RESUMEN

The complement system is a complex network of proteins that plays a crucial role in the innate immune response. One important component of this system is the C5a-C5aR1 complex, which is critical in the recruitment and activation of immune cells. In-depth investigation of the activation mechanism as well as biased signaling of the C5a-C5aR1 system will facilitate the elucidation of C5a-mediated pathophysiology. In this study, we determined the structure of C5a-C5aR1-Gi complex at a high resolution of 3 Å using cryo-electron microscopy (Cryo-EM). Our results revealed the binding site of C5a, which consists of a polar recognition region on the extracellular side and an amphipathic pocket within the transmembrane domain. Furthermore, we found that C5a binding induces conformational changes of C5aR1, which subsequently leads to the activation of G protein signaling pathways. Notably, a key residue (M265) located on transmembrane helix 6 (TM6) was identified to play a crucial role in regulating the recruitment of ß-arrestin driven by C5a. This study provides more information about the structure and function of the human C5a-C5aR1 complex, which is essential for the proper functioning of the complement system. The findings of this study can also provide a foundation for the design of new pharmaceuticals targeting this receptor with bias or specificity.

Asunto(s)

Complemento C5a , Microscopía por Crioelectrón , Receptor de Anafilatoxina C5a , Microscopía por Crioelectrón/métodos , Humanos , Receptor de Anafilatoxina C5a/química , Receptor de Anafilatoxina C5a/metabolismo , Sitios de Unión , Complemento C5a/química , Complemento C5a/metabolismo , Unión Proteica , Transducción de Señal , Conformación Proteica , Modelos Moleculares

RESUMEN

Alzheimer's disease (AD) is the leading cause of dementia in older adults, and the need for effective, sustainable therapeutic targets is imperative. The complement pathway has been proposed as a therapeutic target. C5aR1 inhibition reduces plaque load, gliosis, and memory deficits in animal models, however, the cellular bases underlying this neuroprotection were unclear. Here, we show that the C5aR1 antagonist PMX205 improves outcomes in the Arctic48 mouse model of AD. A combination of single cell and single nucleus RNA-seq analysis of hippocampi derived from males and females identified neurotoxic disease-associated microglia clusters in Arctic mice that are C5aR1-dependent, while microglial genes associated with synapse organization and transmission and learning were overrepresented in PMX205-treated mice. PMX205 also reduced neurotoxic astrocyte gene expression, but clusters associated with protective responses to injury were unchanged. C5aR1 inhibition promoted mRNA-predicted signaling pathways between brain cell types associated with cell growth and repair, while suppressing inflammatory pathways. Finally, although hippocampal plaque load was unaffected, PMX205 prevented deficits in short-term memory in female Arctic mice. In conclusion, C5aR1 inhibition prevents cognitive loss, limits detrimental glial polarization while permitting neuroprotective responses, as well as leaving most protective functions of complement intact, making C5aR1 antagonism an attractive therapeutic strategy for AD.

Asunto(s)

Enfermedad de Alzheimer , Modelos Animales de Enfermedad , Hipocampo , Microglía , Receptor de Anafilatoxina C5a , Transducción de Señal , Animales , Receptor de Anafilatoxina C5a/antagonistas & inhibidores , Receptor de Anafilatoxina C5a/metabolismo , Receptor de Anafilatoxina C5a/genética , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/patología , Enfermedad de Alzheimer/genética , Ratones , Femenino , Masculino , Transducción de Señal/efectos de los fármacos , Microglía/efectos de los fármacos , Microglía/metabolismo , Hipocampo/metabolismo , Hipocampo/patología , Hipocampo/efectos de los fármacos , Neuroglía/efectos de los fármacos , Neuroglía/metabolismo , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Ratones Transgénicos , Humanos , Ratones Endogámicos C57BL

Asunto(s)

Receptor de Anafilatoxina C5a , Humanos , Receptor de Anafilatoxina C5a/metabolismo , Receptor de Anafilatoxina C5a/inmunología , Receptor de Anafilatoxina C5a/genética , Animales , Ratones , Microambiente Tumoral/inmunología , Neoplasias/radioterapia , Neoplasias/inmunología , Neoplasias/metabolismo

RESUMEN

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.

