RESUMO
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-1ß production, both at the transcriptional level and processing of proIL-1ß. Consequently, atherosclerosis-prone mice lacking macrophage-specific C5ar1 had ameliorated cardiovascular disease on a high-cholesterol diet. Conversely, inflammatory gene signatures and IL-1ß 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.
Assuntos
Inflamação/imunologia , Interleucina-1beta/biossíntese , Macrófagos/imunologia , Receptor da Anafilatoxina C5a/imunologia , Animais , Linhagem Celular , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor da Anafilatoxina C5a/deficiênciaRESUMO
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.
Assuntos
alfa-Globulinas/uso terapêutico , AVC Isquêmico/tratamento farmacológico , alfa-Globulinas/administração & dosagem , alfa-Globulinas/metabolismo , Animais , Edema Encefálico/tratamento farmacológico , Edema Encefálico/patologia , Infarto Encefálico/tratamento farmacológico , Infarto Encefálico/patologia , Morte Celular/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Humanos , AVC Isquêmico/metabolismo , AVC Isquêmico/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor da Anafilatoxina C5a/deficiência , Receptor da Anafilatoxina C5a/genética , Receptor da Anafilatoxina C5a/metabolismo , Ativador de Plasminogênio Tecidual/administração & dosagemRESUMO
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.
Assuntos
Plaquetas/metabolismo , Fator Plaquetário 4/metabolismo , Receptor da Anafilatoxina C5a/genética , Receptor da Anafilatoxina C5a/metabolismo , Indutores da Angiogênese , Animais , Ativação do Complemento , Complemento C5a , Inflamação , Camundongos , Camundongos Knockout , Receptor da Anafilatoxina C5a/deficiência , Receptores CXCR3/genética , Transdução de SinaisRESUMO
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.
Assuntos
Complemento C5a/metabolismo , Inflamação/patologia , Pulmão/patologia , Traumatismo Múltiplo/complicações , Traumatismo Múltiplo/metabolismo , Receptor da Anafilatoxina C5a/metabolismo , Choque Hemorrágico/complicações , Choque Hemorrágico/metabolismo , Adulto , Animais , Aptâmeros de Peptídeos/farmacologia , Biomarcadores/metabolismo , Líquido da Lavagem Broncoalveolar , Capilares/patologia , Linhagem Celular , Quimiotaxia/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Alvéolos Pulmonares/patologia , Receptor da Anafilatoxina C5a/deficiênciaRESUMO
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-1ß 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-1ß 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-1ß secretion after PVL exposure and controls S. aureus lung burdens.
Assuntos
Proteínas de Bactérias/antagonistas & inibidores , Toxinas Bacterianas/antagonistas & inibidores , Exotoxinas/antagonistas & inibidores , Células-Tronco Pluripotentes Induzidas/citologia , Leucocidinas/antagonistas & inibidores , Macrófagos/efeitos dos fármacos , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , Receptor da Anafilatoxina C5a/efeitos dos fármacos , Staphylococcus aureus , Animais , Antígeno CD11b/imunologia , Sistemas CRISPR-Cas , Diferenciação Celular , Células Cultivadas , Exotoxinas/deficiência , Técnicas de Introdução de Genes , Humanos , Interleucina-1beta/metabolismo , Antígenos Comuns de Leucócito/fisiologia , Pulmão/imunologia , Pulmão/microbiologia , Macrófagos/citologia , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Monócitos/citologia , Fragmentos de Peptídeos/imunologia , Pneumonia Estafilocócica/imunologia , Subunidades Proteicas , Receptor da Anafilatoxina C5a/deficiência , Receptor da Anafilatoxina C5a/genética , Receptor da Anafilatoxina C5a/fisiologia , Proteínas Recombinantes/metabolismo , Staphylococcus aureus/fisiologiaRESUMO
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.
