is extremely important in tailoring a macrophage innate response to promote anti-tuberculosis immunity in the host. Although the role of toll-like receptors (TLRs) in the regulation of anti-inflammatory and pro-inflammatory responses is known, the detailed molecular mechanisms by which the Mycobacterium tuberculosis bacteria modulate these innate responses are not clearly understood. In this study, we demonstrate that M. tuberculosis heat shock protein 60 (Mtbhsp60, Cpn60.1, and Rv3417c) interacts with both TLR2 and TLR4 receptors, but its interaction with TLR2 leads to clathrin-dependent endocytosis resulting in an increased production of interleukin (IL)-10 and activated p38 MAPK. Blockage of TLR2-mediated endocytosis inhibited IL-10 production but induced production of tumor necrosis factor (TNF)-α and activated ERK1/2. In contrast, upon interaction with TLR4, Mtbhsp60 remained predominantly localized on the cell surface due to poorer endocytosis of the protein that led to decreased IL-10 production and p38 MAPK activation. The Escherichia coli homologue of hsp60 was found to be retained mainly on the macrophage surface upon interaction with either TLR2 or TLR4 that triggered predominantly a pro-inflammatory-type immune response. Our data suggest that cellular localization of Mtbhsp60 upon interaction with TLRs dictates the type of polarization in the innate immune responses in macrophages. This information is likely to help us in tailoring the host protective immune responses against M. tuberculosis.

Partial Restoration of Macrophage Alteration from Diet-Induced Obesity in Response to Porphyromonas gingivalis Infection

Obesity is a chronic inflammatory disease that weakens macrophage innate immune response to infections. Since M1 polarization is crucial during acute infectious diseases, we hypothesized that diet-induced obesity inhibits M1 polarization of macrophages in the response to bacterial infections. Bone marrow macrophages (BMMΦ) from lean and obese mice were exposed to live Porphyromonas gingivalis (P. gingivalis) for three incubation times (1 h, 4 h and 24 h). Flow cytometry analysis revealed that the M1 polarization was inhibited after P. gingivalis exposure in BMMΦ from obese mice when compared with BMMΦ from lean counterparts. Using a computational approach in conjunction with microarray data, we identified switching genes that may differentially control the behavior of response pathways in macrophages from lean and obese mice. The two most prominent switching genes were thrombospondin 1 and arginase 1. Protein expression levels of both genes were higher in obese BMMΦ than in lean BMMΦ after exposure to P. gingivalis. Inhibition of either thrombospondin 1 or arginase 1 by specific inhibitors recovered the M1 polarization of BMMΦ from obese mice after P. gingivalis exposure. These data indicate that thrombospondin 1 and arginase 1 are important bacterial response genes, whose regulation is altered in macrophages from obese mice.

TLR3-Triggered Reactive Oxygen Species Contribute to Inflammatory Responses by Activating Signal Transducer and Activator of Transcription-1

Intracellular reactive oxygen species (ROS) are essential secondary messengers in many signaling cascades governing innate immunity and cellular functions. TLR3 signaling is crucially involved in antiviral innate and inflammatory responses; however, the roles of ROS in TLR3 signaling remain largely unknown. In this study, we show that TLR3-induced ROS generation is required for the activation of NF-κB, IFN-regulatory factor 3, and STAT1-mediated innate immune responses in macrophages. TLR3 induction led to a rapid increase in ROS generation and a physical association between components of the NADPH oxidase (NOX) enzyme complex (NOX2 and p47(phox)) and TLR3 via a Ca(2+)-c-Src tyrosine kinase-dependent pathway. TLR3-induced ROS generation, NOX2, and p47(phox) were required for the phosphorylation and nuclear translocation of STAT1 and STAT2. TLR3-induced activation of STAT1 contributed to the generation of inflammatory mediators, which was significantly attenuated in NOX2- and p47(phox)-deficient macrophages, suggesting a role for ROS-STAT1 in TLR3-mediated innate immune responses. Collectively, these results provide a novel insight into the crucial role that TLR3-ROS signaling plays in innate immune responses by activating STAT1.

Orange juice and hesperidin promote differential innate immune response in macrophages ex vivo.

