RESUMO
Maturation of B cells within germinal centers (GCs) generates diversified B cell pools and high-affinity B cell antigen receptors (BCRs) for pathogen clearance. Increased receptor affinity is achieved by iterative cycles of T cell-dependent, affinity-based B cell positive selection and clonal expansion by mechanisms hitherto incompletely understood. Here we found that, as part of a physiologic program, GC B cells repressed expression of decay-accelerating factor (DAF/CD55) and other complement C3 convertase regulators via BCL6, but increased the expression of C5b-9 inhibitor CD59. These changes permitted C3 cleavage on GC B cell surfaces without the formation of membrane attack complex and activated C3a- and C5a-receptor signals required for positive selection. Genetic disruption of this pathway in antigen-activated B cells by conditional transgenic DAF overexpression or deletion of C3a and C5a receptors limited the activation of mechanistic target of rapamycin (mTOR) in response to BCR-CD40 signaling, causing premature GC collapse and impaired affinity maturation. These results reveal that coordinated shifts in complement regulation within the GC provide crucial signals underlying GC B cell positive selection.
Assuntos
Linfócitos B/imunologia , Ativação do Complemento , Complemento C3a/metabolismo , Complemento C5a/metabolismo , Centro Germinativo/imunologia , Animais , Animais Geneticamente Modificados , Linfócitos B/metabolismo , Antígenos CD55/genética , Antígenos CD55/metabolismo , Antígenos CD59/metabolismo , Linhagem Celular Tumoral , Hematopoiese Clonal/imunologia , Centro Germinativo/citologia , Centro Germinativo/metabolismo , Humanos , Ativação Linfocitária , Camundongos , Tonsila Palatina/citologia , Tonsila Palatina/patologia , Proteínas Proto-Oncogênicas c-bcl-6/metabolismo , Receptor da Anafilatoxina C5a/genética , Receptor da Anafilatoxina C5a/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Receptores de Complemento/genética , Receptores de Complemento/metabolismo , Transdução de Sinais/imunologia , Serina-Treonina Quinases TOR/metabolismoRESUMO
G-protein-coupled receptors (GPCRs), also known as seven transmembrane receptors (7TMRs), typically interact with two distinct signal-transducers, i.e., G proteins and ß-arrestins (ßarrs). Interestingly, there are some non-canonical 7TMRs that lack G protein coupling but interact with ßarrs, although an understanding of their transducer coupling preference, downstream signaling, and structural mechanism remains elusive. Here, we characterize two such non-canonical 7TMRs, namely, the decoy D6 receptor (D6R) and the complement C5a receptor subtype 2 (C5aR2), in parallel with their canonical GPCR counterparts. We discover that D6R and C5aR2 efficiently couple to ßarrs, exhibit distinct engagement of GPCR kinases (GRKs), and activate non-canonical downstream signaling pathways. We also observe that ßarrs adopt distinct conformations for D6R and C5aR2, compared to their canonical GPCR counterparts, in response to common natural agonists. Our study establishes D6R and C5aR2 as ßarr-coupled 7TMRs and provides key insights into their regulation and signaling with direct implication for biased agonism.
Assuntos
Membrana Celular/metabolismo , Conformação Proteica , Transdução de Sinais , beta-Arrestinas/química , Animais , Proteínas de Ligação ao GTP/química , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Ligação Proteica , Domínios Proteicos , Estrutura Secundária de Proteína , Transporte Proteico , Receptor da Anafilatoxina C5a/metabolismoRESUMO
Signaling through the G protein-coupled receptors for the complement fragments C3a and C5a (C3aR and C5aR, respectively) by dendritic cells and CD4(+) cells provides costimulatory and survival signals to effector T cells. Here we found that when signals from C3aR and C5aR were not transduced into CD4(+) cells, signaling via the kinases PI(3)Kγ, Akt and mTOR ceased, activation of the kinase PKA increased, autoinductive signaling by transforming growth factor-ß1 (TGF-ß1) initiated and CD4(+) T cells became Foxp3(+) induced regulatory T cells (iT(reg) cells). Endogenous TGF-ß1 suppressed signaling through C3aR and C5aR by preventing the production of C3a and C5a and upregulating C5L2, an alternative receptor for C5a. The absence of signaling via C3aR and C5aR resulted in lower expression of costimulatory molecules and interleukin 6 (IL-6) and more production of IL-10. The resulting iT(reg) cells exerted robust suppression, had enhanced stability and suppressed ongoing autoimmune disease. Antagonism of C3aR and C5aR can also induce functional human iT(reg) cells.
