Complement protein C1q directs macrophage polarization and limits inflammasome activity during the uptake of apoptotic cells.
ABSTRACT: Deficiency in C1q, the recognition component of the classical complement cascade and a pattern recognition receptor involved in apoptotic cell clearance, leads to lupus-like autoimmune diseases characterized by auto-antibodies to self proteins and aberrant innate immune cell activation likely due to impaired clearance of apoptotic cells. In this study, we developed an autologous system using primary human lymphocytes and human monocyte-derived macrophages (HMDMs) to characterize the effect of C1q on macrophage gene expression profiles during the uptake of apoptotic cells. C1q bound to autologous apoptotic lymphocytes modulated expression of genes associated with JAK/STAT signaling, chemotaxis, immunoregulation, and NLRP3 inflammasome activation in LPS-stimulated HMDMs. Specifically, C1q sequentially induced type I IFNs, IL-27, and IL-10 in LPS-stimulated HMDMs and IL-27 in HMDMs when incubated with apoptotic lymphocyte conditioned media. Coincubation with C1q tails prevented the induction of type I IFNs and IL-27 in a dose-dependent manner, and neutralization of type I IFNs partially prevented IL-27 induction by C1q. Finally, C1q decreased procaspase-1 cleavage and caspase-1-dependent cleavage of IL-1? suggesting a potent inhibitory effect of C1q on inflammasome activation. These results identify specific molecular pathways induced by C1q to suppress macrophage inflammation and provide potential therapeutic targets to control macrophage polarization and thus inflammation and autoimmunity.
Project description:In this study, we developed a unique system using primary human autologous lymphocytes and HMDMs to characterize the effect of C1q on macrophage gene expression profiles during the uptake of apoptotic cells. Our results showed that C1q bound to autologous apoptotic lymphocytes (AL) significantly modulated the response of HMDMs to LPS by increasing expression of cytokines, chemokines and effector molecules associated with immunoregulation and by directly suppressing caspase-1 dependent cleavage of IL-1beta. Overall design: Human monocyte-derived macrophages (HMDMs) were incubated with early apoptotic lymphocytes (EAL), late apoptotic lymphocytes (LAL), C1q-EAL and C1q-LAL for 1h and then and stimulated with 10 ng/ml ultra-pure LPS. After 3 h of LPS stimulation, total RNA was extracted using the Illustra RNAspin Mini Isolation Kit. Gene expression profiles were studied using the Human Gene 1.0 ST array (Affymetrix). Slides were scanned using the Affymetrix GCOS software (performed by the microarray core facility at University of California, Irvine). Data processing and analysis were performed using JMP Genomics 5.0 software (SAS Institue Inc.). Significant differences in gene expression compared to unstimulated HMDMs were identified by ANOVA test using Holm multiple testing method and a false positive rate (alpha error) of 0.05
Project description:Deficiency in complement component C1q is associated with an inability to clear apoptotic cells (efferocytosis) and aberrant inflammation in lupus, and identification of the pathways involved in these processes should reveal important regulatory mechanisms in lupus and other autoimmune or inflammatory diseases. In this study, C1q-dependent regulation of TNF?/IL-6 expression and efferocytosis was investigated using primary mouse bone marrow-derived macrophages and human monocyte-derived macrophages. C1q downregulated LPS-dependent TNF? production in mouse and human macrophages. While prolonged stimulation with C1q (18?h) was required to elicit a dampening of TNF? production from mouse macrophages, the human macrophages responded to C1q with immediate downregulation of TNF?. IL-6 production was unchanged in mouse and upregulated by human macrophages following prolonged stimulation with C1q. Our previous studies indicated that C1q programmed enhanced efferocytosis in mouse macrophages by enhancing expression of Mer tyrosine kinase and its ligand Gas6, a receptor-ligand pair that also inhibits proinflammatory signaling. Here, we demonstrated that C1q-dependent programming of human macrophage efferocytosis required protein synthesis; however, neither Mer nor the related receptor Axl was upregulated in human cells. In addition, while the C1q-collagen-like tails are sufficient for promoting C1q-dependent phagocytosis of antibody-coated targets, the C1q-tails failed to program enhanced efferocytosis or dampen TNF? production. These data further elucidate the mechanisms by which C1q regulates proinflammatory signaling and efferocytosis in macrophages, functions that are likely to influence the progression of autoimmunity and chronic inflammation.
