Decoding anaphylatoxins: unveiling the molecular mechanisms of complement receptor activation and signaling.

Recent advances in cryo-electron microscopy (Cryo-EM) have revolutionized our understanding of the complement C5a/C3a receptors that are crucial in inflammation. A recent report by Yadav et al. has elucidated the activation, ligand binding, selectivity, and signaling bias of these receptors, thereby enhancing structure-guided drug discovery. This paves the way for more effective anti-inflammatory therapies that target these receptors with unprecedented precision.

Functional profiling of the G protein-coupled receptor C3aR1 reveals ligand-mediated biased agonism.

G protein-coupled receptors (GPCRs) are leading druggable targets for several medicines, but many GPCRs are still untapped for their therapeutic potential due to poor understanding of specific signaling properties. The complement C3a receptor 1 (C3aR1) has been extensively studied for its physiological role in C3a-mediated anaphylaxis/inflammation, and in TLQP-21-mediated lipolysis, but direct evidence for the functional relevance of the C3a and TLQP-21 ligands and signal transduction mechanisms are still limited. In addition, C3aR1 G protein coupling specificity is still unclear, and whether endogenous ligands, or drug-like compounds, show ligand-mediated biased agonism is unknown. Here, we demonstrate that C3aR1 couples preferentially to Gi/o/z proteins and can recruit ß-arrestins to cause internalization. Furthermore, we showed that in comparison to C3a63-77, TLQP-21 exhibits a preference toward Gi/o-mediated signaling compared to ß-arrestin recruitment and internalization. We also show that the purported antagonist SB290157 is a very potent C3aR1 agonist, where antagonism of ligand-stimulated C3aR1 calcium flux is caused by potent ß-arrestin-mediated internalization. Finally, ligand-mediated signaling bias impacted cell function as demonstrated by the regulation of calcium influx, lipolysis in adipocytes, phagocytosis in microglia, and degranulation in mast cells. Overall, we characterize C3aR1 as a Gi/o/z-coupled receptor and demonstrate the functional relevance of ligand-mediated signaling bias in key cellular models. Due to C3aR1 and its endogenous ligands being implicated in inflammatory and metabolic diseases, these results are of relevance toward future C3aR1 drug discovery.

Deficiency of Complement C3a and C5a receptors Does Not Prevent Angiotensin II-Induced Hypertension and Hypertensive End-Organ Damage.

BACKGROUND: Complement may drive the pathology of hypertension through effects on innate and adaptive immune responses. Recently an injurious role for the anaphylatoxin receptors C3aR (complement component 3a receptor) and C5aR1 (complement component 5a receptor) in the development of hypertension was shown through downregulation of Foxp3+ (forkhead box protein 3) regulatory T cells. Here, we deepen our understanding of the therapeutic potential of targeting both receptors in hypertension. METHODS: Data from the European Renal cDNA Bank, single cell sequencing and immunohistochemistry were examined in hypertensive patients. The effect of C3aR or C3aR/C5aR1 double deficiency was assessed in two models of Ang II (angiotensin II)-induced hypertension in knockout mice. RESULTS: We found increased expression of C3aR, C5aR1 and Foxp3 cells in kidney biopsies of patients with hypertensive nephropathy. Expression of both receptors was mainly found in myeloid cells. No differences in blood pressure, renal injury (albuminuria, glomerular filtration rate, glomerular and tubulointerstitial injury, inflammation) or cardiac injury (cardiac fibrosis, heart weight, gene expression) between control and mutant mice was discerned in C3aR-/- as well as C3aR/C5aR1-/- double knockout mice. The number of renal Tregs was not decreased in Ang II as well as in DOCA salt induced hypertension. CONCLUSIONS: Hypertensive nephropathy in mice and men is characterized by an increase of renal regulatory T cells and enhanced expression of anaphylatoxin receptors. Our investigations do not corroborate a role for C3aR/C5aR1 axis in Ang II-induced hypertension hence challenging the concept of anaphylatoxin receptor targeting in the treatment of hypertensive disease.

Molecular basis of anaphylatoxin binding, activation, and signaling bias at complement receptors.

