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While serum-circulating complement destroys invading pathogens, intracellularly active complement, termed the “complosome,” functions as a vital orchestrator of cell-metabolic events underlying T cell effector responses. Whether intracellular complement is also nonredundant for the activity of myeloid immune cells is currently unknown. Here, we show that monocytes and macrophages constitutively express complement component (C) 5 and generate autocrine C5a via formation of an intracellular C5 convertase. Cholesterol crystal sensing by macrophages induced C5aR1 signaling on mitochondrial membranes, which shifted ATP production via reverse electron chain flux toward reactive oxygen species generation and anaerobic glycolysis to favor IL-1β production, both at the transcriptional level and processing of pro–IL-1β. Consequently, atherosclerosis-prone mice lacking macrophage-specific C5ar1 had ameliorated cardiovascular disease on a high-cholesterol diet. Conversely, inflammatory gene signatures and IL-1β produced by cells in unstable atherosclerotic plaques of patients were normalized by a specific cell-permeable C5aR1 antagonist. Deficiency of the macrophage cell-autonomous C5 system also protected mice from crystal nephropathy mediated by folic acid. These data demonstrate the unexpected intracellular formation of a C5 convertase and identify C5aR1 as a direct modulator of mitochondrial function and inflammatory output from myeloid cells. Together, these findings suggest that the complosome is a contributor to the biologic processes underlying sterile inflammation and indicate that targeting this system could be beneficial in macrophage-dependent diseases, such as atherosclerosis.

Inter-α inhibitor proteins (IAIPs) are a family of endogenous plasma and extracellular matrix molecules. IAIPs suppress proinflammatory cytokines, limit excess complement activation, and bind extracellular histones to form IAIP-histone complexes, leading to neutralization of histone-associated cytotoxicity in models of sepsis. Many of these detrimental processes also play critical roles in the pathophysiology of ischemic stroke. In this study, we first assessed the clinical relevance of IAIPs in stroke and then tested the therapeutic efficacy of exogenous IAIPs in several experimental stroke models. IAIP levels were reduced in both ischemic stroke patients and in mice subjected to experimental ischemic stroke when compared with controls. Post-stroke administration of IAIP significantly improved stroke outcomes across multiple stroke models, even when given 6 hours after stroke onset. Importantly, the beneficial effects of delayed IAIP treatment were observed in both young and aged mice. Using targeted gene expression analysis, we identified a receptor for complement activation, C5aR1, that was highly suppressed in both the blood and brain of IAIP-treated animals. Subsequent experiments using C5aR1-knockout mice demonstrated that the beneficial effects of IAIPs are mediated in part by C5aR1. These results indicate that IAIP is a potential therapeutic candidate for the treatment of ischemic stroke.

Platelets contribute to the regulation of tissue neovascularization, although the specific factors underlying this function are unknown. Here, we identified the complement anaphylatoxin C5a-mediated activation of C5a receptor 1 (C5aR1) on platelets as a negative regulatory mechanism of vessel formation. We showed that platelets expressing C5aR1 exert an inhibitory effect on endothelial cell functions such as migration and 2D and 3D tube formation. Growth factor- and hypoxia-driven vascularization was markedly increased in C5ar1 -/- mice. Platelet-specific deletion of C5aR1 resulted in a proangiogenic phenotype with increased collateralization, capillarization and improved pericyte coverage. Mechanistically, we found that C5a induced preferential release of CXC chemokine ligand 4 (CXCL4, PF4) from platelets as an important antiangiogenic paracrine effector molecule. Interfering with the C5aR1-CXCL4 axis reversed the antiangiogenic effect of platelets both in vitro and in vivo.In conclusion, we identified a mechanism for the control of tissue neovascularization through C5a/C5aR1 axis activation in platelets and subsequent induction of the antiangiogenic factor CXCL4.

Singular blockade of C5a in experimental models of sepsis is known to confer protection by rescuing lethality and decreasing pro-inflammatory responses. However, the role of inhibiting C5a has not been evaluated in the context of sterile systemic inflammatory responses, like polytrauma and hemorrhagic shock (PT + HS). In our presented study, a novel and highly specific C5a L-aptamer, NoxD21, was used to block C5a activity in an experimental murine model of PT + HS. The aim of the study was to assess early modulation of inflammatory responses and lung damage 4 h after PT + HS induction. NoxD21-treated PT + HS mice displayed greater polymorphonuclear cell recruitment in the lung, increased pro-inflammatory cytokine levels in the bronchoalveolar lavage fluids (BALF) and reduced myeloperoxidase levels within the lung tissue. An in vitro model of the alveolar-capillary barrier was established to confirm these in vivo observations. Treatment with a polytrauma cocktail induced barrier damage only after 16 h, and NoxD21 treatment in vitro did not rescue this effect. Furthermore, to test the exact role of both the cognate receptors of C5a (C5aR1 and C5aR2), experimental PT + HS was induced in C5aR1 knockout (C5aR1 KO) and C5aR2 KO mice. Following 4 h of PT + HS, C5aR2 KO mice had significantly reduced IL-6 and IL-17 levels in the BALF without significant lung damage, and both, C5aR1 KO and C5aR2 KO PT + HS animals displayed reduced MPO levels within the lungs. In conclusion, the C5aR2 could be a putative driver of early local inflammatory responses in the lung after PT + HS.

Staphylococcus aureus causes necrotizing pneumonia by secreting toxins such as leukocidins that target front-line immune cells. The mechanism by which leukocidins kill innate immune cells and trigger inflammation during S. aureus lung infection, however, remains unresolved. Here, we explored human-induced pluripotent stem cell-derived macrophages (hiPSC-dMs) to study the interaction of the leukocidins Panton-Valentine leukocidin (PVL) and LukAB with lung macrophages, which are the initial leukocidin targets during S. aureus lung invasion. hiPSC-dMs were susceptible to the leukocidins PVL and LukAB and both leukocidins triggered NLPR3 inflammasome activation resulting in IL-1β secretion. hiPSC-dM cell death after LukAB exposure, however, was only temporarily dependent of NLRP3, although NLRP3 triggered marked cell death after PVL treatment. CRISPR/Cas9-mediated deletion of the PVL receptor, C5aR1, protected hiPSC-dMs from PVL cytotoxicity, despite the expression of other leukocidin receptors, such as CD45. PVL-deficient S. aureus had reduced ability to induce lung IL-1β levels in human C5aR1 knock-in mice. Unexpectedly, inhibiting NLRP3 activity resulted in increased wild-type S. aureus lung burdens. Our findings suggest that NLRP3 induces macrophage death and IL-1β secretion after PVL exposure and controls S. aureus lung burdens., (©2020 Society for Leukocyte Biology.)

Background: Food-induced anaphylaxis is a serious allergic reaction caused by Fcε-receptor activation on mast cells (MCs). The exact mechanisms breaking oral tolerance and the effector pathways driving food allergy remain elusive. As complement is activated in food-induced anaphylaxis, we aimed to assess the role of C5a in disease pathogenesis., Methods: Oral antigen-induced food-induced anaphylaxis was induced in BALB/c wild-type (wt) and C5ar1 -/- mice. Readouts included diarrhea development, changes in rectal temperature, hematocrit, antigen-specific serum IgE, MCPT-1, and intestinal MC numbers, as well as FcεR1-mediated MC functions including C5a receptor 1 (C5aR1) regulation. Further, histamine-mediated hypothermia and regulation of endothelial tight junctions were determined., Results: Repeated oral OVA challenge resulted in diarrhea, hypothermia, increased hematocrit, high OVA-specific serum IgE, and MCPT-1 levels in wt mice. Male C5ar1 -/- mice were completely whereas female C5ar1 -/- mice were partially protected from anaphylaxis development. Serum MCPT-1 levels were reduced gender-independent, whereas IgE levels were reduced in male but not in female C5ar1 -/- mice. Mechanistically, IgE-mediated degranulation and IL-6 production from C5ar1 -/- BMMCs of both sexes were significantly reduced. Importantly, FcεR1 cross-linking strongly upregulated C5aR1 MC expression in vitro and in vivo. Finally, C5ar1 -/- male mice were largely protected from histamine-induced hypovolemic shock, which was associated with protection from histamine-induced barrier dysfunction in vitro following C5aR targeting., Conclusions: Our findings identify C5aR1 activation as an important driver of IgE-mediated food allergy through regulation of allergen-specific IgE production, FcεR1-mediated MC degranulation, and histamine-driven effector functions preferentially in male mice., (© 2018 EAACI and John Wiley and Sons A/S. Published by John Wiley and Sons Ltd.)