Assuntos
Fatores de Transcrição Forkhead/metabolismo , Receptor da Anafilatoxina C5a/metabolismo , Receptores de Complemento/metabolismo , Transdução de Sinais/imunologia , Linfócitos T Reguladores/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Animais , Comunicação Celular/imunologia , Diferenciação Celular , Classe Ib de Fosfatidilinositol 3-Quinase/imunologia , Classe Ib de Fosfatidilinositol 3-Quinase/metabolismo , Complemento C3a/imunologia , Complemento C3a/metabolismo , Complemento C5a/imunologia , Complemento C5a/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/imunologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Fatores de Transcrição Forkhead/imunologia , Regulação da Expressão Gênica , Interleucina-10/imunologia , Interleucina-10/metabolismo , Interleucina-6/imunologia , Interleucina-6/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas Proto-Oncogênicas c-akt/imunologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor Cross-Talk/imunologia , Receptor da Anafilatoxina C5a/imunologia , Receptores de Quimiocinas/imunologia , Receptores de Quimiocinas/metabolismo , Receptores de Complemento/imunologia , Linfócitos T Reguladores/citologia , Linfócitos T Reguladores/imunologia , Serina-Treonina Quinases TOR/imunologia , Serina-Treonina Quinases TOR/metabolismo , Fator de Crescimento Transformador beta1/imunologiaRESUMO
Demethylation of the T regulatory cell (Treg)-specific demethylation region (TSDR) of the Foxp3 gene is the hallmark of Foxp3+ Treg stability, but the cellular signaling that programs this epigenetic state remains undefined. In this article, we show that suppressed C3a and C5a receptor (C3ar1/C5ar1) signaling in murine Tregs plays an obligate role. Murine C3ar1-/-C5ar1-/- Foxp3+ cells showed increased suppressor of cytokine signaling 1/2/3 expression, vitamin C stabilization, and ten-eleven translocation (TET) 1, TET2, and TET3 expression, all of which are linked to Treg stability. C3ar1-/-C5ar1-/- Foxp3+ cells additionally were devoid of BRD4 signaling that primes Th17 cell lineage commitment. Orally induced OVA-specific C3ar1-/-C5ar1-/- Foxp3+ OT-II Tregs transferred to OVA-immunized wild-type recipients remained >90% Foxp3+ out to 4 mo, whereas identically generated CD55-/- (DAF-/-) Foxp3+ OT-II Tregs (in which C3ar1/C5ar1 signaling is potentiated) lost >75% of Foxp3 expression by 14 d. After 4 mo in vivo, the C3ar1-/-C5ar1-/- Foxp3+ OT-II Tregs fully retained Foxp3 expression even with OVA challenge and produced copious TGF-ß and IL-10. Their TSDR was demethylated comparably with that of thymic Tregs. They exhibited nuclear translocation of NFAT and NF-κB reported to stabilize thymic Tregs by inducing hairpin looping of the TSDR to the Foxp3 promoter. Thus, disabled CD4+ cell C3ar1/C5ar1 signaling triggers the sequential cellular events that lead to demethylation of the Foxp3 TSDR.
Assuntos
Metilação de DNA , Linfócitos T Reguladores , Camundongos , Animais , Fatores de Transcrição/metabolismo , Regulação da Expressão Gênica , Receptor da Anafilatoxina C5a/metabolismo , Proteínas Nucleares/genética , Desmetilação , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismoRESUMO
Chondrocyte ferroptosis induces the occurrence of osteoarthritis (OA). As a key gene of OA, C5a receptor 1 (C5AR1) is related to ferroptosis. Here, we investigated whether C5AR1 interferes with chondrocyte ferroptosis during OA occurrence. C5AR1 was downregulated in PA-treated chondrocytes. Overexpression of C5AR1 increased the cell viability and decreased ferroptosis in chondrocytes. Moreover, Tumor necrosis factor superfamily member 13B (TNFSF13B) was downregulated in PA-treated chondrocytes, and knockdown of TNFSF13B eliminated the inhibitory effect of C5AR1 on ferroptosis in chondrocytes. More importantly, the PI3K/Akt/GSK3ß/Nrf2/HO-1 pathway inhibitor LY294002 reversed the inhibition of C5AR1 or TNFSF13B on ferroptosis in chondrocytes. Finally, we found that C5AR1 alleviated joint tissue lesions and ferroptosis in rats and inhibited the progression of OA in the rat OA model constructed by anterior cruciate ligament transection (ACLT), which was reversed by interfering with TNFSF13B. This study shows that C5AR1 reduces the progression of OA by upregulating TNFSF13B to activate the PI3K/Akt/GSK3ß/Nrf2/HO-1 pathway and thereby inhibiting chondrocyte sensitivity to ferroptosis, indicating that C5AR1 may be a potential therapeutic target for ferroptosis-related diseases.