Asunto(s)

Condrocitos , Ferroptosis , Glucógeno Sintasa Quinasa 3 beta , Factor 2 Relacionado con NF-E2 , Osteoartritis , Fosfatidilinositol 3-Quinasas , Proteínas Proto-Oncogénicas c-akt , Ratas Sprague-Dawley , Receptor de Anafilatoxina C5a , Animales , Ferroptosis/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Condrocitos/metabolismo , Condrocitos/patología , Condrocitos/efectos de los fármacos , Factor 2 Relacionado con NF-E2/metabolismo , Factor 2 Relacionado con NF-E2/genética , Ratas , Osteoartritis/metabolismo , Osteoartritis/patología , Osteoartritis/genética , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Glucógeno Sintasa Quinasa 3 beta/genética , Masculino , Receptor de Anafilatoxina C5a/metabolismo , Receptor de Anafilatoxina C5a/genética , Transducción de Señal , Hemo-Oxigenasa 1/metabolismo , Hemo-Oxigenasa 1/genética , Hemo Oxigenasa (Desciclizante)

RESUMEN

C5a is an integral glycoprotein of the complement system that plays an important role in inflammation and immunity. The physiological concentration of C5a is observed to be elevated under various immunoinflammatory pathophysiological conditions in humans. The pathophysiology of C5a is linked to the "two-site" protein-protein interactions (PPIs) with two genomically related receptors, such as C5aR1 and C5aR2. Therefore, pharmacophores that can potentially block the PPIs between C5a-C5aR1 and C5a-C5aR2 have tremendous potential for development as future therapeutics. Notably, the FDA has already approved antibodies that target the precursors of C5a (Eculizumab, 148 kDa) and C5a (Vilobelimab, 149 kDa) for marketing as complement-targeted therapeutics. In this context, the current study reports the structural characterization of a pair of synthetic designer antibody-like peptides (DePA and DePA1; ≤3.8 kDa) that bind to hotspot regions on C5a and also demonstrates potential traits to neutralize the function of C5a under pathophysiological conditions.

Asunto(s)

Complemento C5a , Péptidos , Receptor de Anafilatoxina C5a , Transducción de Señal , Humanos , Receptor de Anafilatoxina C5a/metabolismo , Receptor de Anafilatoxina C5a/química , Receptor de Anafilatoxina C5a/antagonistas & inhibidores , Complemento C5a/metabolismo , Complemento C5a/química , Péptidos/química , Péptidos/farmacología , Péptidos/metabolismo , Transducción de Señal/efectos de los fármacos , Unión Proteica , Anticuerpos/química , Anticuerpos/metabolismo , Anticuerpos/inmunología , Diseño de Fármacos

RESUMEN

Pregnancy is a fascinating immunological phenomenon because it allows allogeneic fetal and placental tissues to survive inside the mother. As a component of innate immunity with high inflammatory potential, the complement system must be tightly regulated during pregnancy. Dysregulation of the complement system plays a role in pregnancy complications including pre-eclampsia and intrauterine growth restriction. Complement components are also used as biomarkers for pregnancy complications. However, the mechanisms of detrimental role of complement in pregnancy is poorly understood. C5a is the most potent anaphylatoxin and generates multiple immune reactions via two transmembrane receptors, C5aR1 and C5aR2. C5aR1 is pro-inflammatory, but the role of C5aR2 remains largely elusive. Interestingly, murine NK cells have been shown to express C5aR2 without the usual co-expression of C5aR1. Furthermore, C5aR2 appears to regulate IFN-γ production by NK cells in vitro. As IFN-γ produced by uterine NK cells is one of the major factors for the successful development of a vital pregnancy, we investigated the role anaphylatoxin C5a and its receptors in the establishment of pregnancy and the regulation of uterine NK cells by examinations of murine C5ar2-/- pregnancies and human placental samples. C5ar2-/- mice have significantly reduced numbers of implantation sites and a maternal C5aR2 deficiency results in increased IL-12, IL-18 and IFN-γ mRNA expression as well as reduced uNK cell infiltration at the maternal-fetal interface. Human decidual leukocytes have similar C5a receptor expression patterns showing clinical relevance. In conclusion, this study identifies C5aR2 as a key contributor to dNK infiltration and pregnancy success.

Asunto(s)

Células Asesinas Naturales , Ratones Noqueados , Receptor de Anafilatoxina C5a , Útero , Receptor de Anafilatoxina C5a/genética , Receptor de Anafilatoxina C5a/metabolismo , Femenino , Animales , Embarazo , Ratones , Útero/inmunología , Humanos , Células Asesinas Naturales/inmunología , Células Asesinas Naturales/metabolismo , Placenta/inmunología , Placenta/metabolismo , Complemento C5a/inmunología , Complemento C5a/metabolismo , Ratones Endogámicos C57BL , Interferón gamma/metabolismo , Interferón gamma/inmunología