The complement system is a critical part of our innate immune response, and the terminal products of this cascade, anaphylatoxins C3a and C5a, exert their physiological and pathophysiological responses primarily via two GPCRs, C3aR and C5aR1. However, the molecular mechanism of ligand recognition, activation, and signaling bias of these receptors remains mostly elusive. Here, we present nine cryo-EM structures of C3aR and C5aR1 activated by their natural and synthetic agonists, which reveal distinct binding pocket topologies of complement anaphylatoxins and provide key insights into receptor activation and transducer coupling. We also uncover the structural basis of a naturally occurring mechanism to dampen the inflammatory response of C5a via proteolytic cleavage of the terminal arginine and the G-protein signaling bias elicited by a peptide agonist of C3aR identified here. In summary, our study elucidates the innerworkings of the complement anaphylatoxin receptors and should facilitate structure-guided drug discovery to target these receptors in a spectrum of disorders.

SARS-CoV-2 spike protein induces lung endothelial cell dysfunction and thrombo-inflammation depending on the C3a/C3a receptor signalling.

The spike protein of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) can interact with endothelial cells. However, no studies demonstrated the direct effect of the spike protein subunit 1 (S1) in inducing lung vascular damage and the potential mechanisms contributing to lung injury. Here, we found that S1 injection in mice transgenic for human angiotensin converting enzyme 2 (ACE2) induced early loss of lung endothelial thromboresistance at 3 days, as revealed by thrombomodulin loss and von Willebrand factor (vWF) increase. In parallel, vascular and epithelial C3 deposits and enhanced C3a receptor (C3aR) expression were observed. These changes preceded diffuse alveolar damage and lung vascular fibrin(ogen)/platelets aggregates at 7 days, as well as inflammatory cell recruitment and fibrosis. Treatment with C3aR antagonist (C3aRa) inhibited lung C3 accumulation and C3a/C3aR activation, limiting vascular thrombo-inflammation and fibrosis. Our study demonstrates that S1 triggers vascular dysfunction and activates complement system, instrumental to lung thrombo-inflammatory injury. By extension, our data indicate C3aRa as a valuable therapeutic strategy to limit S1-dependent lung pathology.

Cytological and functional effect of complement 3a on Human Scleral Fibroblasts.

PURPOSE: The complement system is considered to play an important role in the progression of myopia, whereas the influence of complement activation on the human scleral fibroblasts (HSFs) remains unknown. Hence, the effect of complement 3a (C3a) on HSFs was investigated in this study. METHODS: HSFs were cultured with exogenous C3a at 0.1 µM for various periods following different measurement protocols, and cells without C3a treatment served as negative control (NC). Cell viability was investigated using the MTS assay after 3 days of C3a treatment. Cell proliferation was evaluated by the 5-Ethynyl-20-Deoxyuridine (EdU) assay following C3a stimulation for 24 hours. Apoptosis was assessed by Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double staining following C3a stimulation for 48 hours and the stained cells were analysed using flow cytometry. The levels of type I collagen and matrix metalloproteinase-2 (MMP-2) were analysed using ELISA following C3a stimulation for 36 and 60 hours. The level of CD59 were analysed using western blot following C3a stimulation for 60 hours. RESULTS: The MTS assay revealed that cell viability was attenuated by 13% and 8% after C3a for 2 and 3 days, respectively (P < 0.05). The EdU assay demonstrated a 9% decrease in proliferation rate for the C3a-treated cells after 24 hours (P < 0.05). The apoptosis analysis revealed an increased percentage of cells in early apoptosis (P = 0.02) and total apoptosis (P = 0.02) in the C3a-treated group. Compared with NC group, the level of MMP-2 was increased by 17.6% (P = 0.002), whereas the levels of type I collagen and CD59 were respectively decreased by 12.5% (P = 0.024) and 21.6% (P = 0.044) with C3a treatment for 60 hours. CONCLUSIONS: These results indicated that C3a-induced complement activation is potentially involved in inducing myopic-associated scleral extracellular matrix remodelling via mediating the proliferation and function of HSFs.