The purpose of this study was to verify the immune response induced by intake of orange juice or hesperidin on macrophages through the secretion of cytokines and nitric oxide. Mice were divided in three groups treated orally with orange juice, hesperidin, or control for 2 weeks. Ex vivo macrophages from all groups of mice were cultured with or without lipopolysaccharide (LPS)-stimuli, and the levels of nitric oxide (NO) and of the cytokines IL-10, IL-12, and TNF-alpha were evaluated. Macrophages of non-LPS-stimulated, orange juice treatment increased IL-12 levels by 143 % and the other cytokines and NO levels were unchanged. Hesperidin treatment increased IL-12 levels by 72 % and strongly decreased the NO secretion. For LPS-stimulated macrophages the orange juice (OJ) treatment decreased TNF-alpha secretion by 100 % and did not alter other cytokines, while NO levels increased 41 %. Hesperidin treatment decreased NO, IL-10, IL-12, and TNF-alpha levels by 56 %, 47 %, 29 %, and 63 %, respectively. In conclusion, OJ and hesperidin showed different immune responses, suggesting that hesperidin displays a suppressive effect on inflammation generated by LPS, while OJ seems to enhance the functions of macrophages associated with antimicrobial activity.

OP0256 Involvement of unfolded protein response and reactive oxygen species pathways in the pathogenesis of tumour necrosis factor receptor- associated periodic syndrome (TRAPS)

BackgroundTRAPS is caused by autosomal dominant mutations in the TNFR1 gene [1]. Many TRAPS mutations are associated with reduced cell surface TNFR1 expression leading to the proposed hypothesis of impaired...

Activating Transcription Factor 3 Contributes to Toll-Like Receptor-Mediated Macrophage Survival via Repression ofBaxandBak

Macrophages play an essential role in the innate immune response to infection and tissue injury. However, excessive macrophage activation may also significantly contribute to chronic inflammatory diseases. The Toll-like receptor (TLR) family are key regulators of innate immune responses in macrophages, and they are able to promote their survival and resistance against apoptosis. We, and others, have shown that the adaptive response gene, activating transcription factor 3 (ATF3), acts as a negative regulator of TLR signaling by repressing transcription of pro-inflammatory cytokines in primary mouse macrophages. Here, we describe a novel role for ATF3 as a component of TLR-mediated survival in macrophages. ATF3-deficient bone marrow macrophages show reduced survival in response to a range of TLR ligands and significantly higher apoptotic rates were observed in response to lipopolysaccharide, indicating that ATF3 is required to suppress apoptosis in macrophages. Furthermore, we show that ATF3 lies downstream of JNK signaling after TLR engagement, resulting in repression of pro-apoptotic Bak and Bax transcription.

A Phosphatidylinositol Species Acutely Generated by Activated Macrophages Regulates Innate Immune Responses

Activation of macrophages with stimuli of the innate immune response results in the intense remodeling of arachidonate-containing phospholipids, leading to the mobilization of large quantities of this fatty acid for conversion into biologically active eicosanoids. As a consequence of this process, the arachidonate levels in membrane phospholipids markedly decrease. We have applied mass spectrometry-based lipid profiling to study the levels of arachidonate-containing phospholipids under inflammatory activation of macrophages. We identify an unusual inositol phospholipid molecule, PI(20:4/20:4), the levels of which do not decrease but actually increase by 300% after activation of the macrophages. PI(20:4/20:4) is formed and degraded rapidly, suggesting a role for this molecule in regulating cell signaling events. Using a metabolipidomic approach consisting in exposing the cells to deuterium-labeled arachidonate at the time they are exposed to stimuli, we show that PI(20:4/20:4) biosynthesis occurs via the sequential incorporation of arachidonate, first into the sn-2 position of a preformed phosphatidylinositol (PI) molecule, followed by the rapid introduction of a second arachidonate moiety into the sn-1 position. Generation requires the participation of cytosolic phospholipase A2α and CoA-dependent acyltransferases. PI(20:4/20:4) formation is also detected in vivo in murine peritonitis exudates. Elevating the intracellular concentration of PI(20:4/20:4) by introducing the lipid into the cells results in enhancement of the microbicidal capacity of macrophages, as measured by reactive oxygen metabolite production and lysozyme release. These findings suggest that PI(20:4/20:4) is a novel bioactive inositol phospholipid molecule that regulates innate immune responses in macrophages.