RESUMEN

Introduction: Complement-mediated damage to the myocardium during acute myocardial infarction (AMI), particularly the late components of the terminal pathway (C5-convertase and C5b-9), have previously been characterized. Unfortunately, only few studies have reported a direct association between dysregulated complement activation and endothelial function. Hence, little attention has been paid to the role of the anaphylatoxin C5a. The endothelial glycocalyx (eGC) together with the cellular actin cortex provide a vasoprotective barrier against chronic vascular inflammation. Changes in their nanomechanical properties (stiffness and height) are recognized as hallmarks of endothelial dysfunction as they correlate with the bioavailability of vasoactive substances, such as nitric oxide (NO). Here, we determined how the C5a:C5aR1 axis affects the eGC and endothelial function in AMI. Methods: Samples of fifty-five patients with ST-elevation myocardial infarction (STEMI) vs. healthy controls were analyzed in this study. eGC components and C5a levels were determined via ELISA; NO levels were quantified chemiluminescence-based. Endothelial cells were stimulated with C5a or patient sera (with/without C5a-receptor1 antagonist "PMX53") and the nanomechanical properties of eGC quantified using the atomic force microscopy (AFM)-based nanoindentation technique. To measure actin cytoskeletal tension regulator activation (RhoA and Rac1) G-LISA assays were applied. Vascular inflammation was examined by quantifying monocyte-endothelium interaction via AFM-based single-cell-force spectroscopy. Results: Serum concentrations of eGC components and C5a were significantly increased during STEMI. Serum and solely C5a stimulation decreased eGC height and stiffness, indicating shedding of the eGC. C5a enhanced RhoA activation, resulting in increased cortical stiffness with subsequent reduction in NO concentrations. Monocyte adhesion to the endothelium was enhanced after both C5a and stimulation with STEMI serum. eGC degradation- and RhoA-induced cortical stiffening with subsequent endothelial dysfunction were attenuated after administering PMX53. Conclusion: This study demonstrates that dysregulated C5a activation during AMI results in eGC damage with subsequent endothelial dysfunction and reduced NO bioavailability, indicating progressively developing vascular inflammation. This could be prevented by antagonizing C5aR1, highlighting the role of the C5a:C5a-Receptor1 axis in vascular inflammation development and endothelial dysfunction in AMI, offering new therapeutic approaches for future investigations.

Asunto(s)

Activación de Complemento , Complemento C5a , Glicocálix , Infarto del Miocardio , Receptor de Anafilatoxina C5a , Humanos , Glicocálix/metabolismo , Glicocálix/patología , Complemento C5a/metabolismo , Infarto del Miocardio/metabolismo , Infarto del Miocardio/inmunología , Infarto del Miocardio/patología , Infarto del Miocardio/fisiopatología , Receptor de Anafilatoxina C5a/metabolismo , Masculino , Persona de Mediana Edad , Femenino , Anciano , Células Endoteliales/metabolismo , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Transducción de Señal , Óxido Nítrico/metabolismo

RESUMEN

Non-alcoholic fatty liver disease (NAFLD) has become the first major chronic liver disease in developed countries. 10-20% of NAFLD patients will progress to non-alcoholic steatohepatitis (NASH), and up to 25% of NASH patients may develop cirrhosis within 10 years. Therefore, it is critical to find key targets that may treat this disease. Here, we identified C5aR1 as a highly-expressed gene in NASH mouse model through analyzing Gene Expression Omnibus (GEO) database and confirmed its higher expression in livers of NASH patients than that of NAFL patients. Meanwhile, we verified its positive correlation with patients' serum alanine transaminase (ALT) and aspartate transaminase (AST) levels. In vivo and in vitro experiments revealed that knocking down C5aR1 in liver significantly reduced liver weight ratio and serum ALT and AST levels and attenuated inflammatory cell infiltration and cell apoptosis in the liver of NASH mice as well as enhanced the efferocytotic ability of liver macrophages, suggesting that C5aR1 may play a crucial role in the efferocytosis of liver macrophages. Furthermore, we also found that the expression levels of nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing protein 3 (NLRP3), caspase-1, IL-1ß and other inflammation-related factors in the liver were significantly reduced. Our work demonstrates a potential mechanism of how C5aR1 deficiency protects against diet-induced NASH by coordinating the regulation of inflammatory factors and affecting hepatic macrophage efferocytosis.