Echinacea purpurea (L.) Moench extract suppresses inflammation by inhibition of C3a/C3aR signaling pathway in TNBS-induced ulcerative colitis rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Echinacea purpurea (L.) Moench (EP) is a perennial herbaceous flowering plant, commonly known as purple conical flower. It was widely used to treat skin inflammation and gastrointestinal diseases. AIM OF STUDY: Ulcerative colitis (UC) is a chronic and nonspecific inflammatory disease. Recent evidence shows that immune disorders are involved in the pathogenesis of UC. To evaluate the protective effect of Echinacea purpurea (L.) Moench exact (EE) on UC and explore the role of complement system in the treatment of UC. MATERIALS AND METHODS: UC model was induced in rats by 2,4,6-trinitrobenzene sulfonic acid (TNBS), and then rats were administered with EE for 10 days. Collect colon tissues for analysis of relevant mechanisms. RESULTS: EE could reduce the weight loss and diarrhea of UC rats. In addition, EE could improve the integrity of intestinal epithelial barrier in UC rats. EE inhibited the level of proinflammatory cytokines and promoted the antioxidation. Furthermore, EE suppressed the expression of C3aR, CFB, CD55, TLR4 and NLRP3. CONCLUSION: These results indicate that EE may achieve therapeutic effect by inhibiting C3a/C3aR signal pathway, suggesting that EE may be used as a medicinal plant to alleviate UC.

Macrophages promote heat stress nephropathy in mice via the C3a-C3aR-TNF pathway.

Heat-stress nephropathy (HSN) is associated with recurrent dehydration. However, the mechanisms underlying HSN remain largely unknown. In this study, we evaluated the role of dehydration in HSN and kidney injury in mice. Firstly, we found that complement was strongly activated in the mice that were exposed to dehydration; and among complement components, the interaction between C3a and its receptor, C3aR, was more closely associated with kidney injury. Then two-month-old mice were intraperitoneally injected with 2% dimethyl sulfoxide (DMSO) or the C3aR inhibitor SB290157 during dehydration. DMSO-treated mice exhibited excessive macrophage infiltration, renal cell apoptosis, and kidney fibrosis. In contrast, SB290157-treated mice had no apparent kidney injury. By fluorescence-activated cell sorting (FACS), we found that SB290157 treatment in mice remarkably inhibited macrophage infiltration and suppressed CCR2 expression in macrophages. In addition, C3a binding to C3aR promoted macrophage polarization toward the M1 phenotype and increased the production of TNF-α, which induced renal tubular epithelial cell (RTEC) apoptosis in vivo and in vitro. Interestingly, C3a treatment failed to directly induce TNF-α production and apoptosis in RTECs. However, TNF-α production in response to C3a treatment was significantly elevated when RTECs were cocultured with macrophages, suggesting that macrophages rather than RTECs are the target of C3a-C3aR interaction. At last, we proved that infusion of macrophages which highly expressed TNF-α would significantly deteriorate HSN in TNF-KO mice when they were exposed to recurrent dehydration. This study uncovers a novel mechanism underlying the pathogenesis of HSN, and a potential pathway to prevent kidney injury during dehydration.

Astrocyte Responses to Complement Peptide C3a are Highly Context-Dependent.

Astrocytes perform a range of homeostatic and regulatory tasks that are critical for normal functioning of the central nervous system. In response to an injury or disease, astrocytes undergo a pronounced transformation into a reactive state that involves changes in the expression of many genes and dramatically changes astrocyte morphology and functions. This astrocyte reactivity is highly dependent on the initiating insult and pathological context. C3a is a peptide generated by the proteolytic cleavage of the third complement component. C3a has been shown to exert neuroprotective effects, stimulate neural plasticity and promote astrocyte survival but can also contribute to synapse loss, Alzheimer's disease type neurodegeneration and blood-brain barrier dysfunction. To test the hypothesis that C3a elicits differential effects on astrocytes depending on their reactivity state, we measured the expression of Gfap, Nes, C3ar1, C3, Ngf, Tnf and Il1b in primary mouse cortical astrocytes after chemical ischemia, after exposure to lipopolysaccharide (LPS) as well as in control naïve astrocytes. We found that C3a down-regulated the expression of Gfap, C3 and Nes in astrocytes after ischemia. Further, C3a increased the expression of Tnf and Il1b in naive astrocytes and the expression of Nes in astrocytes exposed to LPS but did not affect the expression of C3ar1 or Ngf. Jointly, these results provide the first evidence that the complement peptide C3a modulates the responses of astrocytes in a highly context-dependent manner.