Assuntos
Condrócitos , Ferroptose , Glicogênio Sintase Quinase 3 beta , Fator 2 Relacionado a NF-E2 , Osteoartrite , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Ratos Sprague-Dawley , Receptor da Anafilatoxina C5a , Animais , Ferroptose/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Condrócitos/metabolismo , Condrócitos/patologia , Condrócitos/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/metabolismo , Fator 2 Relacionado a NF-E2/genética , Ratos , Osteoartrite/metabolismo , Osteoartrite/patologia , Osteoartrite/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Glicogênio Sintase Quinase 3 beta/genética , Masculino , Receptor da Anafilatoxina C5a/metabolismo , Receptor da Anafilatoxina C5a/genética , Transdução de Sinais , Heme Oxigenase-1/metabolismo , Heme Oxigenase-1/genética , Heme Oxigenase (Desciclizante)RESUMO
Podocytes are essential to maintaining the integrity of the glomerular filtration barrier, but they are frequently affected in lupus nephritis (LN). Here, we show that the significant upregulation of Drp1S616 phosphorylation in podocytes promotes mitochondrial fission, leading to mitochondrial dysfunction and podocyte injury in LN. Inhibition or knockdown of Drp1 promotes mitochondrial fusion and protects podocytes from injury induced by LN serum. In vivo, pharmacological inhibition of Drp1 reduces the phosphorylation of Drp1S616 in podocytes in lupus-prone mice. Podocyte injury is reversed when Drp1 is inhibited, resulting in the alleviation of proteinuria. Mechanistically, complement component C5a (C5a) upregulates the phosphorylation of Drp1S616 and promotes mitochondrial fission in podocytes. Moreover, the expression of C5a receptor 1 (C5aR1) is notably upregulated in podocytes in LN. C5a-C5aR1 axis-controlled phosphorylation of Drp1S616 and mitochondrial fission are substantially suppressed when C5aR1 is knocked down by siRNA. Moreover, lupus-prone mice treated with C5aR inhibitor show reduced phosphorylation of Drp1S616 in podocytes, resulting in significantly less podocyte damage. Together, this study uncovers a novel mechanism by which the C5a-C5aR1 axis promotes podocyte injury by enhancing Drp1-mediated mitochondrial fission, which could have significant implications for the treatment of LN.
Assuntos
Complemento C5a , Dinaminas , Nefrite Lúpica , Dinâmica Mitocondrial , Podócitos , Receptor da Anafilatoxina C5a , Podócitos/metabolismo , Podócitos/patologia , Nefrite Lúpica/metabolismo , Nefrite Lúpica/patologia , Nefrite Lúpica/etiologia , Animais , Receptor da Anafilatoxina C5a/metabolismo , Receptor da Anafilatoxina C5a/genética , Camundongos , Dinaminas/metabolismo , Dinaminas/genética , Complemento C5a/metabolismo , Humanos , Fosforilação , Modelos Animais de Doenças , Mitocôndrias/metabolismo , Transdução de Sinais , FemininoRESUMO
The anaphylatoxin C5a is core effector of complement activation. C5a exerts potent proinflammatory and immunomodulatory actions through interacting with its C5a receptors, C5aR1 and C5aR2, modulating multiple signaling and functional activities of immune cells. Native C5a contains a large N-linked glycosylation site at Asn64, which accounts for up to 25% of its m.w. To date, the vast majority of published studies examining C5a are performed using Escherichia coli-generated recombinant C5a, which is readily available from numerous commercial suppliers, but lacks this glycosylation moiety. However, a plasma-purified "native" form of C5a is also commercially available. The different size and glycosylation of these two C5a versions could have functional implications. Therefore, the current study aimed to compare recombinant human C5a to purified plasma-derived human C5a in driving the signaling and functional activities of human primary macrophages. We found that both versions of C5a displayed similar potencies at triggering C5aR1- and C5aR2-mediated cell signaling, but elicited distinct functional responses in primary human monocyte-derived macrophages. Multiple commercial sources of recombinant C5a, but not the plasma-purified or a synthetic C5a version, induced human monocyte-derived macrophages to produce IL-6 and IL-10 in a C5a receptor-independent manner, which was driven through Syk and NF-κB signaling and apparently not due to endotoxin contamination. Our results, therefore, offer caution against the sole use of recombinant human C5a, particularly in functional/cytokine assays conducted in human primary immune cells, and suggest studies using recombinant human C5a should be paired with C5aR1 inhibitors or purified/synthetic human C5a to confirm relevant findings.
Assuntos
Complemento C5a/metabolismo , Escherichia coli/metabolismo , Macrófagos/imunologia , Plasma/metabolismo , Células Cultivadas , Complemento C5a/genética , Escherichia coli/genética , Glicosilação , Humanos , Interleucina-10/metabolismo , Interleucina-6/metabolismo , Ativação de Macrófagos , NF-kappa B/metabolismo , Receptor da Anafilatoxina C5a/metabolismo , Proteínas Recombinantes/genética , Transdução de SinaisRESUMO
The complement system is a crucial component of the host response to infection and tissue damage. Activation of the complement cascade generates anaphylatoxins including C5a and C3a. C5a exerts a pro-inflammatory effect via the G-protein-coupled receptor C5a anaphylatoxin chemotactic receptor 1 (C5aR1, also known as CD88) that is expressed on cells of myeloid origin. Inhibitors of the complement system have long been of interest as potential drugs for the treatment of diseases such as sepsis, rheumatoid arthritis, Crohn's disease and ischaemia-reperfusion injuries. More recently, a role of C5a in neurodegenerative conditions such as Alzheimer's disease has been identified. Peptide antagonists based on the C5a ligand have progressed to phase 2 trials in psoriasis and rheumatoid arthritis; however, these compounds exhibited problems with off-target activity, production costs, potential immunogenicity and poor oral bioavailability. Several small-molecule competitive antagonists for C5aR1, such as W-54011 and NDT9513727, have been identified by C5a radioligand-binding assays. NDT9513727 is a non-peptide inverse agonist of C5aR1, and is highly selective for the primate and gerbil receptors over those of other species. Here, to study the mechanism of action of C5a antagonists, we determine the structure of a thermostabilized C5aR1 (known as C5aR1 StaR) in complex with NDT9513727. We found that the small molecule bound between transmembrane helices 3, 4 and 5, outside the helical bundle. One key interaction between the small molecule and residue Trp2135.49 seems to determine the species selectivity of the compound. The structure demonstrates that NDT9513727 exerts its inverse-agonist activity through an extra-helical mode of action.