Neuroinflammation and neurodegeneration are common during prion infection, but the mechanisms that underlie these pathological features are not well understood. Several components of innate immunity, such as Toll-like receptor (TLR) 4 and Complement C1q, have been shown to influence prion disease. To identify additional components of innate immunity that might impact prion disease within the central nervous system (CNS), we screened RNA from brains of pre-clinical and clinical 22L-infected mice for alterations in genes associated with innate immunity. Transcription of several genes encoding damage-associated molecular pattern (DAMP) proteins and receptors were increased in the brains of prion-infected mice. To investigate the role of some of these proteins in prion disease of the CNS, we infected mice deficient in DAMP receptor genes Tlr2, C3ar1, and C5ar1 with 22L scrapie. Elimination of TLR2 accelerated disease by a median of 10 days, while lack of C3aR1 or C5aR1 had no effect on disease tempo. Histopathologically, all knockout mouse strains tested were similar to infected control mice in gliosis, vacuolation, and PrPSc deposition. Analysis of proinflammatory markers in the brains of infected knockout mice indicated only a few alterations in gene expression suggesting that C5aR1 and TLR2 signaling did not act synergistically in the brains of prion-infected mice. These results indicate that signaling through TLR2 confers partial neuroprotection during prion infection., Competing Interests: The authors have declared that no competing interests exist.

Rationale: Inflammation and immunity play crucial roles in the development of hypertension. Complement activation-mediated innate immune response is involved in the regulation of hypertension and target-organ damage. However, whether complement-mediated T-cell functions could regulate blood pressure elevation in hypertension is still unclear., Objective: We aim to determine whether C3aR (complement component 3a receptor) and C5aR (complement component 5a receptor) could regulate blood pressure via modulating regulatory T cells (Tregs)., Methods and Results: We showed that angiotensin II (Ang II)-induced hypertension resulted in an elevated expression of C3aR and C5aR in Foxp3 (forkhead box P3)+ Tregs. By using C3aR and C5aR DKO (double knockout) mice, we showed that C3aR and C5aR deficiency together strikingly decreased both systolic and diastolic blood pressure in response to Ang II compared with WT (wild type), single C3aR-deficient (C3aR-/-), or C5aR-deficient (C5aR-/-) mice. Flow cytometric analysis showed that Ang II-induced Treg reduction in the kidney and blood was also blocked in DKO mice. Histological analysis indicated that renal and vascular structure remodeling and damage after Ang II treatment were attenuated in DKO mice compared with WT mice. In vitro, Ang II was able to stimulate C3aR and C5aR expression in cultured CD4+CD25+ natural Tregs. CD3 and CD28 antibody stimuli downregulated Foxp3 expression in WT but not DKO Tregs. More important, depletion of Tregs with CD25 antibody abolished the protective effects against Ang II-induced hypertension and target-organ damage in DKO mice. Adoptive transfer of DKO Tregs showed much more profound protective effects against Ang II-induced hypertension than WT Treg transfer. Furthermore, we demonstrated that C5aR expression in Foxp3+ Tregs was higher in hypertensive patients compared with normotensive individuals., Conclusions: C3aR and C5aR DKO-mediated Treg function prevents Ang II-induced hypertension and target-organ damage. Targeting C3aR and C5aR in Tregs specifically may be an alternative novel approach for hypertension treatment., (© 2018 American Heart Association, Inc.)

Sepsis caused by Neisseria meningitidis (meningococcus) is a rapidly progressing, life-threatening disease. Because its initial symptoms are rather unspecific, medical attention is often sought too late, i.e., when the systemic inflammatory response is already unleashed. This in turn limits the success of antibiotic treatment. The complement system is generally accepted as the most important innate immune determinant against invasive meningococcal disease since it protects the host through the bactericidal membrane attack complex. However, complement activation concomitantly liberates the C5a peptide, and it remains unclear whether this potent anaphylatoxin contributes to protection and/or drives the rapidly progressing immunopathogenesis associated with meningococcal disease. Here, we dissected the specific contribution of C5a receptor 1 (C5aR1), the canonical receptor for C5a, using a mouse model of meningococcal sepsis. Mice lacking C3 or C5 displayed susceptibility that was enhanced by >1,000-fold or 100-fold, respectively, consistent with the contribution of these components to protection. In clear contrast, C5ar1 -/- mice resisted invasive meningococcal infection and cleared N. meningitidis more rapidly than wild-type (WT) animals. This favorable outcome stemmed from an ameliorated inflammatory cytokine response to N. meningitidis in C5ar1 -/- mice in both in vivo and ex vivo whole-blood infections. In addition, inhibition of C5aR1 signaling without interference with the complement bactericidal activity reduced the inflammatory response also in human whole blood. Enticingly, pharmacologic C5aR1 blockade enhanced mouse survival and lowered meningococcal burden even when the treatment was administered after sepsis induction. Together, our findings demonstrate that C5aR1 drives the pathophysiology associated with meningococcal sepsis and provides a promising target for adjunctive therapy. IMPORTANCE The devastating consequences of N. meningitidis sepsis arise due to the rapidly arising and self-propagating inflammatory response that mobilizes antibacterial defenses but also drives the immunopathology associated with meningococcemia. The complement cascade provides innate broad-spectrum protection against infection by directly damaging the envelope of pathogenic microbes through the membrane attack complex and triggers an inflammatory response via the C5a peptide and its receptor C5aR1 aimed at mobilizing cellular effectors of immunity. Here, we consider the potential of separating the bactericidal activities of the complement cascade from its immune activating function to improve outcome of N. meningitidis sepsis. Our findings demonstrate that the specific genetic or pharmacological disruption of C5aR1 rapidly ameliorates disease by suppressing the pathogenic inflammatory response and, surprisingly, allows faster clearance of the bacterial infection. This outcome provides a clear demonstration of the therapeutic benefit of the use of C5aR1-specific inhibitors to improve the outcome of invasive meningococcal disease., (Copyright © 2018 Herrmann et al.)

C5a receptor 1 (C5aR1) is a G protein-coupled receptor for C5a and also an N-linked glycosylated protein. In addition to myeloid cells, C5aR1 is expressed on epithelial cells. In this study, we examined the role of C5aR1 in bacterial adhesion/colonization of renal tubular epithelium and addressed the underlying mechanisms of this role. We show that acute kidney infection was significantly reduced in mice with genetic deletion or through pharmacologic inhibition of C5aR1 following bladder inoculation with uropathogenic E. coli (UPEC). This was associated with reduced expression of terminal α-mannosyl residues (Man; a ligand for type 1 fimbriae of E. coli) on the luminal surface of renal tubular epithelium and reduction of early UPEC colonization in these mice. Confocal microscopy demonstrated that UPEC bind to Man on the luminal surface of renal tubular epithelium. In vitro analyses showed that C5a stimulation enhances Man expression in renal tubular epithelial cells and subsequent bacterial adhesion, which, at least in part, is dependent on TNF-α driven by C5aR1-mediated intracellular signaling. Our findings demonstrate a previously unknown pathogenic role for C5aR1 in acute pyelonephritis, proposing a potentially novel mechanism by which C5a/C5aR1 signaling mediates upregulation of carbohydrate ligands on renal tubules to facilitate UPEC adhesion.