Asunto(s)

Hígado , Macrófagos , Proteína con Dominio Pirina 3 de la Familia NLR , Enfermedad del Hígado Graso no Alcohólico , Fagocitosis , Receptor de Anafilatoxina C5a , Animales , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Enfermedad del Hígado Graso no Alcohólico/patología , Receptor de Anafilatoxina C5a/metabolismo , Receptor de Anafilatoxina C5a/genética , Ratones , Macrófagos/metabolismo , Humanos , Hígado/metabolismo , Hígado/patología , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Masculino , Modelos Animales de Enfermedad , Ratones Endogámicos C57BL , Apoptosis , Alanina Transaminasa/sangre , Alanina Transaminasa/metabolismo , Aspartato Aminotransferasas/sangre , Aspartato Aminotransferasas/metabolismo , Eferocitosis

RESUMEN

As another receptor for complement activation product C5a, C5aR2 has been paid much attention these years. Although controversial and complex, its specific signals or roles in modulating the classic receptor C5aR1 have been investigated and gradually revealed. The hypothesis of the heterodimer of C5aR1 and C5aR2 has also been suggested and observed under extremely high C5a concentrations. In this article, we tried to investigate whether C5aR2 would affect C5aR1 expression under normal or inflammatory conditions in WT and C5ar2 -/- mice of C57BL/6 background. We focused on the innate immune cells-neutrophils and macrophages. The mRNA levels of C5ar1 in normal kidney, liver, and the mRNA or protein levels of naïve-bone marrow and peripheral blood leukocytes and peritoneal Mφs were comparable between WT and C5ar2 -/- mice, indicating the technique of C5aR2 knockout did not affect the transcription of its neighboring gene C5aR1. However, the mean fluorescence intensity of surface C5aR1 on naïve circulating C5ar2 -/- neutrophils detected by FACS was reduced, which might be due to the reduced internalization of C5aR1 on C5ar2 -/- neutrophils. In the peritonitis model induced by i.p. injection of thioglycollate, more neutrophils were raised after 10 hr in C5ar2 -/- peritoneal cavity, indicating the antagonism of C5aR2 on C5aR1 signal in neutrophil chemotaxis. After 3 days of thioglycollate injection, the mainly infiltrating macrophages were comparable between WT and C5ar2 -/- mice, but the C5ar1 mRNA and surface or total C5aR1 protein expression were both reduced in C5ar2 -/- macrophages, combined with our previous study of reduced chemokines and cytokines expression in C5ar2 -/- peritoneal macrophages, indicating that C5aR2 in macrophages may cooperate with C5aR1 inflammatory signals. Our article found C5aR2 deficiency lessened C5aR1 distribution and expression in neutrophils and macrophages with different functions, indicating C5aR2 might function differently in different cells.

Asunto(s)

Macrófagos , Neutrófilos , Peritonitis , Receptor de Anafilatoxina C5a , Animales , Ratones , Complemento C5a/metabolismo , Complemento C5a/inmunología , Modelos Animales de Enfermedad , Inmunidad Innata , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Neutrófilos/inmunología , Neutrófilos/metabolismo , Peritonitis/inmunología , Receptor de Anafilatoxina C5a/metabolismo , Receptor de Anafilatoxina C5a/genética

RESUMEN

BACKROUND: Complement activation may facilitate hypertension through its effects on immune responses. The anaphylatoxin C5a, a major inflammatory effector, binds to the C5a receptors 1 and 2 (C5aR1, C5aR2). We have recently shown that C5aR1-/- mice have reduced hypertensive renal injury. The role of C5aR2 in hypertension is unknown. METHODS: For examination of C5aR2 expression on infiltrating and resident renal cells a tandem dye Tomato-C5aR2 knock-in reporter mouse was used. Human C5aR2 expression was analyzed in a single-cell RNAseq data set from the kidneys of hypertensive patients. Finally, we examined the effect of angiotensin II-induced hypertension in C5aR2-deficient mice. RESULTS: Flow cytometric analysis of leukocytes isolated from kidneys of the reporter mice showed that dendritic cells are the major C5aR2-expressing population (34%) followed by monocyte/macrophages (30%) and neutrophils (14%). Using confocal microscopy C5aR2 was not detected in resident renal or cardiac cells. In the human kidney, C5aR2 was also mainly found in monocytes, macrophages, and dendritic cells with a significantly higher expression in hypertension (P < 0.05). Unilateral nephrectomy was performed followed by infusion of Ang II (0.75 ng/g/min) and a high salt diet in wildtype (n = 18) and C5aR2-deficient mice (n = 14). Blood pressure, renal injury (albuminuria, glomerular filtration rate, glomerular and tubulointerstitial injury, inflammation), and cardiac injury (cardiac fibrosis, heart weight, gene expression) did not differ between hypertensive wildtype and C5aR2-/- mice. CONCLUSIONS: In summary, C5aR2 is mainly expressed in myeloid cells in the kidney in mice and humans but its deficiency has no effect on Ang II-induced hypertensive injury.