An anticomplement homogeneous polysaccharide from Hedyotis diffusa attenuates lipopolysaccharide-induced acute lung injury and inhibits neutrophil extracellular trap formation.

BACKGROUND: Owing to the involvement of the overactivated complement system in acute lung injury (ALI) development, anticomplement components may attenuate ALI. Hedyotis diffusa is a traditional Chinese medicine for treating lung heat and its crude polysaccharides (HDP) exhibit significant anticomplement activity in vitro. PURPOSE: To obtain an anticomplement homogeneous polysaccharide from HDP and verify its therapeutic effect and mechanism on ALI. METHODS: Diethylaminoethyl-52 (DEAE-52) cellulose and gel permeation columns were used to isolate a homogeneous polysaccharide HD-PS-3, which was then characterized using nuclear magnetic resonance (NMR) and methylation analysis. In vitro, the anticomplement activities of HD-PS-3 through classical and alternative pathways were determined using a hemolytic test. The therapeutic effects of HDP and HD-PS-3 on ALI were evaluated in lipopolysaccharide (LPS) intratracheal instilled mice. Hematoxylin and eosin (H&E) staining, enzyme-linked immunosorbent assay (ELISA), and immunohistochemical staining were used to assess histological changes, measure cytokine levels, and evaluate the degree of complement component 3c (C3c) deposition and neutrophil infiltration, respectively. ELISA, western blotting, and immunofluorescence were used to analyze neutrophil extracellular trap (NET) formation. RESULTS: From HDP, 1.5 g of the homogeneous polysaccharide HD-PS-3 was obtained. HD-PS-3 was an acidic heteropolysaccharide with an acetyl group, which was composed of →4,6)-α-Glcp-(1→, →3,4)-α-Glcp-(1→, →4)-α-Glcp-(1→, →4,6)-α-Galp-(1→, →5)-α-Araf-(1→, α-Rhap-(1→, α-Araf-(1→, α-GlcpA-(1→, →4)-ß-Manp-(1→, ß-Manp-(1→ and →3)-ß-Manp-(1→. The in vitro results suggest that HD-PS-3 exhibited anticomplement activity with CH50 and AP50 values of 115 ± 12 µg/ml and 307 ± 11 µg/ml, respectively. After confirming the efficacy of HDP (200 mg/kg) in attenuating lung injury, the effect of HD-PS-3 on ALI was also investigated. HD-PS-3 (75 and 150 mg/kg) attenuated LPS-induced ALI as well, evidenced by lung pathology, lung injury scores, protein concentration, leukocyte counts, tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) contents in bronchoalveolar lavage fluid (BALF). Mechanistically, HD-PS-3 inhibited complement activation, manifested in reduced pulmonary C3c deposition in lung tissue and complement component 3a (C3a) content in BALF. Neutrophil recruitment was also reduced by HD-PS-3, with significantly reduced pulmonary neutrophil infiltration and lower levels of C-X-C motif chemokine ligand 1 (CXCL1) and myeloperoxidase (MPO) in BALF. In addition, HD-PS-3 reduced the levels of MPO-DNA complex in BALF, decreased citrullinated histone H3 (Cit H3) expression and NET formation (colocalization of MPO, Cit H3, and DNA) in lung tissue. CONCLUSION: An anticomplement homogeneous polysaccharide HD-PS-3 was isolated from H. diffusa. HD-PS-3 exhibited a therapeutic effect against ALI, and the mechanism might be related to its inhibitory effects on complement activation, neutrophil recruitment, and NET formation.

C3aR contributes to unilateral ureteral obstruction-induced renal interstitial fibrosis via the activation of the NLRP3 inflammasome.