Hypertension is one of the most common risk factors for the development heart failure in the general population. Inflammation plays a central role in this adverse remodeling and eventually to the development of heart failure. Circulating levels of Complement factor 5a (C5a) are increased in hypertensive patients and the C5a receptor is associated with the presence of cardiac fibrosis and inflammation in an experimental hypertension model. To test if C5aR is involved in adverse cardiac remodeling following pressure-overload, we induced transverse aortic constriction (TAC) in wildtype and C5a receptor deficient mice (C5aR -/- ). Six weeks after TAC, C5aR-/- animals showed a similar degree of cardiac hypertrophy and decrease in cardiac function as wild type mice (End Systolic Volume; 50.30±5.32 µl vs. 55.81±8.16 µl). In addition, other features of adverse cardiac remodeling like cardiomyocyte cell size (WGA staining), fibrosis (picrosirius red staining) or collagen degradation (matrix metalloproteinase activity assay) did not differ either. In conclusion, full body C5aR deficiency does not affect adverse cardiac remodeling after pressure-overload. However, our finding are in contrast with C5a inhibition studies. Our observations do present the role of C5a-C5aR in adverse cardiac remodeling and heart failure as controversial at the least.

Background: Pharmacologic inhibition of C5aR1, a receptor for the complement activation proinflammatory fragment, C5a, suppressed pathology and cognitive deficits in Alzheimer's disease (AD) mouse models. To validate that the effect of the antagonist was specifically via C5aR1 inhibition, mice lacking C5aR1 were generated and compared in behavior and pathology. In addition, since C5aR1 is primarily expressed on cells of the myeloid lineage, and only to a lesser extent on endothelial cells and neurons in brain, gene expression in microglia isolated from adult brain at multiple ages was compared across all genotypes., Methods: C5aR1 knock out mice were crossed to the Arctic AD mouse model, and characterized for pathology and for behavior performance in a hippocampal dependent memory task. CX3CR1 GFP and CCR2 RFP reporter mice were bred to C5aR1 sufficient and knockout wild type and Arctic mice to enable sorting of microglia (GFP-positive, RFP-negative) isolated from adult brain at 2, 5, 7 and 10 months of age followed by RNA-seq analysis., Results: A lack of C5aR1 prevented behavior deficits at 10 months, although amyloid plaque load was not altered. Immunohistochemical analysis showed no CCR2 + monocytes/macrophages near the plaques in the Arctic brain with or without C5aR1. Microglia were sorted from infiltrating monocytes (GFP and RFP-positive) for transcriptome analysis. RNA-seq analysis identified inflammation related genes as differentially expressed, with increased expression in the Arctic mice relative to wild type and decreased expression in the Arctic/C5aR1KO relative to Arctic. In addition, phagosomal-lysosomal gene expression was increased in the Arctic mice relative to wild type but further increased in the Arctic/C5aR1KO mice. A decrease in neuronal complexity was seen in hippocampus of 10 month old Arctic mice at the time that correlates with the behavior deficit, both of which were rescued in the Arctic/C5aR1KO., Conclusions: These data are consistent with microglial polarization in the absence of C5aR1 signaling reflecting decreased induction of inflammatory genes and enhancement of degradation/clearance pathways, which is accompanied by preservation of CA1 neuronal complexity and hippocampal dependent cognitive function. These results provide links between microglial responses and loss of cognitive performance and, combined with the previous pharmacological approach to inhibit C5aR1 signaling, support the potential of this receptor as a novel therapeutic target for AD in humans.

This study investigated the role of the alternative receptor for complement activation fragment C5a, C5aR2, in secondary inflammatory pathology after contusive spinal cord injury (SCI) in mice. C5ar2 -/- mice exhibited decreased intraparenchymal tumor necrosis factor alpha and interleukin-6 acutely post-injury, but these reductions did not translate into improved outcomes. We show that loss of C5aR2 leads to increased lesion volumes, reduced myelin sparing, and significantly worsened recovery from SCI in C5ar2 -/- animals compared to wild-type (WT) controls. Loss of C5aR2 did not alter leukocyte mobilization from the bone marrow in response to SCI, and neutrophil recruitment/presence at the lesion site was also not different between genotypes. Acute treatment of SCI mice with the selective C5aR1 antagonist, PMX205, improved SCI outcomes, compared to vehicle controls, and, importantly, fully alleviated the worsened recovery of C5ar2 -/- mice compared to their WT counterparts. Collectively, these findings indicate that C5aR2 is neuroprotective and a novel target to restrain injurious C5a signaling after a major neurotraumatic event.

C3 glomerulopathy is a potentially life-threatening disease of the kidney caused by dysregulated alternative pathway complement activation. The specific complement mediator(s) responsible for kidney injury in C3 glomerulopathy are yet to be defined and no specific therapy is currently available. We previously developed a mouse model of lethal C3 glomerulopathy with factor H and properdin gene double mutations. Therefore, we used this model to examine the role of C5 and C5a receptor (C5aR) in the pathogenesis of the disease. Disease severity in these factor H/properdin double-mutant mice was found to be correlated with plasma C5 levels, and prophylactic anti-C5 mAb therapy was effective in preventing lethal C3 glomerulopathy. When given to these double-mutant mice that had already developed active disease with severe proteinuria, anti-C5 mAb treatment also prevented death in half of the mice. Deficiency of C5aR significantly reduced disease severity, suggesting that C5aR-mediated inflammation contributed to C3 glomerulopathy. Thus, C5 and C5aR have a critical role in C3 glomerulopathy. Hence, early intervention targeting these pathways may be an effective therapeutic strategy for patients with C3 glomerulopathy., (Copyright © 2016 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Background: Recipients of vascularized composite allografts require aggressive and lifelong immunosuppression, and because the surgery is usually performed in nonlife-threatening situations, the development of strategies to minimize immunosuppression is especially pertinent for this procedure. We investigated how complement affects acute graft injury, alloimmunity, and immunosuppressive therapy., Methods: Vascularized composite allografts were transplanted from Balb/C to C57BL/6 mice that were complement deficient (C3 or double C3a Receptor (R)/C5aR), or treated with a targeted complement inhibitor (CR2-Crry). Allografts were analyzed for acute inflammation and injury, subacute T cell response, and survival in the absence and presence of cyclosporine A (CsA) therapy., Results: Allografts in C3-deficient or CR2-Crry-treated recipients were protected from skin and muscle ischemia-reperfusion injury (IRI). C3aR/C5aR-deficient recipients were more modestly protected. IgM and C3d colocalized within allografts from wild type and C3aR/C5aR-deficient recipients indicating IgM-mediated complement activation, and C3d deposition was almost absent in allografts from C3-deficient and CR2-Crry-treated recipients. Inflammatory cell infiltration and P-selectin expression was also significantly reduced in C3-deficient and CR2-Crry-treated recipients. Acute treatment with CR2-Crry or with 3 mg/kg per day CsA modestly, but significantly increased median allograft survival from 5.8 to 7.4 and 7.2 days, respectively. However, combined acute CR2-Crry treatment and CsA therapy increased mean graft survival to 17.2 days. Protection was associated with significantly reduced T cell infiltration of allografts and Tc1 cells in recipient spleens., Conclusions: Complement-mediated IRI augments graft allogenicity, and appropriate complement inhibition ameliorates IRI, decreases alloimmune priming and allows more immune-sparing CsA dosing.

Gaucher disease is caused by mutations in GBA1, which encodes the lysosomal enzyme glucocerebrosidase (GCase). GBA1 mutations drive extensive accumulation of glucosylceramide (GC) in multiple innate and adaptive immune cells in the spleen, liver, lung and bone marrow, often leading to chronic inflammation. The mechanisms that connect excess GC to tissue inflammation remain unknown. Here we show that activation of complement C5a and C5a receptor 1 (C5aR1) controls GC accumulation and the inflammatory response in experimental and clinical Gaucher disease. Marked local and systemic complement activation occurred in GCase-deficient mice or after pharmacological inhibition of GCase and was associated with GC storage, tissue inflammation and proinflammatory cytokine production. Whereas all GCase-inhibited mice died within 4-5 weeks, mice deficient in both GCase and C5aR1, and wild-type mice in which GCase and C5aR were pharmacologically inhibited, were protected from these adverse effects and consequently survived. In mice and humans, GCase deficiency was associated with strong formation of complement-activating GC-specific IgG autoantibodies, leading to complement activation and C5a generation. Subsequent C5aR1 activation controlled UDP-glucose ceramide glucosyltransferase production, thereby tipping the balance between GC formation and degradation. Thus, extensive GC storage induces complement-activating IgG autoantibodies that drive a pathway of C5a generation and C5aR1 activation that fuels a cycle of cellular GC accumulation, innate and adaptive immune cell recruitment and activation in Gaucher disease. As enzyme replacement and substrate reduction therapies are expensive and still associated with inflammation, increased risk of cancer and Parkinson disease, targeting C5aR1 may serve as a treatment option for patients with Gaucher disease and, possibly, other lysosomal storage diseases.