Assuntos
Hipersensibilidade Alimentar/etiologia , Imunoglobulina E/sangue , Receptor da Anafilatoxina C5a/genética , Fatores Sexuais , Anafilaxia , Animais , Degranulação Celular , Quimases/sangue , Feminino , Masculino , Mastócitos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Receptor da Anafilatoxina C5a/deficiência , Receptores de IgE/imunologiaRESUMO
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.
Assuntos
Hipertensão/imunologia , Receptor da Anafilatoxina C5a/deficiência , Receptores de Complemento 3b/deficiência , Linfócitos T Reguladores/imunologia , Angiotensina II/toxicidade , Animais , Células Cultivadas , Hipertensão/etiologia , Hipertensão/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB CRESUMO
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.
Assuntos
Neisseria meningitidis/imunologia , Neisseria meningitidis/patogenicidade , Receptor da Anafilatoxina C5a/metabolismo , Sepse/microbiologia , Sepse/fisiopatologia , Animais , Atividade Bactericida do Sangue , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Knockout , Receptor da Anafilatoxina C5a/deficiência , Análise de SobrevidaRESUMO
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.
Assuntos
Neuroproteção , Doenças Priônicas/patologia , Receptor 2 Toll-Like/metabolismo , Anafilatoxinas/análise , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Quimiocinas/metabolismo , Proteínas do Sistema Complemento/metabolismo , Citocinas/metabolismo , Suscetibilidade a Doenças , Expressão Gênica , Imunidade Inata/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Doenças Priônicas/metabolismo , Doenças Priônicas/veterinária , RNA/genética , RNA/metabolismo , Receptor da Anafilatoxina C5a/deficiência , Receptor da Anafilatoxina C5a/genética , Receptor da Anafilatoxina C5a/metabolismo , Receptores de Complemento/deficiência , Receptores de Complemento/genética , Receptores de Complemento/metabolismo , Índice de Gravidade de Doença , Transdução de Sinais , Receptor 2 Toll-Like/deficiência , Receptor 2 Toll-Like/genéticaRESUMO
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.
Assuntos
Infecções por Escherichia coli/metabolismo , Pielonefrite/microbiologia , Receptor da Anafilatoxina C5a/fisiologia , Infecções Urinárias/metabolismo , Escherichia coli Uropatogênica , Doença Aguda , Animais , Aderência Bacteriana/fisiologia , Complemento C5a/imunologia , Citocinas/biossíntese , Mediadores da Inflamação/metabolismo , Rim/metabolismo , Rim/microbiologia , Camundongos Knockout , Microscopia Confocal , Pielonefrite/metabolismo , Pielonefrite/prevenção & controle , Receptor da Anafilatoxina C5a/deficiência , Receptor da Anafilatoxina C5a/metabolismo , Regulação para Cima/imunologiaRESUMO
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.
Assuntos
Cardiomegalia/patologia , Receptor da Anafilatoxina C5a/genética , Remodelação Ventricular , Actinas/metabolismo , Animais , Colágeno/genética , Colágeno/metabolismo , Constrição Patológica , Fibrose , Leucócitos/citologia , Leucócitos/metabolismo , Metaloproteinases da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Miocárdio/patologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Receptor da Anafilatoxina C5a/deficiênciaRESUMO
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. CX3CR1GFP and CCR2RFP 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.
Assuntos
Doença de Alzheimer/metabolismo , Hipocampo/metabolismo , Inflamação/metabolismo , Microglia/metabolismo , Receptor da Anafilatoxina C5a/metabolismo , Doença de Alzheimer/patologia , Animais , Cognição , Hipocampo/patologia , Humanos , Inflamação/patologia , Camundongos , Camundongos Knockout , Microglia/patologia , Receptor da Anafilatoxina C5a/deficiência , Transdução de Sinais/fisiologiaRESUMO
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.