Assuntos
Benzodioxóis/química , Benzodioxóis/metabolismo , Imidazóis/química , Imidazóis/metabolismo , Receptor da Anafilatoxina C5a/antagonistas & inibidores , Receptor da Anafilatoxina C5a/química , Animais , Benzodioxóis/farmacologia , Sítios de Ligação , Cristalografia por Raios X , Agonismo Inverso de Drogas , Células HEK293 , Humanos , Imidazóis/farmacologia , Modelos Moleculares , Mutação , Estabilidade Proteica , Estrutura Secundária de Proteína , Receptor da Anafilatoxina C5a/genética , Receptor da Anafilatoxina C5a/metabolismo , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/metabolismo , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismoRESUMO
As per the classical view of the coagulation system, it functions solely in plasma to maintain hemostasis. An experimental approach modeling vascular reconstitution was used to show that vascular endothelial cells (ECs) endogenously synthesize coagulation factors during angiogenesis. Intracellular thrombin generated from this synthesis promotes the mitotic function of vascular endothelial cell growth factor A (VEGF-A). The thrombin concurrently cleaves C5a from EC-synthesized complement component C5 and unmasks the tethered ligand for EC-expressed protease-activated receptor 4 (PAR4). The two ligands jointly trigger EC C5a receptor-1 (C5ar1) and PAR4 signaling, which together promote VEGF receptor 2 growth signaling. C5ar1 is functionally associated with PAR4, enabling C5a or thrombin to elicit Gαi and/or Gαq signaling. EC coagulation factor and EC complement component synthesis concurrently down-regulate with contact inhibition. The connection of these processes with VEGF receptor 2 signaling provides new insights into mechanisms underlying angiogenesis. Knowledge of endogenous coagulation factor/complement component synthesis and joint PAR4/C5ar1 signaling could be applied to other cell types.
Assuntos
Fatores de Coagulação Sanguínea/biossíntese , Células Endoteliais/metabolismo , Neovascularização Fisiológica , Receptor da Anafilatoxina C5a/metabolismo , Receptores Ativados por Proteinase/metabolismo , Transdução de Sinais , Animais , Fatores de Coagulação Sanguínea/genética , Feminino , Masculino , Camundongos , Camundongos Knockout , Receptor da Anafilatoxina C5a/genética , Receptores Ativados por Proteinase/genéticaRESUMO
The current pandemic of coronavirus disease (COVID-19) caused by SARS-CoV-2 is a significant global health challenge. A recent study by Carvelli and colleagues now demonstrates the involvement of complement C5a and its receptor C5aR1 in disease progression and suggests that blockade of the C5a-C5aR1 axis may represent a potential therapeutic strategy against COVID-19.