Project description:BACKGROUND:The role of the alternative complement pathway and its mediation by retinal microglia and macrophages, is well-established in the pathogenesis of Age-Related Macular Degeneration (AMD). However, the contribution of the classical complement pathway towards the progression of retinal degenerations is not fully understood, including the role of complement component 1q (C1q) as a critical activator molecule of the classical pathway. Here, we investigated the contribution of C1q to progressive photoreceptor loss and neuroinflammation in retinal degenerations. METHODS:Wild-type (WT), C1qa knockout (C1qa-/-) and mice treated with a C1q inhibitor (ANX-M1; Annexon Biosciences), were exposed to photo-oxidative damage (PD) and were observed for progressive lesion development. Retinal function was assessed by electroretinography, followed by histological analyses to assess photoreceptor degeneration. Retinal inflammation was investigated through complement activation, macrophage recruitment and inflammasome expression using western blotting, qPCR and immunofluorescence. C1q was localised in human AMD donor retinas using immunohistochemistry. RESULTS:PD mice had increased levels of C1qa which correlated with increasing photoreceptor cell death and macrophage recruitment. C1qa-/- mice did not show any differences in photoreceptor loss or inflammation at 7 days compared to WT, however at 14 days after the onset of damage, C1qa-/- retinas displayed less photoreceptor cell death, reduced microglia/macrophage recruitment to the photoreceptor lesion, and higher visual function. C1qa-/- mice displayed reduced inflammasome and IL-1? expression in microglia and macrophages in the degenerating retina. Retinal neutralisation of C1q, using an intravitreally-delivered anti-C1q antibody, reduced the progression of retinal degeneration following PD, while systemic delivery had no effect. Finally, retinal C1q was found to be expressed by subretinal microglia/macrophages located in the outer retina of early AMD donor eyes, and in mouse PD retinas. CONCLUSIONS:Our data implicate subretinal macrophages, C1q and the classical pathway in progressive retinal degeneration. We demonstrate a role of local C1q produced by microglia/macrophages as an instigator of inflammasome activation and inflammation. Crucially, we have shown that retinal C1q neutralisation during disease progression may slow retinal atrophy, providing a novel strategy for the treatment of complement-mediated retinal degenerations including AMD.
Project description:Failure to efficiently clear apoptotic cells is linked to defects in development and the onset of autoimmunity. Complement component C1q is required for efficient engulfment of apoptotic cells in mice and humans; however, the molecular mechanisms leading to C1q-dependent engulfment are not fully understood. In this study, we used primary mouse macrophages to identify and characterize a novel molecular mechanism for macrophage-mediated C1q-dependent engulfment of apoptotic cells. We found that macrophage activation with C1q resulted in cycloheximide-sensitive enhanced engulfment, indicating a requirement for de novo protein synthesis. To investigate the cycloheximide-sensitive pathway, C1q-elicited macrophage transcripts were identified by microarray. C1q triggered the expression of Mer tyrosine kinase (Mer) and the Mer ligand growth arrest-specific 6: a receptor-ligand pair that mediates clearance of apoptotic cells. Full-length native C1q, and not the collagen-like tail or heat-denatured protein, stimulated Mer expression. This novel pathway is specific to C1q because mannose-binding lectin, a related collectin, failed to upregulate Mer expression and function. Soluble Mer-Fc fusion protein inhibited C1q-dependent engulfment of apoptotic cells, indicating a requirement for Mer. Moreover, Mer-deficient macrophages failed to respond to C1q with enhanced engulfment. Our results suggest that C1q elicits a macrophage phenotype specifically tailored for apoptotic cell clearance, and these data are consistent with the established requirement for C1q in prevention of autoimmunity.
Project description:Acute lung injury and acute respiratory distress syndrome (ALI/ARDS) is characterized by uncontrolled progressive lung inflammation. Macrophages serve a key role in the pathogenesis of ALI/ARDS. Macrophage pyroptosis is a process of cell death releasing the proinflammatory cytokines interleukin (IL)?1? and IL?18. It was hypothesized that macrophage pyroptosis may partially account for the uncontrolled lung inflammation of ALI/ARDS. In the present study, greater macrophage pyroptosis in lipopolysaccharide (LPS)?treated macrophages and the ALI/ARDS mouse model was observed. The expression of nucleotide?binding domain, leucine?rich?containing family, pyrin domain?containing (NLRP)3 and IL?1? and cleavage of caspase?1 were significantly elevated following LPS treatment accompanied by greater activation of p38 mitogen?activated protein kinase (MAPK) signaling in vitro and in vivo. However, blocking p38 MAPK signaling through the inhibitor SB203580 significantly suppressed the acute lung injury and excessive lung inflammation in vivo, consistent with the reduced expression of the NLRP3 inflammasome and IL?1? and cleavage of caspase?1. Pretreatment of the rat NR8383 macrophage cell line with SB203580 significantly decreased the population of caspase?1+PI+ pyroptotic cells and expression of NLRP3/IL?1?. However, a larger population of Annexin V+PI? apoptotic cells was observed following blocking of the p38 MAPK signaling pathway. The results indicated that blockage of p38 MAPK signaling pathway skewed macrophage cell death from proinflammatory pyroptosis towards non?inflammatory apoptosis. These effects may contribute to attenuated acute lung injury and excessive inflammation in the SB203580?treated mice. The results may provide a novel therapeutic strategy for the treatment of uncontrolled lung inflammation in patients with ALI/ARDS.