Assuntos
Proteínas do Sistema Complemento/imunologia , Doença de Gaucher/imunologia , Doença de Gaucher/patologia , Glucosilceramidas/imunologia , Glucosilceramidas/metabolismo , Inflamação/imunologia , Inflamação/patologia , Animais , Células Apresentadoras de Antígenos/citologia , Células Apresentadoras de Antígenos/imunologia , Autoanticorpos/imunologia , Ativação do Complemento , Complemento C5a/biossíntese , Complemento C5a/imunologia , Proteínas do Sistema Complemento/biossíntese , Citocinas/biossíntese , Citocinas/imunologia , Modelos Animais de Doenças , Feminino , Doença de Gaucher/metabolismo , Doença de Gaucher/prevenção & controle , Glucosilceramidase/antagonistas & inibidores , Glucosilceramidase/deficiência , Glucosilceramidase/genética , Glucosiltransferases/biossíntese , Glucosiltransferases/metabolismo , Humanos , Imunoglobulina G/imunologia , Inflamação/metabolismo , Inflamação/prevenção & controle , Masculino , Camundongos , Receptor da Anafilatoxina C5a/deficiência , Receptor da Anafilatoxina C5a/imunologia , Receptor da Anafilatoxina C5a/metabolismo , Linfócitos T/citologia , Linfócitos T/imunologiaRESUMO
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.
Assuntos
Neuroproteção , Receptor da Anafilatoxina C5a/fisiologia , Traumatismos da Medula Espinal/tratamento farmacológico , Traumatismos da Medula Espinal/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Peptídeos Cíclicos/farmacologia , Receptor da Anafilatoxina C5a/antagonistas & inibidores , Receptor da Anafilatoxina C5a/deficiência , Traumatismos da Medula Espinal/imunologiaRESUMO
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.
Assuntos
Anticorpos Monoclonais/farmacologia , Complemento C3/metabolismo , Complemento C5/antagonistas & inibidores , Inativadores do Complemento/farmacologia , Via Alternativa do Complemento/efeitos dos fármacos , Glomerulonefrite/prevenção & controle , Rim/efeitos dos fármacos , Insuficiência Renal/prevenção & controle , Animais , Complemento C3/imunologia , Complemento C5/imunologia , Complemento C5/metabolismo , Fator H do Complemento/deficiência , Fator H do Complemento/genética , Modelos Animais de Doenças , Predisposição Genética para Doença , Glomerulonefrite/genética , Glomerulonefrite/imunologia , Glomerulonefrite/metabolismo , Rim/imunologia , Rim/metabolismo , Rim/patologia , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Properdina/deficiência , Properdina/genética , Proteinúria/imunologia , Proteinúria/metabolismo , Proteinúria/prevenção & controle , Receptor da Anafilatoxina C5a/deficiência , Receptor da Anafilatoxina C5a/genética , Insuficiência Renal/genética , Insuficiência Renal/imunologia , Insuficiência Renal/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de TempoRESUMO
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.
Assuntos
Inativadores do Complemento/farmacologia , Aloenxertos Compostos/efeitos dos fármacos , Aloenxertos Compostos/transplante , Ciclosporina/farmacologia , Sobrevivência de Enxerto/efeitos dos fármacos , Membro Posterior/efeitos dos fármacos , Membro Posterior/transplante , Imunossupressores/farmacologia , Proteínas Recombinantes de Fusão/farmacologia , Traumatismo por Reperfusão/prevenção & controle , Alotransplante de Tecidos Compostos Vascularizados , Animais , Quimiotaxia de Leucócito/efeitos dos fármacos , Ativação do Complemento/efeitos dos fármacos , Complemento C3/deficiência , Complemento C3/genética , Aloenxertos Compostos/irrigação sanguínea , Aloenxertos Compostos/imunologia , Genótipo , Membro Posterior/irrigação sanguínea , Membro Posterior/imunologia , Masculino , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Animais , Fenótipo , Receptor da Anafilatoxina C5a/deficiência , Receptor da Anafilatoxina C5a/genética , Receptores Acoplados a Proteínas G/deficiência , Receptores Acoplados a Proteínas G/genética , Traumatismo por Reperfusão/etiologia , Traumatismo por Reperfusão/imunologia , Traumatismo por Reperfusão/metabolismo , Baço/efeitos dos fármacos , Baço/imunologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Fatores de Tempo , Alotransplante de Tecidos Compostos Vascularizados/efeitos adversosRESUMO
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.