Asunto(s)

Angiotensina II , Hipertensión , Riñón , Receptor de Anafilatoxina C5a , Animales , Receptor de Anafilatoxina C5a/metabolismo , Receptor de Anafilatoxina C5a/genética , Hipertensión/metabolismo , Hipertensión/fisiopatología , Humanos , Riñón/patología , Riñón/metabolismo , Riñón/inmunología , Masculino , Ratones Noqueados , Modelos Animales de Enfermedad , Ratones Endogámicos C57BL , Ratones , Presión Sanguínea , Enfermedades Renales/metabolismo , Enfermedades Renales/patología , Enfermedades Renales/inducido químicamente , Hipertensión Renal , Nefritis

RESUMEN

Patients with autosomal dominant polycystic kidney disease (ADPKD), a genetic disease due to mutations of the PKD1 or PKD2 gene, show signs of complement activation in the urine and cystic fluid, but their pathogenic role in cystogenesis is unclear. We tested the causal relationship between complement activation and cyst growth using a Pkd1KO renal tubular cell line and newly generated conditional Pkd1-/- C3-/- mice. Pkd1-deficient tubular cells have increased expression of complement-related genes (C3, C5, CfB, C3ar, and C5ar1), while the gene and protein expression of complement regulators DAF, CD59, and Crry is decreased. Pkd1-/- C3-/- mice are unable to fully activate the complement cascade and are characterized by a significantly slower kidney cystogenesis, preserved renal function, and reduced intrarenal inflammation compared with Pkd1-/- C3+/+ controls. Transgenic expression of the cytoplasmic C-terminal tail of Pkd1 in Pkd1KO cells lowered C5ar1 expression, restored Daf levels, and reduced cell proliferation. Consistently, both DAF overexpression and pharmacological inhibition of C5aR1 (but not C3aR) reduced Pkd1KO cell proliferation. In conclusion, the loss of Pkd1 promotes unleashed activation of locally produced complement by downregulating DAF expression in renal tubular cells. Increased C5a formation and C5aR1 activation in tubular cells promotes cyst growth, offering a new therapeutic target.

Asunto(s)

Antígenos CD55 , Complemento C3 , Ratones Noqueados , Riñón Poliquístico Autosómico Dominante , Animales , Riñón Poliquístico Autosómico Dominante/genética , Riñón Poliquístico Autosómico Dominante/patología , Riñón Poliquístico Autosómico Dominante/metabolismo , Ratones , Antígenos CD55/genética , Antígenos CD55/metabolismo , Complemento C3/genética , Complemento C3/metabolismo , Receptor de Anafilatoxina C5a/metabolismo , Receptor de Anafilatoxina C5a/genética , Modelos Animales de Enfermedad , Activación de Complemento , Canales Catiónicos TRPP/genética , Canales Catiónicos TRPP/metabolismo , Humanos , Proliferación Celular , Masculino , Línea Celular , Receptores de Complemento 3b/genética , Receptores de Complemento 3b/metabolismo

RESUMEN

Although Poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi) have been approved in multiple diseases, including BRCA1/2 mutant breast cancer, responses are usually transient requiring the deployment of combination therapies for optimal efficacy. Here we thus explore mechanisms underlying sensitivity and resistance to PARPi using two intrinsically PARPi sensitive (T22) and resistant (T127) syngeneic murine breast cancer models in female mice. We demonstrate that tumor associated macrophages (TAM) potentially contribute to the differential sensitivity to PARPi. By single-cell RNA-sequencing, we identify a TAM_C3 cluster, expressing genes implicated in anti-inflammatory activity, that is enriched in PARPi resistant T127 tumors and markedly decreased by PARPi in T22 tumors. Rps19/C5aR1 signaling is selectively elevated in TAM_C3. C5aR1 inhibition or transferring C5aR1hi cells increases and decreases PARPi sensitivity, respectively. High C5aR1 levels in human breast cancers are associated with poor responses to immune checkpoint blockade. Thus, targeting C5aR1 may selectively deplete pro-tumoral macrophages and engender sensitivity to PARPi and potentially other therapies.