There is accumulating evidence during sepsis that cardiomyocyte (CM) homeostasis is compromised, resulting in cardiac dysfunction. An important role for complement in these outcomes is now demonstrated. Addition of C5a to electrically paced CMs caused prolonged elevations of intracellular Ca(2+) concentrations during diastole, together with the appearance of spontaneous Ca(2+) transients. In polymicrobial sepsis in mice, we found that three key homeostasis-regulating proteins in CMs were reduced: Na(+)/K(+)-ATPase, which is vital for effective action potentials in CMs, and two intracellular Ca(2+) concentration regulatory proteins, that is, sarcoplasmic/endoplasmic reticulum calcium ATPase 2 and the Na(+)/Ca(2+) exchanger. Sepsis caused reduced mRNA levels and reductions in protein concentrations in CMs for all three proteins. The absence of either C5a receptor mitigated sepsis-induced reductions in the three regulatory proteins. Absence of either C5a receptor (C5aR1 or C5aR2) diminished development of defective systolic and diastolic echocardiographic/Doppler parameters developing in the heart (cardiac output, left ventricular stroke volume, isovolumic relaxation, E' septal annulus, E/E' septal annulus, left ventricular diastolic volume). We also found in CMs from septic mice the presence of defective current densities for Ik1, l-type calcium channel, and Na(+)/Ca(2+) exchanger. These defects were accentuated in the copresence of C5a. These data suggest complement-related mechanisms responsible for development of cardiac dysfunction during sepsis., Competing Interests: The authors declare no commercial or financial conflicts of interest related to the studies., (Copyright © 2016 by The American Association of Immunologists, Inc.)

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-1β. In this study, we investigate whether C5a modulates lipopolysaccharide- (LPS-) induced NLRP3 inflammasome activation in myeloid cells. Appearance of plasma IL-1β during endotoxemia was reduced in C5aR1(-/-) mice when compared to wild-type mice. In vitro, C5a significantly enhanced LPS-induced production of IL-1β in bone marrow Ly6C-high inflammatory monocytes, accompanied by augmented intracellular pro-IL-1β 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-1β, which was accompanied by attenuated levels of pro-IL-1β, 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-1β response to LPS challenge in vivo, our results suggest overall that C5a augments physiologic inflammasome responses.

Atherogenic processes and vascular remodelling after arterial injury are controlled and driven by a plethora of factors amongst which the activation of the complement system is pivotal. Recently, we reported a clear correlation between high expressions of the second receptor for complement anaphylatoxin C5a, the C5a receptor-like 2 (C5L2, C5aR2), with high pro-inflammatory cytokine expression in advanced human atherosclerotic plaques. This prompted us to speculate that C5aR2 might have a functional role in atherosclerosis. We, therefore, investigated the role of C5aR2 in atherosclerosis and vascular remodelling. Here, we demonstrate that C5ar2 deletion, in atherosclerosis-prone mice, attenuates atherosclerotic as well as neointimal plaque formation, reduces macrophages and CD3+ T cells and induces features of plaque stability, as analysed by histomorphometry and quantitative immunohistochemistry. As a possible underlying mechanism, C5ar2-deficient plaques showed significantly reduced expression of C5a receptor (C5ar1), Tnf-α as well as Vcam-1, as determined by qPCR and quantitative immunohistochemistry. In addition, in vitro mechanistic studies revealed a reduction of these pro-inflammatory and pro-atherosclerotic mediators in C5ar2-deficient macrophages. Finally, blocking C5ar1 with antagonist JPE1375, in C5ar2(-/-)/Apoe(-/-) mice, led to a further reduction in neointimal plaque formation with reduced inflammation. In conclusion, C5ar2 deficiency attenuates atherosclerosis and neointimal plaque formation after arterial injury. This identifies C5aR2 as a promising target to reduce atherosclerosis and restenosis after vascular interventions.

Complement 5a (C5a), a potent immune mediator generated by complement activation, promotes tumor growth; however, its role in tumor metastasis remains unclear. We demonstrate that C5a contributes to tumor metastases by modulating tumor inflammation in hepatic metastases of colon cancer. Colon cancer cell lines generate C5a under serum-free conditions, and C5a levels increase over time in a murine syngeneic colon cancer hepatic metastasis model. Furthermore, in the absence of C5a receptor or upon pharmacological inhibition of C5a production with an anti-C5 monoclonal antibody, tumor metastasis is severely impaired. A lack of C5a receptor in colon cancer metastatic foci reduces the infiltration of macrophages, neutrophils, and dendritic cells, and the role for C5a receptor on these cells were further verified by bone marrow transplantation experiments. Moreover, C5a signaling increases the expression of the chemokine monocyte chemoattractant protein-1 and the anti-inflammatory molecules arginase-1, interleukin 10, and transforming growth factor β, but is inversely correlated with the expression of pro-inflammatory molecules, which suggests a mechanism for the role of C5a in the inflammatory microenvironment required for tumor metastasis. Our results indicate a new and potentially promising therapeutic application of complement C5a inhibitor for the treatment of malignant tumors., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Aims: Early reperfusion is mandatory for the treatment of acute myocardial infarction. This process, however, also induces additional loss of viable myocardium, called ischaemia-reperfusion (IR) injury. Complement activation plays an important role in IR injury, partly through binding of C5a to its major receptor (C5aR). We investigated the role of C5aR on infarct size and cardiac function in a model for myocardial IR injury., Methods and Results: BALB/c (WT) mice and C5aR(-/-) mice underwent coronary occlusion for 30 min, followed by reperfusion. Infarct size, determined 24 h after IR, was reduced in C5aR(-/-) mice compared with WT mice (28.5 ± 2.1 vs. 35.7 ± 2.5%, P = 0.017). Bone marrow (BM) chimaera experiments showed that this effect was due to the absence of C5aR on circulating leucocytes, since a similar reduction in infarct size was observed in WT mice with C5aR-deficient BM cells (25.3 ± 2.2 vs. 34.6 ± 2.8%, P < 0.05), but not in C5aR(-/-) mice with WT BM cells. Reduced infarct size was associated with fewer neutrophils, T cells, and macrophages in the infarcted area 24 h after IR in C5aR(-/-) mice, and also with lower levels of Caspase-3/7 indicating less inflammation and apoptosis. Echocardiography 4 weeks after IR showed an improved ejection fraction in C5aR(-/-) mice (25.8 ± 5.5 vs. 19.2 ± 5.4%, P < 0.001)., Conclusion: The absence of C5aR on circulating leucocytes reduces infarct size, is associated with reduced leucocyte infiltration and with less apoptosis in the infarcted myocardium, and improves cardiac function in a mouse model of myocardial IR injury. Selective blocking of C5aR might be a promising strategy to prevent myocardial IR injury., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2014. For permissions please email: journals.permissions@oup.com.)

There is considerable evidence that influenza A virus (IAV) promotes adherence, colonization, and superinfection by S. pneumoniae (Spn) and contributes to the pathogenesis of otitis media (OM). The complement system is a critical innate immune defense against both pathogens. To assess the role of the complement system in the host defense and the pathogenesis of acute pneumococcal OM following IAV infection, we employed a well-established transtympanically-induced mouse model of acute pneumococcal OM. We found that antecedent IAV infection enhanced the severity of acute pneumococcal OM. Mice deficient in complement C1qa (C1qa-/-) or factor B (Bf -/-) exhibited delayed viral and bacterial clearance from the middle ear and developed significant mucosal damage in the eustachian tube and middle ear. This indicates that both the classical and alternative complement pathways are critical for the oto-immune defense against acute pneumococcal OM following influenza infection. We also found that Spn increased complement activation following IAV infection. This was characterized by sustained increased levels of anaphylatoxins C3a and C5a in serum and middle ear lavage samples. In contrast, mice deficient in the complement C5a receptor (C5aR) demonstrated enhanced bacterial clearance and reduced severity of OM. Our data support the concept that C5a-C5aR interactions play a significant role in the pathogenesis of acute pneumococcal OM following IAV infection. It is possible that targeting the C5a-C5aR axis might prove useful in attenuating acute pneumococcal OM in patients with influenza infection.