Project description:C5a is an inflammatory mediator generated by complement activation that positively regulates various arms of immune defense, including Toll-like receptor 4 (TLR4) signaling. The NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome is activated by pathogen products and cellular/tissue damage products and is a major contributor of IL-1?. In this study, we investigate whether C5a modulates lipopolysaccharide- (LPS-) induced NLRP3 inflammasome activation in myeloid cells. Appearance of plasma IL-1? during endotoxemia was reduced in C5aR1(-/-) mice when compared to wild-type mice. In vitro, C5a significantly enhanced LPS-induced production of IL-1? in bone marrow Ly6C-high inflammatory monocytes, accompanied by augmented intracellular pro-IL-1? expression. This effect was abolished during p38 blockade by SB 203580 and in the absence of C5aR1. Conversely, C5a suppressed LPS-induced macrophage production of IL-1?, which was accompanied by attenuated levels of pro-IL-1?, NLRP3, and caspase-1 expression. C5a's suppressive effects were negated during phosphoinositide 3-kinase (PI3K) inhibition by wortmannin but were largely preserved in the absence of C5aR1. Thus, C5a bidirectionally amplifies TLR4-mediated NLRP3 inflammasome activation in monocytes while suppressing this pathway in macrophages. However, as C5aR1 deficiency attenuates the IL-1? response to LPS challenge in vivo, our results suggest overall that C5a augments physiologic inflammasome responses.
Project description:Occupational exposure to respirable crystalline silica (cSiO2) has been etiologically linked to human autoimmunity. Intranasal instillation with cSiO2 triggers profuse inflammation in the lung and onset of autoimmunity in lupus-prone mice; however, dietary supplementation with the omega-3 polyunsaturated fatty acid docosahexaenoic acid (DHA) abrogates these responses. Inflammasome activation, IL-1 cytokine release, and death in alveolar macrophages following cSiO2 exposure are early and critical events that likely contribute to triggering premature autoimmune pathogenesis by this particle. Here we tested the hypothesis that DHA suppresses cSiO2-induced NLRP3 inflammasome activation, IL-1 cytokine release, and cell death in the macrophage. The model used was the murine macrophage RAW 264.7 cell line stably transfected with the inflammasome adapter protein ASC (RAW-ASC). Following priming with LPS, both the canonical activator nigericin and cSiO2 elicited robust inflammasome activation in RAW-ASC cells, as reflected by IL-1? release and caspase-1 activation. These responses were greatly diminished or absent in wild-type RAW cells. In contrast to IL-1?, cSiO2 induced IL-1? release in both RAW-ASC and to a lesser extent in RAW-WT cells after LPS priming. cSiO2-driven effects in RAW-ASC cells were confirmed in bone-marrow derived macrophages. Pre-incubating RAW-ASC cells with 10 and 25 ?M DHA for 24 h enriched this fatty acid in the phospholipids by 15- and 25-fold, respectively, at the expense of oleic acid. DHA pre-incubation suppressed inflammasome activation and release of IL-1? and IL-1? by nigericin, cSiO2, and two other crystals - monosodium urate and alum. DHA's suppressive effects were linked to inhibition of LPS-induced Nlrp3, Il1b, and Il1a transcription, potentially through the activation of PPAR?. Finally, nigericin-induced death was inflammasome-dependent, indicative of pyroptosis, and could be inhibited by DHA pretreatment. In contrast, cSiO2-induced death was inflammasome-independent and not inhibited by DHA. Taken together, these findings indicate that DHA suppresses cSiO2-induced inflammasome activation and IL-1 cytokine release in macrophages by acting at the level of priming, but was not protective against cSiO2-induced cell death.