Assuntos
Betacoronavirus/imunologia , Complemento C5a/imunologia , Infecções por Coronavirus/imunologia , Pneumonia Viral/imunologia , Receptor da Anafilatoxina C5a/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Betacoronavirus/efeitos dos fármacos , Betacoronavirus/fisiologia , COVID-19 , Complemento C5a/metabolismo , Infecções por Coronavirus/terapia , Infecções por Coronavirus/virologia , Modelos Animais de Doenças , Humanos , Pandemias , Pneumonia Viral/terapia , Pneumonia Viral/virologia , Receptor da Anafilatoxina C5a/metabolismo , Receptores Acoplados a Proteínas G/imunologia , Receptores Acoplados a Proteínas G/metabolismo , SARS-CoV-2 , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologiaRESUMO
After restoration of spontaneous circulation (ROSC) following cardiac arrest, complements can be activated and excessive autophagy can contribute to the brain ischemia-reperfusion (I/R) injury. Mild hypothermia (HT) protects against brain I/R injury after ROSC, but the mechanisms have not been fully elucidated. Here, we found that HT significantly inhibited the increases in serum NSE, S100ß, and C5a, as well as neurologic deficit scores, TUNEL-positive cells, and autophagic vacuoles in the pig brain cortex after ROSC. The C5a receptor 1 (C5aR1) mRNA and the C5a, C5aR1, Beclin 1, LC3-II, and cleaved caspase-3 proteins were significantly increased, but the P62 protein and the PI3K/Akt/mTOR pathway-related proteins were significantly reduced in pigs after ROSC or neuronal oxygen-glucose deprivation/reoxygenation. HT could significantly attenuate the above changes in NT-treated neurons. Furthermore, C5a treatment induced autophagy and apoptosis and reduced the PI3K/Akt/mTOR pathway-related proteins in cultured neurons, which could be reversed by C5aR1 antagonist PMX205. Our findings demonstrated that C5a could bind to C5aR1 to induce neuronal autophagy during the brain I/R injury, which was associated with the inhibited PI3K/Akt/mTOR pathway. HT could inhibit C5a-induced neuronal autophagy by regulating the C5a-C5aR1 interaction and the PI3K/Akt/mTOR pathway, which might be one of the neuroprotective mechanisms underlying I/R injury. The C5a receptor 1 (C5aR1) mRNA and the C5a, C5aR1, Beclin 1, LC3-II, and cleaved caspase-3 proteins were significantly increased, but the P62 protein and the PI3K/Akt/mTOR pathway-related proteins were significantly reduced in pigs after ROSC or neuronal oxygen-glucose deprivation/reoxygenation. Mild hypothermia (HT) could significantly attenuate the above changes in NT-treated neurons. Furthermore, C5a treatment induced autophagy and apoptosis and reduced the PI3K/Akt/mTOR pathway-related proteins in cultured neurons, which could be reversed by C5aR1 antagonist PMX205. Proposed mechanism by which HT protects against brain I/R injury by repressing C5a-C5aR1-induced excessive autophagy. Complement activation in response to brain I/R injury generates C5a that can interact with C5aR1 to inactivate mTOR, probably through the PI3K-AKT pathway, which can finally lead to autophagy activation. The excessively activated autophagy ultimately contributes to cell apoptosis and brain injury. HT may alleviate complement activation and then reduce C5a-induced autophagy to protect against brain I/R injury. HT, mild hypothermia; I/R, ischemia reperfusion.
Assuntos
Parada Cardíaca , Hipotermia , Fármacos Neuroprotetores , Traumatismo por Reperfusão , Animais , Suínos , Caspase 3/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Hipotermia/metabolismo , Proteína Beclina-1/metabolismo , Receptor da Anafilatoxina C5a/metabolismo , Fármacos Neuroprotetores/farmacologia , Serina-Treonina Quinases TOR/metabolismo , Encéfalo/metabolismo , Traumatismo por Reperfusão/complicações , Traumatismo por Reperfusão/metabolismo , Oxigênio/metabolismo , Parada Cardíaca/metabolismo , Parada Cardíaca/terapia , Autofagia , RNA Mensageiro/metabolismo , Glucose/metabolismoRESUMO
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
BACKGROUND: The complement system is part of the innate immune system that clears pathogens and cellular debris. In the healthy brain, complement influences neurodevelopment and neurogenesis, synaptic pruning, clearance of neuronal blebs, recruitment of phagocytes, and protects from pathogens. However, excessive downstream complement activation that leads to generation of C5a, and C5a engagement with its receptor C5aR1, instigates a feed-forward loop of inflammation, injury, and neuronal death, making C5aR1 a potential therapeutic target for neuroinflammatory disorders. C5aR1 ablation in the Arctic (Arc) model of Alzheimer's disease protects against cognitive decline and neuronal injury without altering amyloid plaque accumulation. METHODS: To elucidate the effects of C5a-C5aR1 signaling on AD pathology, we crossed Arc mice with a C5a-overexpressing mouse (ArcC5a+) and tested hippocampal memory. RNA-seq was performed on hippocampus and cortex from Arc, ArcC5aR1KO, and ArcC5a+ mice at 2.7-10 months and age-matched controls to assess mechanisms involved in each system. Immunohistochemistry was used to probe for protein markers of microglia and astrocytes activation states. RESULTS: ArcC5a+ mice had accelerated cognitive decline compared to Arc. Deletion of C5ar1 delayed or prevented the expression of some, but not all, AD-associated genes in the hippocampus and a subset of pan-reactive and A1 reactive astrocyte genes, indicating a separation between genes induced by amyloid plaques alone and those influenced by C5a-C5aR1 signaling. Biological processes associated with AD and AD mouse models, including inflammatory signaling, microglial cell activation, and astrocyte migration, were delayed in the ArcC5aR1KO hippocampus. Interestingly, C5a overexpression also delayed the increase of some AD-, complement-, and astrocyte-associated genes, suggesting the possible involvement of neuroprotective C5aR2. However, these pathways were enhanced in older ArcC5a+ mice compared to Arc. Immunohistochemistry confirmed that C5a-C5aR1 modulation in Arc mice delayed the increase in CD11c-positive microglia, while not affecting other pan-reactive microglial or astrocyte markers. CONCLUSION: C5a-C5aR1 signaling in AD largely exerts its effects by enhancing microglial activation pathways that accelerate disease progression. While C5a may have neuroprotective effects via C5aR2, engagement of C5a with C5aR1 is detrimental in AD models. These data support specific pharmacological inhibition of C5aR1 as a potential therapeutic strategy to treat AD.