Asunto(s)

Neoplasias de la Mama , Resistencia a Antineoplásicos , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Receptor de Anafilatoxina C5a , Macrófagos Asociados a Tumores , Animales , Femenino , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Resistencia a Antineoplásicos/genética , Resistencia a Antineoplásicos/efectos de los fármacos , Ratones , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Neoplasias de la Mama/metabolismo , Humanos , Macrófagos Asociados a Tumores/efectos de los fármacos , Macrófagos Asociados a Tumores/inmunología , Macrófagos Asociados a Tumores/metabolismo , Línea Celular Tumoral , Receptor de Anafilatoxina C5a/antagonistas & inhibidores , Receptor de Anafilatoxina C5a/metabolismo , Receptor de Anafilatoxina C5a/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Macrófagos/metabolismo , Macrófagos/efectos de los fármacos

RESUMEN

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.

Asunto(s)

Complemento C5a , Dinaminas , Nefritis Lúpica , Dinámicas Mitocondriales , Podocitos , Receptor de Anafilatoxina C5a , Podocitos/metabolismo , Podocitos/patología , Nefritis Lúpica/metabolismo , Nefritis Lúpica/patología , Nefritis Lúpica/etiología , Animales , Receptor de Anafilatoxina C5a/metabolismo , Receptor de Anafilatoxina C5a/genética , Ratones , Dinaminas/metabolismo , Dinaminas/genética , Complemento C5a/metabolismo , Humanos , Fosforilación , Modelos Animales de Enfermedad , Mitocondrias/metabolismo , Transducción de Señal , Femenino

RESUMEN

The complement system constitutes an integral component of the innate immune system and plays a critical role in adaptive immunity. Activation of this system engenders the production of complement peptide fragments, including C5a, which engage G-protein coupled receptors predominantly expressed in immune-associated cells, such as neutrophils, initiating pro-inflammatory responses. Intriguingly, our investigation has unveiled the presence of C5a receptor 1 (C5aR1) expression within skeletal muscle, a key metabolic tissue and primary target of insulin. Herein, we demonstrate that C5aR1 activation by C5a in differentiated human skeletal muscle cells elicits acute suppression of insulin signalling. This suppression manifests as impaired insulin-dependent association between IRS1 and the p85 subunit of PI3-kinase, a 50% reduction in Akt phosphorylation, and a 60% decline in insulin-stimulated glucose uptake. This impairment in insulin signalling is associated with a three-fold elevation in intramyocellular diacylglycerol (DAG) levels and a two-fold increase in cytosolic calcium content, which promote PKC-mediated IRS1 inhibition via enhanced phosphorylation at IRS1 Ser1101. Significantly, our findings demonstrate that structurally diverse C5aR1 antagonists, along with genetic deletion or stable silencing of C5aR1 by 80% using short-hairpin RNA, effectively attenuate repression of insulin signalling by C5a in LHCN-M2 human skeletal myotubes. These results underscore the potential of heightened C5aR1 activation, characteristic of obesity and chronic inflammatory conditions, to detrimentally impact insulin function within skeletal muscle cells. Additionally, the study suggests that agents targeting the C5a-C5aR axis, originally devised for mitigating complement-dependent inflammatory conditions, may offer therapeutic avenues to ameliorate immune-driven insulin resistance in key peripheral metabolic tissues, including skeletal muscle.

Asunto(s)

Factores Inmunológicos , Insulina , Receptor de Anafilatoxina C5a , Humanos , Factores Inmunológicos/metabolismo , Insulina/fisiología , Músculo Esquelético/metabolismo , Receptor de Anafilatoxina C5a/metabolismo , Transducción de Señal

RESUMEN

The complement system mediates diverse regulatory immunological functions. C5aR2, an enigmatic receptor for anaphylatoxin C5a, has been shown to modulate PRR-dependent pro-inflammatory cytokine secretion in human macrophages. However, the specific downstream targets and underlying molecular mechanisms are less clear. In this study, CRISPR-Cas9 was used to generate macrophage models lacking C5aR2, which were used to probe the role of C5aR2 in the context of PRR stimulation. cGAS and STING-induced IFN-ß secretion was significantly increased in C5aR2 KO THP-1 cells and C5aR2-edited primary human monocyte-derived macrophages, and STING and IRF3 expression were increased, albeit not significantly, in C5aR2 KO cell lines implicating C5aR2 as a regulator of the IFN-ß response to cGAS-STING pathway activation. Transcriptomic analysis by RNAseq revealed that nucleic acid sensing and antiviral signalling pathways were significantly up-regulated in C5aR2 KO THP-1 cells. Altogether, these data suggest a link between C5aR2 and nucleic acid sensing in human macrophages. With further characterisation, this relationship may yield therapeutic options in interferon-related pathologies.