Necrotizing and crescentic GN (NCGN) with a paucity of glomerular immunoglobulin deposits is associated with ANCA. The most common ANCA target antigens are myeloperoxidase (MPO) and proteinase 3. In a manner that requires activation of the alternative complement pathway, passive transfer of antibodies to mouse MPO (anti-MPO) induces a mouse model of ANCA NCGN that closely mimics human disease. Here, we confirm the importance of C5aR/CD88 in the mediation of anti-MPO-induced NCGN and report that C6 is not required. We further demonstrate that deficiency of C5a-like receptor (C5L2) has the reverse effect of C5aR/CD88 deficiency and results in more severe disease, indicating that C5aR/CD88 engagement enhances inflammation and C5L2 engagement suppresses inflammation. Oral administration of CCX168, a small molecule antagonist of human C5aR/CD88, ameliorated anti-MPO-induced NCGN in mice expressing human C5aR/CD88. These observations suggest that blockade of C5aR/CD88 might have therapeutic benefit in patients with ANCA-associated vasculitis and GN.

Objective: Transfusion of aged blood has been associated with increased morbidity and mortality in critically ill patients. During storage, erythrocytes release increasing numbers of microvesicles (red blood cell-derived microvesicles [RBC-MV]). We hypothesized that RBC-MV mediate some of the deleterious effects of aged blood transfusions., Approach and Results: We established a murine transfusion model using RBC-MV purified from aged mouse erythrocytes. Injection of RBC-MV into healthy mice had no effect. However, they aggravated pulmonary leukocyte sequestration and peripheral blood leukopenia induced by lipopolysaccharides. Lipopolysaccharide-induced proinflammatory cytokines were significantly increased in plasma after RBC-MV injection. These effects were not seen in C5aR-deficient mice. In vitro, RBC-MV bound C3 fragments after incubation with plasma but failed to bind immunoglobulins, C1q, or mannose-binding lectin. Preventing thrombin generation inhibited complement activation in vitro and in vivo and reversed the proinflammatory effects of RBC-MV in lipopolysaccharide-primed mice. Finally, the RBC-MV-induced phenotype was recapitulated using phosphatidylserine-expressing liposomes, suggesting that surface expression of phosphatidylserine by RBC-MV was mechanistically involved., Conclusions: These results point toward a thrombin-dependent mechanism of complement activation by RBC-MV independent of the classical, lectin, or alternative pathway. Besides identifying RBC-MV as potential mediators of transfusion-related morbidity, our findings may be relevant for other inflammatory disorders involving intravascular microvesicle release, for example, sickle cell disease or thrombotic microangiopathy.

Premature babies are particularly vulnerable to brain injury. In this study we focus on cortical brain damage associated with long-term cognitive, behavioral, attentional or socialization deficits in children born preterm. Using a mouse model of preterm birth (PTB), we demonstrated that complement component C5a contributes to fetal cortical brain injury. Disruption of cortical dendritic and axonal cytoarchitecture was observed in PTB-mice. Fetuses deficient in C5aR (-/-) did not show cortical brain damage. Treatment with antibody anti-C5, that prevents generation of C5a, also prevented cortical fetal brain injury in PTB-mice. C5a also showed a detrimental effect on fetal cortical neuron development and survival in vitro. Increased glutamate release was observed in cortical neurons in culture exposed to C5a. Blockade of C5aR prevented glutamate increase and restored neurons dendritic and axonal growth and survival. Similarly, increased glutamate levels - measured by (1)HMRS - were observed in vivo in PTB-fetuses compared to age-matched controls. The blockade of glutamate receptors prevented C5a-induced abnormal growth and increased cell death in isolated fetal cortical neurons. Simvastatin and pravastatin prevented cortical fetal brain developmental and metabolic abnormalities -in vivo and in vitro. Neuroprotective effects of statins were mediated by Akt/PKB signaling pathways. This study shows that complement activation plays a crucial role in cortical fetal brain injury in PTL and suggests that complement inhibitors and statins might be good therapeutic options to improve neonatal outcomes in preterm birth., (© 2013.)

The complement system is a potent component of the innate immune response, promoting inflammation and orchestrating defense against pathogens. However, dysregulation of complement is critical to several autoimmune and inflammatory syndromes. Elevated expression of the proinflammatory cytokine IL-1β is often linked to such diseases. In this study, we reveal the mechanistic link between complement and IL-1β secretion using murine dendritic cells. IL-1β secretion occurs following intracellular caspase-1 activation by inflammasomes. We show that complement elicits secretion of both IL-1β and IL-18 in vitro and in vivo via the NLRP3 inflammasome. This effect depends on the inflammasome components NLRP3 and ASC, as well as caspase-1 activity. Interestingly, sublethal complement membrane attack complex formation, but not the anaphylatoxins C3a and C5a, activated the NLRP3 inflammasome in vivo. These findings provide insight into the molecular processes underlying complement-mediated inflammation and highlight the possibility of targeting IL-1β to control complement-induced disease and pathological inflammation.

The pathways underlying dendritic cell (DC) activation in allergic asthma are incompletely understood. Here we demonstrate that adoptive transfer of ovalbumin-pulsed wild-type (wt) but not of C5a receptor-deficient (C5aR⁻/⁻) bone marrow (BM)-derived DCs (BMDCs) induced mixed T helper type 2 (Th2)/Th17 maladaptive immunity, associated with severe airway hyperresponsiveness, mucus production, and mixed eosinophilic/neutrophilic inflammation. Mechanistically, antigen uptake, processing, and CD11b expression were reduced in C5aR⁻/⁻ BMDCs. Further, interleukin (IL)-1β, -6, and -23 production were impaired resulting in reduced Th17 cell differentiation, associated with accelerated activated T-cell death in vitro and in vivo. Surprisingly, we found an increased frequency of CD11b(hi)CD11c(int)Gr1⁺F4/80⁺ cells, expressing arginase and nitric oxide synthase in C5aR⁻/⁻ BM preparations. Intratracheal administration of ovalbumin-pulsed wt DCs and sorted CD11b(hi)CD11c(int)Gr1⁺F4/80⁺ C5aR⁻/⁻ cells reduced Th2 immune responses in vivo. Together, we uncover novel roles for C5aR in Th17 differentiation, T-cell survival, and differentiation of a DC-suppressor population controlling Th2 immunity in experimental allergic asthma.

The protective immunity induced by the whole-killed parasite vaccine against malarial blood-stage infection is dependent on the CD4(+) T cell response. However, the mechanism underlying this robust CD4(+) T cell response elicited by the whole-killed parasite vaccine is still largely unknown. In this study, we observe that immunization with Plasmodium yoelii-parasitized RBC lysate activates complement C5 and generates C5a. However, the protective efficacy against P. yoelii 17XL challenge is considerably reduced, and the malaria-specific CD4(+) T cell activation and memory T cell differentiation are largely suppressed in the C5aR-deficient (C5aR(-/-)) mice. An adoptive transfer assay demonstrates that the reduced protection of C5aR(-/-) mice is closely associated with the severely impaired CD4(+) T cell response. This is further confirmed by the fact that administration of C5aR antagonist significantly reduces the protective efficacy of the immunized B cell-deficient mice. Further study indicates that the defective CD4(+) T cell response in C5aR(-/-) mice is unlikely involved in the expansion of CD4(+)CD25(+)Foxp3(+) T cells, but strongly linked to a defect in dendritic cell (DC) maturation and the ability to allostimulate CD4(+) T cells. These results demonstrate that C5aR signaling is essential for the optimal induction of the malaria-specific CD4(+) T cell response by the whole-killed parasite vaccine through modulation of DCs function, which provides us with new clues to design an effective blood-stage subunit vaccine and helps us to understand the mechanism by which the T cell response is regulated by the complement system.