AIMS: Complement component 3a and its receptor (C3a/C3aR) and nucleotide-binding oligomerization domain-like receptor protein-3 (NLRP3) inflammasome are involved in the pathogenesis of renal interstitial fibrosis (RIF). However, the mechanisms have not been clearly illuminated. This study aimed to elucidate the roles of C3aR and the NLRP3 inflammasome involved in unilateral ureteral obstruction (UUO)-induced renal interstitial fibrosis. MAIN METHODS: UUO models were established using male C57BL/6 wild-type (WT) mice and age-matched C3aR-deficient mice. MCC950, an inhibitor of the NLRP3 inflammasome, was intraperitoneally injected in UUO mice. Blood samples were collected to quantify serum creatinine and urea. Kidney samples were collected for hematoxylin-eosin (HE), Masson, and immunohistochemistry staining, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay, and Western blotting. KEY FINDINGS: Renal function, renal fibrosis, and renal inflammation in WT mice were aggravated with longer periods of UUO. C3aR deficiency improved renal function and attenuated renal fibrosis and the activation of the NLRP3 inflammasome in UUO mice. Renal function and renal fibrosis in UUO mice were attenuated after NLRP3 inflammasome inhibition; however, the expression of C3aR did not change. SIGNIFICANCE: Our data revealed that C3aR may aggravate RIF by regulating the activation of the NLRP3 inflammasome (particularly regulating inflammasome assembly) in renal tubular epithelial cells in the UUO model.

Complement C3a and C3a Receptor Activation Mediates Podocyte Injuries in the Mechanism of Primary Membranous Nephropathy.

BACKGROUND: The complement system is highly activated in primary membranous nephropathy (MN). Identifying the complement components that damage podocytes has important therapeutic implications. This study investigated the role of C3a and the C3a receptor (C3aR) in the pathogenesis of MN. METHODS: C3aR expression in kidneys and circulating levels of C3a of MN patients were examined. Human podocyte damage was assessed after exposure to MN plasma +/- C3aR blockade (SB290157, JR14a). C3aR antagonists were administered to rats with Heymann nephritis on day 0 or after proteinuria. Clinical and pathologic parameters, specific IgG and complement activation, and podocyte injuries were then assessed. RESULTS: In the glomeruli, C3aR staining merged well with podocin. Overexpression of C3aR correlated positively with proteinuria, serum creatinine, and no response to treatments. Human podocytes exposed to MN plasma showed increased expression of PLA2R, C3aR, and Wnt3/ß-catenin, reduced expression of synaptopodin and migration function, downregulated Bcl-2, and decreased cell viability. C3aR antagonists could block these effects. In Heymann nephritis rats, C3aR blockade attenuated proteinuria, electron-dense deposition, foot process width, and glomerular basement membrane thickening in glomeruli. The increased plasma C3a levels and overexpression of C3aR were also alleviated. Specific, but not total, IgG levels decreased, with less deposition of rat IgG in glomeruli and subsequent reduction of C1q, factor B, and C5b-9. CONCLUSION: C3a anaphylatoxin is a crucial effector of complement-mediated podocyte damage in MN. The C3aR antagonist may be a potentially viable treatment for this disease.

Immunosuppression participated in complement activation-mediated inflammatory injury caused by 4-octylphenol via TLR7/IκBα/NF-κB pathway in common carp (Cyprinus carpio) gills.

4-octylphenol (4-OP), a toxic estrogenic environmental pollutant, can threaten aquatic animal and human health. However, toxic effect of 4-OP on fish has not been reported. To investigate molecular mechanism of gill poisoning caused by 4-OP exposure, a carp 4-OP poisoning model was established, and then blood and gills were collected on day 60. The results demonstrated that gill was a target organ attacked by 4-OP, and exposure to 4-OP caused carp gill inflammatory injury. There were 1605 differentially expressed genes (DEGs, including 898 up-regulated DEGs and 707 down-regulated DEGs). KEGG and GO were used to further analyze obtained 1605 DEGs, indicating that complement activation, immune response, and inflammatory response participated in the mechanism of 4-OP-caused carp gill inflammatory injury. Our data at transcription level further revealed that 4-OP caused complement activation through triggering complement component 3a/complement component 3a receptor (C3a/C3aR) axis and complement component 5a/complement component 5a receptor 1 (C5a/C5aR1) axis, induced immunosuppression through the imbalances of T helper (Th) 1/Th2 cells and regulatory T (Treg)/Th17 cells, as well as caused inflammatory injury via toll like receptor 7/inhibitor kappa B alpha/nuclear factor-kappa B (TLR7/IκBα/NF-κB) pathway. Taken together, immunosuppression participated in complement activation-mediated inflammatory damage in carp gills after 4-OP treatment. The findings of this study will provide pioneering information and theoretical support for the mechanism of 4-OP poisoning, and will provide reference for the assessment of estrogenic environmental pollution risk.