Assuntos
Doença de Alzheimer , Fenômenos Biológicos , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Animais , Camundongos , Microglia/metabolismo , Placa Amiloide/metabolismo , Receptor da Anafilatoxina C5a/genética , Receptor da Anafilatoxina C5a/metabolismo , Transdução de SinaisRESUMO
The complement activation fragment C5a is a potent proinflammatory mediator that is increasingly recognized as an immune modulator. C5a acts through two C5a receptors, C5aR1 (C5aR, CD88) and C5aR2 (C5L2, GPR77), to powerfully modify multiple aspects of immune cell function. Although C5aR1 is generally acknowledged to be proinflammatory and immune-activating, the potential roles played by C5aR2 remain poorly defined. Despite studies demonstrating C5aR2 can modulate C5aR1 in human cells, it is not yet known whether C5aR2 functionality is limited to, or requires, C5aR1 activation or influences immune cells more broadly. The present study, therefore, aimed to characterize the roles of C5aR2 on the signaling and function of primary human monocyte-derived macrophages, using a C5aR2 agonist (Ac-RHYPYWR-OH; P32) to selectively activate the receptor. We found that although C5aR2 activation with P32 by itself was devoid of any detectable MAPK signaling activities, C5aR2 agonism significantly dampened C5aR1-, C3aR-, and chemokine-like receptor 1 (CMKLR1)-mediated ERK signaling and altered intracellular calcium mobilization mediated by these receptors. Functionally, selective C5aR2 activation also downregulated cytokine production triggered by various TLRs (TLR2, TLR3, TLR4, and TLR7), C-type lectin receptors (Dectin-1, Dectin-2, and Mincle), and the cytosolic DNA sensor stimulator of IFN genes (STING). Surprisingly, activity at the C-type lectin receptors was particularly powerful, with C5aR2 activation reducing Mincle-mediated IL-6 and TNF-α generation by 80-90%. In sum, this study demonstrates that C5aR2 possesses pleiotropic functions in primary human macrophages, highlighting the role of C5aR2 as a powerful regulator of innate immune function.
Assuntos
Macrófagos/metabolismo , Receptor da Anafilatoxina C5a/metabolismo , Transdução de Sinais/fisiologia , Células Cultivadas , Humanos , Interferons/metabolismo , Interleucina-6/metabolismo , Lectinas Tipo C/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Monócitos/metabolismo , Receptores de Quimiocinas/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Neutrophil infiltration to ischemic tissues following reperfusion worsens injury. A key driver of neutrophil recruitment and activation is the complement factor C5a, which signals through two receptors, C5aR1 and C5aR2. In this study, we used a neutrophil-dependent mouse model of intestinal ischemia-reperfusion (IR) injury to investigate the underexplored role of C5aR2 in neutrophil mobilization, recruitment, and disease outcomes. We show that intestinal IR induces rapid neutrophil mobilization along with a concomitant reduction in plasma C5a levels that is driven by both C5aR1 and C5aR2. Intestinal IR in C5aR2-/- mice led to worsened intestinal damage and increased neutrophil infiltration. Inhibition of C5aR1 signaling in C5aR2-/- mice with PMX53 prevented neutrophil accumulation and reduced IR pathology, suggesting a key requirement for enhanced neutrophil C5aR1 activation in the absence of C5aR2 signaling. Interestingly, C5aR2 deficiency also reduced circulating neutrophil numbers after IR, as well as following G-CSF-mediated bone marrow mobilization, which was independent of C5aR1, demonstrating that C5aR2 has unique and distinct functions from C5aR1 in neutrophil egress. Despite enhanced tissue injury in C5aR2-/- IR mice, there were significant reductions in intestinal proinflammatory cytokines, highlighting complicated dual protective/pathogenic roles for C5aR2 in pathophysiology. Collectively, we show that C5aR2 is protective in intestinal IR by inhibiting C5aR1-mediated neutrophil recruitment to the ischemic tissue. This is despite the potentially local pathogenic effects of C5aR2 in increasing intestinal proinflammatory cytokines and enhancing circulating neutrophil numbers in response to mobilizing signals. Our data therefore suggest that this balance between the dual pro- and anti-inflammatory roles of C5aR2 ultimately dictates disease outcomes.