Project description:BACKGROUND: Type I interferons (IFNs), including IFN-alpha (IFNA) and IFN-beta (IFNB), have anti-inflammatory properties and are used to treat patients with autoimmune and inflammatory disorders. However, little is known of the role of IFN-tau (IFNT), a type I IFN produced by ruminant animals for inflammation. Because IFNB has recently been shown to inhibit nucleotide-binding oligomerization domain-like receptor, pyrin domain-containing 3 (NLRP3) inflammasome activation and subsequent secretion of the potent inflammatory cytokine interleukin (IL)-1?, we examined the effects of ruminant IFNT on NLRP3 inflammasome-mediated IL-1? secretion in human THP-1 macrophages. METHODS AND RESULTS: IFNT dose-dependently inhibited IL-1? secretion induced by nano-silica, a well-known activators of NLRP3 inflammasomes, in human macrophages primed with lipopolysaccharide (LPS, TLR4 agonist) and Pam3CSK4 (TLR1/2 agonist). IFNT also suppressed phagocytosis of nano-silica and reactive oxygen species (ROS) generation. Western blot analysis showed that IFNT inhibited both pro-IL-1? and mature IL-1?. In addition, real-time RT-PCR analysis showed that IFNT suppressed IL-1? mRNA expression induced by LPS and Pam3CSK4. Although nano-silica particles did not induce IL-10 secretion, IFNT induced IL-10 secretion in a dose-dependent manner. Furthermore, IFNT-suppressed IL-1? secretion was restored by anti-IL-10 neutralizing antibody. CONCLUSIONS: Ruminant IFNT inhibits NLRP3 inflammasome-driven IL-1? secretion in human macrophages via multiple pathways, including the uptake of nano-silica particles, generation of ROS, and IL-10-mediated inhibition of pro-IL-1? induction. It may be a therapeutic alternative to IFNA and IFNB.
Project description:Inflammasome is an intracellular signaling complex of the innate immune system. Activation of inflammasomes promotes the secretion of interleukin 1? (IL-1?) and IL-18 and triggers pyroptosis. Caspase-1 and -11 (or -4/5 in human) in the canonical and non-canonical inflammasome pathways, respectively, are crucial for inflammasome-mediated inflammatory responses. Here we report that gasdermin D (GSDMD) is another crucial component of inflammasomes. We discovered the presence of GSDMD protein in nigericin-induced NLRP3 inflammasomes by a quantitative mass spectrometry-based analysis. Gene deletion of GSDMD demonstrated that GSDMD is required for pyroptosis and for the secretion but not proteolytic maturation of IL-1? in both canonical and non-canonical inflammasome responses. It was known that GSDMD is a substrate of caspase-1 and we showed its cleavage at the predicted site during inflammasome activation and that this cleavage was required for pyroptosis and IL-1? secretion. Expression of the N-terminal proteolytic fragment of GSDMD can trigger cell death and N-terminal modification such as tagging with Flag sequence disrupted the function of GSDMD. We also found that pro-caspase-1 is capable of processing GSDMD and ASC is not essential for GSDMD to function. Further analyses of LPS plus nigericin- or Salmonella typhimurium-treated macrophage cell lines and primary cells showed that apoptosis became apparent in Gsdmd(-/-) cells, indicating a suppression of apoptosis by pyroptosis. The induction of apoptosis required NLRP3 or other inflammasome receptors and ASC, and caspase-1 may partially contribute to the activation of apoptotic caspases in Gsdmd(-/-) cells. These data provide new insights into the molecular mechanisms of pyroptosis and reveal an unexpected interplay between apoptosis and pyroptosis.
Project description:Overactivation of the innate immune response underlies many forms of liver injury including that caused by hepatotoxins. Recent studies have demonstrated that macrophage autophagy regulates innate immunity and resultant tissue inflammation. Although hepatocyte autophagy has been shown to modulate hepatic injury, little is known about the role of autophagy in hepatic macrophages during the inflammatory response to acute toxic liver injury. Our aim therefore was to determine whether macrophage autophagy functions to down regulate hepatic inflammation.Mice with a LysM-CRE-mediated macrophage knockout of the autophagy gene ATG5 were examined for their response to toxin-induced liver injury from D-galactosamine/lipopolysaccharide (GalN/LPS).Knockout mice had increased liver injury from GalN/LPS as determined by significant increases in serum alanine aminotransferase, histological evidence of liver injury, positive terminal deoxynucleotide transferase-mediated deoxyuridine triphosphate nick end-labeling, caspase activation and mortality as compared to littermate controls. Levels of proinflammatory tumor necrosis factor and interleukin (IL)-6 hepatic mRNA and serum protein were unchanged, but serum IL-1? was significantly increased in knockout mice. The increase in serum IL-1? was secondary to elevated hepatic caspase 1 activation and inflammasome-mediated cleavage of pro-IL-1? to its active form. Cultured hepatic macrophages from GalN/LPS-treated knockout mice had similarly increased IL-1? production. Dysregulation of IL-1? was the mechanism of increased liver injury as an IL-1 receptor antagonist prevented injury in knockout mice in concert with decreased neutrophil activation.Macrophage autophagy functions to limit acute toxin-induced liver injury and death by inhibiting the generation of inflammasome-dependent IL-1?.