Assuntos
Coinfecção/imunologia , Miócitos Cardíacos/imunologia , Miócitos Cardíacos/patologia , Sepse/imunologia , Sepse/fisiopatologia , Animais , Cálcio/metabolismo , Canais de Cálcio Tipo L/imunologia , Coinfecção/microbiologia , Coinfecção/fisiopatologia , Complemento C5a/imunologia , Citoplasma/química , Citoplasma/metabolismo , Coração/fisiopatologia , Camundongos , Miócitos Cardíacos/microbiologia , Receptor da Anafilatoxina C5a/deficiência , Receptor da Anafilatoxina C5a/imunologia , Receptor da Anafilatoxina C5a/fisiologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/imunologia , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismo , Sepse/complicaçõesRESUMO
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-1ß. In this study, we investigate whether C5a modulates lipopolysaccharide- (LPS-) induced NLRP3 inflammasome activation in myeloid cells. Appearance of plasma IL-1ß during endotoxemia was reduced in C5aR1(-/-) mice when compared to wild-type mice. In vitro, C5a significantly enhanced LPS-induced production of IL-1ß in bone marrow Ly6C-high inflammatory monocytes, accompanied by augmented intracellular pro-IL-1ß 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-1ß, which was accompanied by attenuated levels of pro-IL-1ß, 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-1ß response to LPS challenge in vivo, our results suggest overall that C5a augments physiologic inflammasome responses.
Assuntos
Inflamassomos/metabolismo , Macrófagos/metabolismo , Monócitos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Receptor da Anafilatoxina C5a/metabolismo , Animais , Western Blotting , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/metabolismo , Complemento C5a/metabolismo , Complemento C5a/farmacologia , Modelos Animais de Doenças , Endotoxemia/metabolismo , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Inflamassomos/efeitos dos fármacos , Interleucina-1beta/metabolismo , Macrófagos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Monócitos/efeitos dos fármacos , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Receptor da Anafilatoxina C5a/deficiência , Receptor da Anafilatoxina C5a/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
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.
Assuntos
Doenças da Aorta/prevenção & controle , Apolipoproteínas E/deficiência , Aterosclerose/prevenção & controle , Lesões das Artérias Carótidas/prevenção & controle , Receptor da Anafilatoxina C5a/deficiência , Animais , Aorta/metabolismo , Aorta/patologia , Doenças da Aorta/genética , Doenças da Aorta/metabolismo , Doenças da Aorta/patologia , Apolipoproteínas E/genética , Aterosclerose/genética , Aterosclerose/metabolismo , Aterosclerose/patologia , Complexo CD3/metabolismo , Lesões das Artérias Carótidas/genética , Lesões das Artérias Carótidas/metabolismo , Lesões das Artérias Carótidas/patologia , Artéria Carótida Primitiva/metabolismo , Artéria Carótida Primitiva/patologia , Células Cultivadas , Modelos Animais de Doenças , Predisposição Genética para Doença , Mediadores da Inflamação/metabolismo , Macrófagos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neointima , Fenótipo , Placa Aterosclerótica , Receptor da Anafilatoxina C5a/genética , Receptor da Anafilatoxina C5a/metabolismo , Ruptura Espontânea , Linfócitos T/metabolismo , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Molécula 1 de Adesão de Célula Vascular/genética , Molécula 1 de Adesão de Célula Vascular/metabolismo , Remodelação VascularRESUMO
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 ß, 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.