Assuntos
Isquemia Mesentérica/imunologia , Infiltração de Neutrófilos , Receptor da Anafilatoxina C5a/metabolismo , Traumatismo por Reperfusão/imunologia , Animais , Complemento C5a/análise , Complemento C5a/metabolismo , Modelos Animais de Doenças , Humanos , Jejuno/citologia , Jejuno/imunologia , Jejuno/patologia , Masculino , Isquemia Mesentérica/sangue , Isquemia Mesentérica/complicações , Isquemia Mesentérica/patologia , Camundongos , Camundongos Knockout , Receptor da Anafilatoxina C5a/genética , Traumatismo por Reperfusão/sangue , Traumatismo por Reperfusão/patologiaRESUMO
In the metastasis-targeted organs, angiogenesis is essential for the progression of dormant micrometastases to rapidly growing and clinically overt lesions. However, we observed changes suggesting angiogenic switching in the mouse lungs prior to arrival of tumor cells (i.e., in the premetastatic niche) in the models of breast carcinoma. This angiogenic switching appears to be caused by myeloid-derived suppressor cells recruited to the premetastatic lungs through complement C5a receptor 1 signaling. These myeloid cells are known to secrete several proangiogenic factors in tumors, including IL-1ß and matrix metalloproteinase-9, and we found upregulation of these genes in the premetastatic lungs. Blockade of C5a receptor 1 synergized with antiangiogenic Listeria monocytogenes-based vaccines to decrease the lung metastatic burden by reducing vascular density and improving antitumor immunity in the lungs. This was mediated even when growth of primary breast tumors was not affected by these treatments. This work provides initial evidence that angiogenesis contributes to the premetastatic niche in rapidly progressing cancers and that inhibiting this process through immunotherapy is beneficial for reducing or even preventing metastasis.
Assuntos
Vacinas Anticâncer/administração & dosagem , Neoplasias Pulmonares/terapia , Neoplasias Mamárias Experimentais/terapia , Células Supressoras Mieloides/imunologia , Neovascularização Patológica/terapia , Inibidores da Angiogênese/farmacologia , Inibidores da Angiogênese/uso terapêutico , Animais , Antineoplásicos Imunológicos/farmacologia , Antineoplásicos Imunológicos/uso terapêutico , Linhagem Celular Tumoral , Terapia Combinada/métodos , Complemento C5a/imunologia , Complemento C5a/metabolismo , Feminino , Humanos , Imunoterapia/métodos , Listeria monocytogenes/imunologia , Pulmão/irrigação sanguínea , Pulmão/imunologia , Pulmão/patologia , Neoplasias Pulmonares/irrigação sanguínea , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/secundário , Neoplasias Mamárias Experimentais/imunologia , Neoplasias Mamárias Experimentais/patologia , Metaloproteinase 9 da Matriz/metabolismo , Camundongos , Camundongos Knockout , Células Supressoras Mieloides/metabolismo , Metástase Neoplásica/imunologia , Metástase Neoplásica/terapia , Neovascularização Patológica/imunologia , Receptor da Anafilatoxina C5a/antagonistas & inibidores , Receptor da Anafilatoxina C5a/genética , Receptor da Anafilatoxina C5a/metabolismo , Microambiente Tumoral/imunologiaRESUMO
Complement factor C5a was originally identified as a powerful promoter of inflammation through activation of the C5a receptor 1 (C5ar1). Recent evidence suggests involvement of C5a not only in pro- but also in anti-inflammatory signaling. The present study aims to unveil the role of C5ar1 as potential therapeutic target in a murine sepsis model. Our study discloses a significantly increased survival in models of mild to moderate but not severe sepsis of C5ar1-deficient mice. The decreased mortality of C5ar1-deficient mice is accompanied by improved pathogen clearance and largely preserved liver function. C5ar1-deficient mice exhibited a significantly increased production of the pro-inflammatory mediator interferon-γ (IFN-γ) and a decreased production of the anti-inflammatory cytokine interleukin-10 (IL-10). Together, these data uncover C5a signaling as a mediator of immunosuppressive processes during sepsis and describe the C5ar1 and related changes of the IFN-γ to IL-10 ratio as markers for the immunological (dys)function accompanying sepsis.
Assuntos
Biomarcadores , Suscetibilidade a Doenças/imunologia , Imunomodulação , Receptor da Anafilatoxina C5a/metabolismo , Sepse/metabolismo , Animais , Citocinas/metabolismo , Modelos Animais de Doenças , Imunidade Inata , Imunomodulação/efeitos dos fármacos , Mediadores da Inflamação/metabolismo , Camundongos , Camundongos Knockout , Terapia de Alvo Molecular , Fenótipo , Receptor da Anafilatoxina C5a/antagonistas & inibidores , Receptor da Anafilatoxina C5a/genética , Sepse/diagnóstico , Sepse/tratamento farmacológico , Sepse/etiologiaRESUMO
The deposition of IgG autoantibodies in peripheral tissues and the subsequent activation of the complement system, which leads to the accumulation of the anaphylatoxin C5a in these tissues, is a common hallmark of diverse autoimmune diseases, including rheumatoid arthritis (RA) and pemphigoid diseases (PDs). C5a is a potent chemoattractant for granulocytes and mice deficient in its precursor C5 or its receptor C5aR1 are resistant to granulocyte recruitment and, consequently, to tissue inflammation in several models of autoimmune diseases. However, the mechanism whereby C5a/C5aR regulates granulocyte recruitment in these diseases has remained elusive. Mechanistic studies over the past five years into the role of C5a/C5aR1 in the K/BxN serum arthritis mouse model have provided novel insights into the mechanisms C5a/C5aR1 engages to initiate granulocyte recruitment into the joint. It is now established that the critical actions of C5a/C5aR1 do not proceed in the joint itself, but on the luminal endothelial surface of the joint vasculature, where C5a/C5aR1 mediate the arrest of neutrophils on the endothelium by activating ß2 integrin. Then, C5a/C5aR1 induces the release of leukotriene B4 (LTB4) from the arrested neutrophils. The latter, subsequently, initiates by autocrine/paracrine actions via its receptor BLT1 the egress of neutrophils from the blood vessel lumen into the interstitial. Compelling evidence suggests that this C5a/C5aR1-LTB4/BLT1 axis driving granulocyte recruitment in arthritis may represent a more generalizable biological principle critically regulating effector cell recruitment in other IgG autoantibody-induced diseases, such as in pemphigoid diseases. Thus, dual inhibition of C5a and LTB4, as implemented in nature by the lipocalin coversin in the soft-tick Ornithodoros moubata, may constitute a most effective therapeutic principle for the treatment of IgG autoantibody-driven diseases.