Asunto(s)

Interferón beta , Macrófagos , Proteínas de la Membrana , Ácidos Nucleicos , Receptor de Anafilatoxina C5a , Humanos , Interferón beta/metabolismo , Macrófagos/metabolismo , Ácidos Nucleicos/metabolismo , Nucleotidiltransferasas/metabolismo , Transducción de Señal , Receptor de Anafilatoxina C5a/metabolismo , Proteínas de la Membrana/metabolismo

RESUMEN

Previous studies have shown that silica nanoparticles (SiNPs) exposure can affect the respiratory, cardiovascular, reproductive and other systems, with the lung being the primary target organ for the direct effect, causing damage with a central feature of pulmonary inflammation and fibrosis. However, the underlying mechanisms of pulmonary fibrosis due to SiNPs are not fully understood. The aim of the study was to investigate the role of complement anaphylatoxin C5a in SiNPs-induced pulmonary fibrosis. A mouse model of SiNPs-induced pulmonary fibrosis was established, and pulmonary fibrosis-related indicators, epithelial-to-mesenchymal transition (EMT), C5a/C5aR1 and high mobility group protein B1 (HMGB1) proteins were measured. An in vitro study using the human lung epithelial cell line BEAS-2B investigated whether C5a leads to epithelial-to-mesenchymal trans-differentiation. In vivo studies revealed that SiNPs-induced pulmonary fibrosis mainly manifested as EMT trans-differentiation in airway epithelial cells, which subsequently led to excessive deposition of extracellular matrix (ECM). Furthermore, we found that C5a and C5aR1 proteins were also increased in SiNPs-induced pulmonary fibrosis tissue. In vitro studies also showed that C5a directly activated HMGB1/RAGE signaling and induced EMT in BEAS-2B cells. Finally, treatment of SiNPs-exposed mice with the C5aR1 inhibitor PMX205 effectively reduced C5aR1 levels and inhibited the activation of HMGB1/RAGE signaling and the expression of EMT-related proteins, culminating in a significant alleviation of pulmonary fibrosis. Taken together, our results suggest that C5a/C5aR1 is the main signaling pathway for SiNPs-induced pulmonary fibrosis, which induces EMT in airway epithelial cells via the HMGB1/RAGE axis.

Asunto(s)

Proteína HMGB1 , Nanopartículas , Fibrosis Pulmonar , Humanos , Animales , Ratones , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/metabolismo , Proteína HMGB1/metabolismo , Dióxido de Silicio/toxicidad , Células Epiteliales/metabolismo , Receptor de Anafilatoxina C5a/metabolismo , Complemento C5a/metabolismo

RESUMEN

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.

Asunto(s)

Metilación de ADN , Linfocitos T Reguladores , Ratones , Animales , Factores de Transcripción/metabolismo , Regulación de la Expresión Génica , Receptor de Anafilatoxina C5a/metabolismo , Proteínas Nucleares/genética , Desmetilación , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo

RESUMEN

Introduction: The function of the second receptor for the complement cleavage product C5a, C5aR2, is poorly understood and often neglected in the immunological context. Using mice with a global deficiency of C5aR2, we have previously reported an important role of this receptor in the pathogenesis of the neutrophil-driven autoimmune disease epidermolysis bullosa acquisita (EBA). Based on in vitro analyses, we hypothesized that the absence of C5aR2 specifically on neutrophils is the cause of the observed differences. Here, we report the generation of a new mouse line with a LysM-specific deficiency of C5aR2. Methods: LysM-specific deletion of C5aR2 was achieved by crossing LysMcre mice with tdTomato-C5ar2fl/fl mice in which the tdTomato-C5ar2 gene is flanked by loxP sites. Passive EBA was induced by subcutaneous injection of rabbit anti-mouse collagen type VII IgG. The effects of targeted deletion of C5ar2 on C5a-induced effector functions of neutrophils were examined in in vitro assays. Results: We confirm the successful deletion of C5aR2 at both the genetic and protein levels in neutrophils. The mice appeared healthy and the expression of C5aR1 in bone marrow and blood neutrophils was not negatively affected by LysM-specific deletion of C5aR2. Using the antibody transfer mouse model of EBA, we found that the absence of C5aR2 in LysM-positive cells resulted in an overall amelioration of disease progression, similar to what we had previously found in mice with global deficiency of C5aR2. Neutrophils lacking C5aR2 showed decreased activation after C5a stimulation and increased expression of the inhibitory Fcγ receptor FcγRIIb. Discussion: Overall, with the data presented here, we confirm and extend our previous findings and show that C5aR2 in neutrophils regulates their activation and function in response to C5a by potentially affecting the expression of Fcγ receptors and CD11b. Thus, C5aR2 regulates the finely tuned interaction network between immune complexes, Fcγ receptors, CD11b, and C5aR1 that is important for neutrophil recruitment and sustained activation. This underscores the importance of C5aR2 in the pathogenesis of neutrophil-mediated autoimmune diseases.