Objective: To investigate the impact of whole body C5a receptor (C5aR) deficiency on energy metabolism and fat storage., Design: Male wildtype (WT) and C5aR knockout (C5aRKO) mice were fed a low fat (CHOW) or a high fat high sucrose diet-induced obesity (DIO) diet for 14 weeks. Body weight and food intake were measured weekly. Indirect calorimetry, dietary fatload clearance, insulin and glucose tolerance tests were also evaluated. Liver, muscle and adipose tissue mRNA gene expression were measured by RT-PCR., Results: At week one and 12, C5aRKO mice on DIO had increased oxygen consumption. After 12 weeks, although food intake was comparable, C5aRKO mice had lower body weight (-7% CHOW, -12% DIO) as well as smaller gonadal (-38% CHOW, -36% DIO) and inguinal (-29% CHOW, -30% DIO) fat pads than their WT counterparts. Conversely, in WT mice, C5aR was upregulated in DIO vs CHOW diets in gonadal adipose tissue, muscle and liver, while C5L2 mRNA expression was lower in C5aRKO on both diet. Furthermore, blood analysis showed lower plasma triglyceride and non-esterified fatty acid levels in both C5aRKO groups, with faster postprandial triglyceride clearance after a fatload. Additionally, C5aRKO mice showed lower CD36 expression in gonadal and muscle on both diets, while DGAT1 expression was higher in gonadal (CHOW) and liver (CHOW and DIO) and PPARγ was increased in muscle and liver., Conclusion: These observations point towards a role (either direct or indirect) for C5aR in energy expenditure and fat storage, suggesting a dual role for C5aR in metabolism as well as in immunity.

Thymus-derived (natural) CD4(+) FoxP3(+) regulatory T cells (nT reg cells) are required for immune homeostasis and self-tolerance, but must be stringently controlled to permit expansion of protective immunity. Previous findings linking signals transmitted through T cell-expressed C5a receptor (C5aR) and C3a receptor (C3aR) to activation, differentiation, and expansion of conventional CD4(+)CD25(-) T cells (T conv cells), raised the possibility that C3aR/C5aR signaling on nT reg cells could physiologically modulate nT reg cell function and thereby further impact the induced strength of T cell immune responses. In this study, we demonstrate that nT reg cells express C3aR and C5aR, and that signaling through these receptors inhibits nT reg cell function. Genetic and pharmacological blockade of C3aR/C5aR signal transduction in nT reg cells augments in vitro and in vivo suppression, abrogates autoimmune colitis, and prolongs allogeneic skin graft survival. Mechanisms involve C3a/C5a-induced phosphorylation of AKT and, as a consequence, phosphorylation of the transcription factor Foxo1, which results in lowered nT reg cell Foxp3 expression. The documentation that C3a/C3aR and C5a/C5aR modulate nT reg cell function via controlling Foxp3 expression suggests targeting this pathway could be exploited to manipulate pathogenic or protective T cell responses.

The complement system is an important part of innate immunity. Complement activation generates a set of effector molecules with diverse biological functions. C5a is a crucial terminal component of the complement cascade. Several reports suggest that C5a can support nociceptive sensitization and inflammation in various models, including models of incisional pain. However, information concerning the differential effects of C5a on specific modalities of nociception, the role of C5a in supporting neutrophil infiltration, secondary nociceptive mediator generation, and the location of the relevant populations of C5a receptors supporting incisional sensitization are needed. In these studies we utilized C5a receptor-null mice (C5aR(-/-)) and matched controls to study nociceptive changes after hind paw incision. Heat hyperalgesia and mechanical allodynia were measured for 4 days after incision. We also followed hind paw edema, wound area neutrophil infiltration using the myeloperoxidase assay, and interleukin-1β and nerve growth factor levels using both enzyme-linked immunosorbent assay and immunohistochemical techniques. The main findings were: (1) Heat vs mechanical nociceptive sensitization after incision were differentially reduced in C5aR(-/-) mice, with thermal sensitization affected throughout the postincisional period but mechanical sensitization affected only at later time points; (2) Edema developed after incision in wild-type mice but only slightly and transiently in C5aR(-/-) mice, and (3) Deletion of C5aR blocked interleukin-1β and nerve growth factor production near the wound site. These findings demonstrate that the complement system component C5a is a novel biomarker and mediator associated with postsurgical nociceptive processing. C5aR may provide a novel target for the control of pain and inflammation after surgery., (Copyright © 2011 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.)

Pneumococcal meningitis is the most common and severe form of bacterial meningitis. Fatality rates are substantial, and long-term sequelae develop in about half of survivors. Disease outcome has been related to the severity of the proinflammatory response in the subarachnoid space. The complement system, which mediates key inflammatory processes, has been implicated as a modulator of pneumococcal meningitis disease severity in animal studies. Additionally, SNPs in genes encoding complement pathway proteins have been linked to susceptibility to pneumococcal infection, although no associations with disease severity or outcome have been established. Here, we have performed a robust prospective nationwide genetic association study in patients with bacterial meningitis and found that a common nonsynonymous complement component 5 (C5) SNP (rs17611) is associated with unfavorable disease outcome. C5 fragment levels in cerebrospinal fluid (CSF) of patients with bacterial meningitis correlated with several clinical indicators of poor prognosis. Consistent with these human data, C5a receptor-deficient mice with pneumococcal meningitis had lower CSF wbc counts and decreased brain damage compared with WT mice. Adjuvant treatment with C5-specific monoclonal antibodies prevented death in all mice with pneumococcal meningitis. Thus, our results suggest C5-specific monoclonal antibodies could be a promising new antiinflammatory adjuvant therapy for pneumococcal meningitis.

The C5a anaphylatoxin receptor (C5aR; CD88) is activated as part of the complement cascade and exerts important inflammatory, antimicrobial, and regulatory functions, at least in part, via crosstalk with TLRs. However, the periodontal pathogen Porphyromonas gingivalis can control C5aR activation by generating C5a through its own C5 convertase-like enzymatic activity. In this paper, we show that P. gingivalis uses this mechanism to proactively and selectively inhibit TLR2-induced IL-12p70, whereas the same pathogen-instigated C5aR-TLR2 crosstalk upregulates other inflammatory and bone-resorptive cytokines (IL-1β, IL-6, and TNF-α). In vivo, the ability of P. gingivalis to manipulate TLR2 activation via the C5a-C5aR axis allowed it to escape IL-12p70-dependent immune clearance and to cause inflammatory bone loss in a murine model of experimental periodontitis. In the latter regard, C5aR-deficient or TLR2-deficient mice were both resistant to periodontal bone loss, in stark contrast with wild-type control mice, which is consistent with the interdependent interactions of C5aR and TLR2 in P. gingivalis immune evasion and induction of bone-resorptive cytokines. In conclusion, P. gingivalis targets C5aR to promote its adaptive fitness and cause periodontal disease. Given the current availability of safe and effective C5aR antagonists, pharmacological blockade of C5aR could act therapeutically in human periodontitis and reduce associated systemic risks.

Interaction between C5a, a product of complement activation, and its receptor (C5aR) upregulates antigen-specific T cell responses by modulating the activation of antigen-presenting cells and T cells. Whether this C5a-C5aR interaction contributes to the immune responses that promote renal allograft rejection is unknown. Here, we found that deficiency of C5aR in both graft and recipient reduced allospecific T cell responses and prolonged renal allograft survival. In addition, lack of C5aR impaired the function of donor and recipient antigen-presenting cells and inhibited the response of recipient T cells to allostimulation. Furthermore, deficiency of C5aR in both graft and recipient reduced early inflammation in the grafts, with less cellular infiltration around the vessels and fewer F4/80 positive cells in the peritubular interstitium. These data demonstrate that C5aR is critical for a full adaptive immune response and mediates renal allograft rejection. Engagement of C5aR on dendritic cells and T cells modulates their function, enhancing allospecific T cell responses that lead to allograft rejection. Targeting C5a signaling may have therapeutic potential for T cell-mediated graft rejection.