Assuntos
Doenças Autoimunes/imunologia , Complemento C5a/metabolismo , Inflamação/imunologia , Neutrófilos/imunologia , Receptor da Anafilatoxina C5a/metabolismo , Animais , Autoanticorpos/metabolismo , Autoimunidade , Movimento Celular , Modelos Animais de Doenças , Humanos , Leucotrieno B4/metabolismo , Camundongos , Ativação de Neutrófilo , Transdução de SinaisRESUMO
The complement system is well appreciated for its role as an important effector of innate immunity that is activated by the classical, lectin or alternative pathway. C5a is one important mediator of the system that is generated in response to canonical and non-canonical C5 cleavage by circulating or cell-derived proteases. In addition to its function as a chemoattractant for neutrophils and other myeloid effectors, C5a and its sister molecule C3a have concerted roles in cell homeostasis and surveillance. Through activation of their cognate G protein coupled receptors, C3a and C5a regulate multiple intracellular pathways within the mitochondria and the lysosomal compartments that harbor multiple enzymes critical for protein, carbohydrate and lipid metabolism. Genetic mutations of such lysosomal enzymes or their receptors can result in the compartmental accumulation of specific classes of substrates in this organelle summarized as lysosomal storage diseases (LSD). A frequent LSD is Gaucher disease (GD), caused by autosomal recessively inherited mutations in GBA1, resulting in functional defects of the encoded enzyme, acid ß-glucosidase (glucocerebrosidase, GCase). Such mutations promote excessive accumulation of ß-glucosylceramide (GC or GL1) in innate and adaptive immune cells frequently associated with chronic inflammation. Recently, we uncovered an unexpected link between the C5a and C5a receptor 1 (C5aR1) axis and the accumulation of GL1 in experimental and clinical GD. Here, we will review the pathways of complement activation in GD, its role as a mediator of the inflammatory response, and its impact on glucosphingolipid metabolism. Further, we will discuss the potential role of the C5a/C5aR1 axis in GL1-specific autoantibody formation and as a novel therapeutic target in GD.
Assuntos
Complemento C5a/metabolismo , Doença de Gaucher/imunologia , Glucosilceramidase/genética , Inflamação/imunologia , Doenças por Armazenamento dos Lisossomos/imunologia , Animais , Autoanticorpos/metabolismo , Doença de Gaucher/genética , Glucosilceramidas/metabolismo , Humanos , Receptor da Anafilatoxina C5a/metabolismoRESUMO
Diabetic kidney disease (DKD) is a severe DM complication. While complement C5 up-regulation and gut dysbiosis are found in T2DM, their roles in DKD are unclear. Here, we investigated the effect of C5 on the gut microbiota during DKD development. Renal C5a/C5a receptor (C5aR) expression changes were measured in T2DM patients and db/db mice. Db/db mice were treated with a C5aR antagonist (C5aRA), and renal function, gut microbiota and renal genome changes were analysed. The effects of C5a and short-chain fatty acids (SCFAs) on the signal transducer and activator of transcription 3 (STAT3) pathway were examined in vitro. C5a was up-regulated in glomerular endothelial cells (GECs) of T2DM patients and db/db mice. Although glucose and lipid metabolism were unchanged, C5aR blockade alleviated renal dysfunction, ECM deposition, macrophage infiltration and proinflammatory factor expression in db/db mice. C5aRA partly reversed the declines in gut microbiota diversity and abundance and gut SCFA levels in db/db mice. C5aRA down-regulated the expression of many immune response-related genes, such as STAT3, in db/db mouse kidneys. C5aRA and SCFAs suppressed C5a-induced STAT3 activation in human renal glomerular endothelial cells (HRGECs). Based on our results, C5 hyperactivation promotes DKD by activating STAT3 in GECs and impairing the gut-kidney axis, suggesting that this hyperactivation is a potential target for the treatment of DKD.