Impact of Host Membrane Pore Formation by the Yersinia pseudotuberculosis Type III Secretion System on the Macrophage Innate Immune Response

Type III secretion systems (T3SSs) are used by Gram-negative pathogens to form pores in host membranes and deliver virulence-associated effector proteins inside host cells. In pathogenic Yersinia, the T3SS pore-forming proteins are YopB and YopD. Mammalian cells recognize the Yersinia T3SS, leading to a host response that includes secretion of the inflammatory cytokine interleukin-1β (IL-1β), Toll-like receptor (TLR)-independent expression of the stress-associated transcription factor Egr1 and the inflammatory cytokine tumor necrosis factor alpha (TNF-α), and host cell death. The known Yersinia T3SS effector proteins are dispensable for eliciting these responses, but YopB is essential. Three models describe how the Yersinia T3SS might trigger inflammation: (i) mammalian cells sense YopBD-mediated pore formation, (ii) innate immune stimuli gain access to the host cytoplasm through the YopBD pore, and/or (iii) the YopB-YopD translocon itself or its membrane insertion is proinflammatory. To test these models, we constructed a Yersinia pseudotuberculosis mutant expressing YopD devoid of its predicted transmembrane domain (YopD(ΔTM)) and lacking the T3SS cargo proteins YopHEMOJTN. This mutant formed pores in macrophages, but it could not mediate translocation of effector proteins inside host cells. Importantly, this mutant did not elicit rapid host cell death, IL-1β secretion, or TLR-independent Egr1 and TNF-α expression. These data suggest that YopBD-mediated translocation of unknown T3SS cargo leads to activation of host pathways influencing inflammation, cell death, and response to stress. As the YopD(ΔTM) Y. pseudotuberculosis mutant formed somewhat smaller pores with delayed kinetics, an alternative model is that the wild-type YopB-YopD translocon is specifically sensed by host cells.

Biofilm-derived Legionella pneumophila evades the innate immune response in macrophages

Legionella pneumophila, the causative agent of Legionnaire's disease, replicates in human alveolar macrophages to establish infection. There is no human-to-human transmission and the main source of infection is L. pneumophila biofilms established in air conditioners, water fountains, and hospital equipments. The biofilm structure provides protection to the organism from disinfectants and antibacterial agents. L. pneumophila infection in humans is characterized by a subtle initial immune response, giving time for the organism to establish infection before the patient succumbs to pneumonia. Planktonic L. pneumophila elicits a strong immune response in murine, but not in human macrophages enabling control of the infection. Interactions between planktonic L. pneumophila and murine or human macrophages have been studied for years, yet the interface between biofilm-derived L. pneumophila and macrophages has not been explored. Here, we demonstrate that biofilm-derived L. pneumophila replicates significantly more in murine macrophages than planktonic bacteria. In contrast to planktonic L. pneumophila, biofilm-derived L. pneumophila lacks flagellin expression, do not activate caspase-1 or -7 and trigger less cell death. In addition, while planktonic L. pneumophila is promptly delivered to lysosomes for degradation, most biofilm-derived bacteria were enclosed in a vacuole that did not fuse with lysosomes in murine macrophages. This study advances our understanding of the innate immune response to biofilm-derived L. pneumophila and closely reproduces the natural mode of infection in human.

Mouse macrophage innate immune response to chikungunya virus infection

BackgroundInfection with Chikungunya alphavirus (CHIKV) can cause severe arthralgia and chronic arthritis in humans with persistence of the virus in perivascular macrophages of the synovial membrane by mechanisms largely ill-characterized.FindingsWe herein analysed the innate immune response (cytokine and programmed cell death) of RAW264.7 mouse macrophages following CHIKV infection. We found that the infection was restrained to a small percentage of cells and was not associated with a robust type I IFN innate immune response (IFN-α4 and ISG56). TNF-α, IL-6 and GM-CSF expression were upregulated while IFN-γ, IL-1α, IL-2, IL-4, IL-5, IL-10 or IL-17 expression could not be evidenced prior to and after CHIKV exposure. Although CHIKV is known to drive apoptosis in many cell types, we found no canonical signs of programmed cell death (cleaved caspase-3, -9) in infected RAW264.7 cells.ConclusionThese data argue for the capacity of CHIKV to infect and drive a specific innate immune response in RAW264.7 macrophage cell which seems to be polarized to assist viral persistence through the control of apoptosis and IFN signalling.