Asunto(s)

Enfermedades Autoinmunes , Epidermólisis Ampollosa Adquirida , Animales , Ratones , Complemento C5a/metabolismo , Activación Neutrófila , Neutrófilos , Receptor de Anafilatoxina C5a/genética , Receptor de Anafilatoxina C5a/metabolismo , Receptores de Complemento/metabolismo , Receptores de IgG/metabolismo

RESUMEN

Complement activation and Rab GTPase trafficking are commonly observed in inflammatory responses. Recruitment of innate immune cells to sites of infection or injury and secretion of inflammatory chemokines are promoted by complement component 5a (C5a) that activates the cell surface protein C5a receptor1 (C5aR1). Persistent activation can lead to a myriad of inflammatory and autoimmune diseases. Here, we demonstrate that the mechanism of C5a induced chemotaxis of human monocyte-derived macrophages (HMDMs) and their secretion of inflammatory chemokines are controlled by Rab5a. We find that C5a activation of the G protein coupled receptor C5aR1 expressed on the surface of HMDMs, recruits ß-arrestin2 via Rab5a trafficking, then activates downstream phosphatidylinositol 3-kinase (PI3K)/Akt signaling that culminates in chemotaxis and secretion of pro-inflammatory chemokines from HMDMs. High-resolution lattice light-sheet microscopy on live cells showed that C5a activates C5aR1-GFP internalization and colocalization with Rab5a-tdTomato but not with dominant negative mutant Rab5a-S34N-tdTomato in HEK293 cells. We found that Rab5a is significantly upregulated in differentiated HMDMs and internalization of C5aR1 is dependent on Rab5a. Interestingly, while knockdown of Rab5a inhibited C5aR1-mediated Akt phosphorylation, it did not affect C5aR1-mediated ERK1/2 phosphorylation or intracellular calcium mobilization in HMDMs. Functional analysis using transwell migration and µ-slide chemotaxis assays indicated that Rab5a regulates C5a-induced chemotaxis of HMDMs. Further, C5aR1 was found to mediate interaction of Rab5a with ß-arrestin2 but not with G proteins in HMDMs. Furthermore, C5a-induced secretion of pro-inflammatory chemokines (CCL2, CCL3) from HMDMs was attenuated by Rab5a or ß-arrestin2 knockdown or by pharmacological inhibition with a C5aR1 antagonist or a PI3K inhibitor. These findings reveal a C5a-C5aR1-ß-arrestin2-Rab5a-PI3K signaling pathway that regulates chemotaxis and pro-inflammatory chemokine secretion in HMDMs and suggests new ways of selectively modulating C5a-induced inflammatory outputs.

Asunto(s)

Quimiocinas , Quimiotaxis , Macrófagos , Receptor de Anafilatoxina C5a , Proteínas de Unión al GTP rab5 , Humanos , beta-Arrestinas/metabolismo , Quimiocinas/metabolismo , Complemento C5a/metabolismo , Células HEK293 , Macrófagos/metabolismo , Transporte de Proteínas , Proteínas de Unión al GTP rab5/metabolismo , Receptor de Anafilatoxina C5a/metabolismo

RESUMEN

Complement activation is thought to underline the pathologic progression of obesity-related metabolic disorders; however, its role in adaptive thermogenesis has scarcely been explored. Here, we identify complement C3a receptor (C3aR) and C5a receptor (C5aR) as critical switches to control adipocyte browning and energy balance in male mice. Loss of C3aR and C5aR in combination, more than individually, increases cold-induced adipocyte browning and attenuates diet-induced obesity in male mice. Mechanistically, loss of C3aR and C5aR increases regulatory T cell (Treg) accumulation in the subcutaneous white adipose tissue during cold exposure or high-fat diet. Activated Tregs produce adenosine, which is converted to inosine by adipocyte-derived adenosine deaminases. Inosine promotes adipocyte browning in a manner dependent on activating adenosine A2a receptor. These data reveal a regulatory mechanism of complement in controlling adaptive thermogenesis and suggest that targeting the C3aR/C5aR pathways may represent a therapeutic strategy in treating obesity-related metabolic diseases.

Asunto(s)

Receptor de Anafilatoxina C5a , Transducción de Señal , Animales , Masculino , Ratones , Adipocitos , Dieta , Obesidad , Receptor de Anafilatoxina C5a/metabolismo