Alzheimer's disease (AD), a progressive neurodegenerative disease characterized by the accumulation of amyloid-beta protein and neuronal loss, is the leading cause of age-related dementia in the world today. The disease is also associated with neuroinflammation, robust activation of astrocytes and microglia, and evidence of activation of the complement system, localized with both fibrillar amyloid-beta (fAbeta) plaques and tangles. The observations are consistent with a complement-dependent component of AD progression. We have previously shown that inhibition of the major complement receptor for C5a (CD88) with the antagonist PMX205 results in a significant reduction in pathology in two mouse models of AD. To further characterize the role of complement in AD-related neuroinflammation, we examined the age- and disease-associated expression of CD88 in brain of transgenic mouse models of AD and the influence of PMX205 on the presence of various complement activation products using flow cytometry, western blot, and immunohistochemistry. CD88 was found to be up-regulated in microglia, in the immediate vicinity of amyloid plaques. While thioflavine plaque load and glial recruitment is significantly reduced after treatment with PMX205, C1q remains co-localized with fAbeta plaques and C3 is still expressed by the recruited astrocytes. Thus, with PMX205, potentially beneficial activities of these early complement components may remain intact, while detrimental activities resulting from C5a-CD88 interaction are inhibited. This further supports the targeted inhibition of specific complement mediated activities as an approach for AD therapy.

C5a is a proinflammatory mediator that has recently been shown to regulate adaptive immune responses. Here we demonstrate that C5a receptor (C5aR) signaling in DC affects the development of Treg and Th17 cells. Genetic ablation or pharmacological targeting of the C5aR in spleen-derived DC results in increased production of TGF-beta leading to de novo differentiation of Foxp3(+) Treg within 12 h after co-incubation with CD4(+) T cells from DO11.10/RAG2(-/-) mice. Stimulation of C5aR(-/-) DC with OVA and TLR2 ligand Pam(3)CSK(4) increased TGF-beta production and induced high levels of IL-6 and IL-23 but only minor amounts of IL-12 leading to differentiation of Th cells producing IL-17A and IL-21. Th17 differentiation was also found in vivo after adoptive transfer of CD4(+) Th cell into C5aR(-/-) mice immunized with OVA and Pam(3)CSK(4). The altered cytokine production of C5aR(-/-) DC was associated with low steady state MHC class II expression and an impaired ability to upregulate CD86 and CD40 in response to TLR2. Our data suggest critical roles for C5aR in Treg and Th17-cell differentiation through regulation of DC function.

It is known that C3 is required for optimal expansion of T cells during acute viral infections. However, it is not yet determined whether T cell responses to intracellular bacterial infections require C3. Therefore, we have investigated the requirement for C3 to elicit potent T cell responses to Listeria monocytogenes (LM). We show that expansion of Ag-specific CD8 and CD4 T cells during a primary response to LM was markedly reduced in the absence of C3 activity. Further studies indicated that, unlike in an influenza virus infection, the regulation of LM-specific T cell responses by C3 might not involve the downstream effector C5a. Moreover, reduced T cell responses to LM was not linked to defective maturation of dendritic cells or developmental anomalies in the peripheral T cell compartment of C3-deficient mice. Experiments involving adoptive transfer of C3-deficient CD8 T cells into the C3-sufficient environment of wild-type mice showed that these T cells do not have intrinsic proliferative defects, and a paracrine source of C3 will suffice for clonal expansion of CD8 T cells in vivo. However, stimulation of purified C3-deficient CD8 T cells by plastic-immobilized anti-CD3 showed that C3 promotes T cell proliferation directly, independent of its effects on APC. On the basis of these findings, we propose that diminished T cell responses to LM in C3-deficient mice might be at least in part due to lack of direct effects of C3 on T cells. These studies have furthered our understanding of C3-mediated regulation of T cell immunity to intracellular pathogens.

Toll-like receptors (TLRs) and complement are 2 major components of innate immunity that provide a first-line host defense and shape the adaptive immune responses. We show here that coincidental activation of complement and several TLRs in mice led to the synergistic production of serum factors that promoted T-helper cell 17 (Th17) differentiation from anti-CD3/CD28 or antigen-stimulated T cells. Although multiple TLR-triggered cytokines were regulated by complement, Th17 cell-promoting activity in the serum was correlated with interleukin (IL)-6 induction, and antibody neutralization of IL-6 abrogated the complement effect. By using both in vitro and in vivo approaches, we examined in more detail the mechanism and physiologic implication of complement/TLR4 interaction on Th17-cell differentiation. We found that the complement effect required C5a receptor, was evident at physiologically relevant levels of C5a, and could be demonstrated in cultured peritoneal macrophages as well as in the setting of antigen immunization. Importantly, despite an inhibitory effect of complement on IL-23 production, complement-promoted Th17 cells were functionally competent in causing autoimmunity in an adoptive transfer model of experimental autoimmune encephalomyelitis. Collectively, these data establish a link between complement/TLR interaction and Th17-cell differentiation and provide new insight into the mechanism of action of complement in autoimmunity.

Our recent studies have shown that immune cell-produced complement provides costimulatory and survival signals to naive CD4(+) T cells. Whether these signals are similarly required during effector cell expansion and what molecular pathways link locally produced complement to T-cell survival were not clarified. To address this, we stimulated monoclonal and polyclonal T cells in vitro and in vivo with antigen-presenting cells (APCs) deficient in the complement regulatory protein, decay accelerating factor (DAF), and/or the complement component C3. We found that T-cell expansion induced by DAF-deficient APCs was augmented with diminished T-cell apoptosis, whereas T-cell expansion induced by C3(-/-) APCs was reduced because of enhanced T-cell apoptosis. These effects were traced to locally produced C5a, which through binding to T cell-expressed C5aR, enhanced expression of Bcl-2 and prevented Fas up-regulation. The results show that C5aR signal transduction in T cells is important to allow optimal T-cell expansion, as well as to maintain naive cell viability, and does so by suppressing programmed cell death.

C5 deficient mice are highly resistant to experimental autoimmune myasthenia gravis (EAMG) despite intact immune response to acetylcholine receptor (AChR), validating the pivotal role played by membrane attack complex (MAC, C5b-9) in neuromuscular junction destruction. To distinguish the significance of C5a from that of C5b in EAMG pathogenesis, C5a receptor (C5aR) knockout (KO) and wild-type (WT) mice were immunized with AChR to induce pathogenic anti-AChR antibodies. In contrast with C5 deficient mice, C5aR KO mice were equally susceptible to EAMG as WT mice and exhibited comparable antibody and lymphocyte proliferation response to AChR implicating that C5a is not involved in EAMG development.

The host response to intravascular, Gram-negative bacteria includes profound immunologic, hematologic and physiologic changes. Numerous host defense mechanisms are activated by Gram-negative bacteria, including the complement system. Activation of the complement system leads to cleavage of C5 with subsequent generation of the C5a anaphylatoxin peptide. C5a mediates potent, proinflammatory activities by binding to the C5a receptor (C5aR, CD88). In this study, we report the targeted disruption of the murine C5aR gene (C5aR-/- mice) and define the role of the C5aR in a model of Gram-negative bacteremia. Following an intravenous infusion of heat-killed Escherichia coli, the C5aR-/- mice were completely protected from the mortality suffered by their wild-type littermates (P<0.001). The C5aR-/- mice were also significantly (P=0.008) more resistant to mortality following an intravenous infusion of purified E. coli endotoxin compared to the wild-type littermates. In addition, the C5aR-/- mice were resistant to the thrombocytopenia and hemoconcentration observed in wild-type animals. Lethality in the wild-type mice was reversed by pre-treatment with either the histamine antagonist diphenhydramine or triprolidine. The wild-type littermates were also rescued following pre-treatment with the basophil and mast cell-stabilizing agent - cromolyn sodium. Collectively, these data demonstrate that not only is the absence of the C5aR protective in E. coli bacteremia, but that C5aR-dependent histamine release plays a major role in shock induced by Gram-negative septicemia. Moreover, they provide additional in vivo evidence that C3a and C5a have divergent biological functions in Gram-negative bacteremia and shock.

The complement system, an important part of the innate immune system, provides protection against invading pathogens, in part through its proinflammatory activities. Although most complement proteins are synthesized locally in the brain and the relevant complement receptors are expressed on resident brain cells, little is known about brain-specific role(s) of the complement system. C3a and C5a, complement-derived peptides with anaphylatoxic properties, have been implicated in noninflammatory functions, such as tissue regeneration and neuroprotection. Recently, we have shown that signaling through C3a receptor (C3aR) is involved in the regulation of neurogenesis. In the present study, we assessed basal neurogenesis in mice lacking C5a receptor (C5aR(-/-)) and mice expressing C3a and C5a, respectively in the CNS under the control of glial fibrillary acidic protein (GFAP) promoter (C3a/GFAP and C5a/GFAP, respectively) and thus without the requirement for complement activation. We did not observe any difference among C5aR(-/-), C3a/GFAP and C5a/GFAP mice and their respective controls in the number of newly formed neuroblasts and newly formed neurons in the subventricular zone (SVZ) of lateral ventricles and hippocampal dentate gyrus, the two neurogenic niches in the adult brain, or the olfactory bulb, the final destination of new neurons formed in the SVZ. Our results indicate that signaling through C5aR is not involved in basal neurogenesis in adult mice and that basal neurogenesis in adult C3a/GFAP and C5a/GFAP mice is not altered., (2007 Wiley-Liss, Inc.)

This article addresses how quantitative models such as the one proposed in the companion article can be used to study cellular network perturbations such as knockdowns and pharmacological perturbations in a predictive manner. Using the kinetic model for cytosolic calcium dynamics in RAW 264.7 cells developed in the companion article, the calcium response to complement 5a (C5a) for the knockdown of seven proteins (C5a receptor; G-beta-2; G-alpha,i-2,3; regulator of G-protein signaling-10; G-protein coupled receptor kinase-2; phospholipase C beta-3; arrestin) is predicted and validated against the data from the Alliance for Cellular Signaling. The knockdown responses provide insights into how altered expressions of important proteins in disease states result in intermediate measurable phenotypes. Long-term response and long-term dose response have also been predicted, providing insights into how the receptor desensitization, internalization, and recycle result in tolerance. Sensitivity analysis of long-term response shows that the mechanisms and parameters in the receptor recycle path are important for long-term calcium dynamics.

Recent findings indicate that complement activation--involving specifically C3 and C5--contributes to antiphospholipid (aPL)-mediated thrombosis. Two complement effector pathways are initiated by the cleavage of C5, C5a and C5b, which leads to the formation of the C5b-9 membrane attack complex. To delineate and distinguish the role of C5a from the C5b-9 membrane attack complex seeded by C5b, we examined the in vivo effects (thrombosis) of aPL on C5a receptor-deficient (C5aR-/-) mice. C5aR-/- and C5aR+/+ mice were injected with IgM or with IgG from two different patients with APS (IgM-APS or IgG-APS) or with control IgM or IgG (IgM-NHS or IgG-NHS) twice. Complement fixing activity of the Ig fractions and anticardiolipin activity in the sera of the mice were determined by enzyme-linked immunosorbent assay. Surgical procedures to study thrombus dynamics were performed. IgM-APS but not IgG-APS fixed C1q to cardiolipin-coated plates. IgM-APS significantly enhanced thrombus size in C5aR+/+ mice compared to C5aR+/+ mice treated with IgM-NHS (3198 +/- 2361 microm2 versus 585 +/- 460 microm2). C5aR-/- mice treated with IgM-APS showed a significant reduction in thrombi size as compared with C5aR+/+ mice (676 +/- 690 microm2 versus 3198 +/- 2361 microm2; P = 0.001). IgG-APS enhanced thrombus formation significantly in C5aR+/+ when compared to IgG-NHS-treated mice (3507 +/- 965 microm2 versus 1321 +/- 798 microm2), and these effects were not altered in C5aR-/- mice (3400 +/- 1681 microm2). The data indicate that C5aR-/- mice are protected from the thrombogenic effects of some aPL antibodies.

The in vitro macrophage response to zymosan has been attributed to Toll-like receptor 2 (TLR2). Whether TLR2 is obligatory for the zymosan-induced in vivo response has not been assessed. The importance of this question is underscored by the fact that zymosan activates complement in a cell-independent manner. We have investigated whether the in vitro observation of TLR2 as the dominant zymosan receptor on macrophages would translate to an experimental peritonitis model in vivo. We have treated mice with zymosan, resulting in significant leukocyte (primarily neutrophil) accumulation in the peritoneum at 4 h. Zymosan-mediated leukocyte recruitment was TLR2 independent, but was predominantly dependent on the complement components, C3 and C5a with a minor contribution from LTB4. Peritoneal neutrophilia was 50% mast cell dependent and this defect was reproduced using C5a receptor (C5aR)-deficient mast cells in mast cell-deficient mice, suggesting that C5aR is responsible for mast cell activation following zymosan challenge. By 24 h, the response to zymosan involved primarily monocyte recruitment and was C3 and C5aR independent. Taken together, these studies indicate that the in vivo inflammatory response to zymosan does not necessarily mimic the TLR2 dependence observed in vitro, and that complement plays a dominant role in early, but not late, zymosan-mediated peritonitis.

The development and progression of systemic lupus erythematosus (SLE) is strongly associated with complement activation and deposition. To characterize the role of C5a and its receptor (C5aR) in SLE, C5aR-deficient mice were backcrossed nine generations onto the lupus-like MRL(lpr) genetic background. Evidence is presented that C5aR modulates both renal injury and T cell responses in MRL(lpr) mouse. C5aR-deficient MRL(lpr) mice had prolonged viability, with a mean survival time of 33.0 wk compared with 22.6 wk in control mice. Renal injury was also attenuated in the C5aR-/- MRL(lpr) mice. At 20 wk of age C5aR-/- MRL(lpr) mice had a complete absence of glomerular crescents and marked reductions in glomerular hypercellularity. There was no difference in the degree of glomerular C3 deposition; however, IgG deposits were reduced in the C5aR-/- MRL(lpr) mice. The reduction in glomerular injury was also associated with a four-fold decrease in renal CD4+ T cell infiltrates. Whereas there were modest differences in total IgG anti-dsDNA antibody titers, C5aR-deficient mice had 3.5-fold higher levels of IgG1 and 15-fold lower levels of IgG2a anti-dsDNA antibody titers compared to controls. The differences in anti-dsDNA IgG subclasses were associated with reduced CD4+ Th-1 responses in the C5aR-/- MRL(lpr) mice, including diminished production of IL-12p70, IFN-gamma, and increased expression of the Th-2 transcription factor GATA-3. These findings indicate that the C5aR plays a major role in modulating complement-dependent renal injury and T helper cell Th-1 responses in the MRL(lpr) mouse.

Background: MC(T) and MC(TC) types of human mast cells (MCs) are distinguished from one another on the basis of the protease compositions of their secretory granules, but their functional and developmental relationships have been uncertain., Objective: These studies better define the functional properties and developmental relationship of MC(T) and MC(TC) cells., Methods: Mast cells were dispersed from human skin and lung, purified with anti-Kit antibody, and separated into CD88+ and CD88- populations by cell sorting. These cells were evaluated by immunocytochemistry with antitryptase and antichymase mAbs; for chymase and tryptase mRNA by real-time RT-PCR; for conversion of MC(T) to MC(TC) cells during cell culture with recombinant human stem cell factor and recombinant human IL-6; and for degranulation and leukotriene C 4 (LTC 4 ) secretion when stimulated with anti-FcepsilonRI, substance P, C5a, and compound 48/80., Results: Mature MC(T) and MC(TC) cells were separated from one another on the basis of selective expression of CD88, the C5aR, on MC(TC) cells. Lung MC(T) cells had negligible levels of chymase mRNA and retained their MC(T) phenotype in culture. Mature MC(TC) cells from skin and lung degranulated in response to FcepsilonRI cross-linking, C5a, compound 48/80, and substance P. Lung MC(TC) cells released LTC 4 on activation, but no LTC 4 was detected when skin-derived MC(TC) cells were activated. MC(T) cells from lung degranulated and released LTC 4 in response to anti-FcepsilonRI and substance P, but not to C5a and compound 48/80., Conclusion: These observations functionally distinguish MC(T) from MC(TC) types of human mast cells and suggest important differences that may affect their participation in diseases such as asthma and urticaria.