Adipsin of the Alternative Complement Pathway Is a Potential Predictor for Preeclampsia in Early Pregnancy.

Objective: The concentrations of complement proteins (adipsin, C3a, and C5a) and soluble endoglin (sENG) in the plasma were measured in this study, and their value as early-pregnancy predictors and potential diagnostic marker of preeclampsia was assessed, respectively. Experimental Design: Plasma samples were obtained from healthy and preeclampsia pregnant women before delivery for a cross-sectional study. Plasma samples were collected from healthy and preeclampsia pregnant women throughout pregnancy and postpartum for a follow-up study. Enzyme-linked immunosorbent assays were used to detect plasma levels of several complement proteins (adipsin, C3a, and C5a) and sENG. Results: The plasma levels of adipsin, C5a, and sENG were significantly increased before delivery in pregnant women with preeclampsia. During pregnancy, the plasma adipsin, C5a, and sENG levels were increased from the third trimester in healthy pregnant women; plasma adipsin levels remained stable after delivery, while C3a levels increased in the second trimester and remained stable afterward. Furthermore, levels of adipsin, C5a, and sENG were higher in preeclampsia patients at different stages of pregnancy; the C3a level presents a similar change and no difference was found in the third trimester. In the first trimester, receiver-operating curve (ROC) curve analysis showed that adipsin (AUC, 0.83 ± 0.06, P=0.001) and sENG (AUC, 0.74 ± 0.09, P=0.021) presented high value as predictors of early pregnancy. Conclusions: Adipsin is likely a novel plasma biomarker to monitor the increased risk of preeclampsia in early pregnancy. Moreover, the increased plasma levels of adipsin, C5a, and sENG before delivery may be associated with preeclampsia.

The Inflammatory Feed-Forward Loop Triggered by the Complement Component C3 as a Potential Target in Endometriosis.

The complement system is a major component of humoral innate immunity, acting as a first line of defense against microbes via opsonization and lysis of pathogens. However, novel roles of the complement system in inflammatory and immunological processes, including in cancer, are emerging. Endometriosis (EM), a benign disease characterized by ectopic endometrial implants, shows certain unique features of cancer, such as the capacity to invade surrounding tissues, and in severe cases, metastatic properties. A defective immune surveillance against autologous tissue deposited in the peritoneal cavity allows immune escape for endometriotic lesions. There is evidence that the glandular epithelial cells found in endometriotic implants produce and secrete the complement component C3. Here, we show, using immunofluorescence and RT-qPCR, the presence of locally synthesized C3 in the ectopic endometriotic tissue, but not in the eutopic tissue. We generated a murine model of EM via injection of minced uterine tissue from a donor mouse into the peritoneum of recipient mice. The wild type mice showed greater amount of cyst formation in the peritoneum compared to C3 knock-out mice. Peritoneal washings from the wild type mice with EM showed more degranulated mast cells compared to C3 knock-out mice, consistent with higher C3a levels in the peritoneal fluid of EM patients. We provide evidence that C3a participates in an auto-amplifying loop leading to mast cell infiltration and activation, which is pathogenic in EM. Thus, C3 can be considered a marker of EM and its local synthesis can promote the engraftment of the endometriotic cysts.

Targeting Complement C3a Receptor to Improve Outcome After Ischemic Brain Injury.

Ischemic stroke is a major cause of disability. No efficient therapy is currently available, except for the removal of the occluding blood clot during the first hours after symptom onset. Loss of function after stroke is due to cell death in the infarcted tissue, cell dysfunction in the peri-infarct region, as well as dysfunction and neurodegeneration in remote brain areas. Plasticity responses in spared brain regions are a major contributor to functional recovery, while secondary neurodegeneration in remote regions is associated with depression and impedes the long-term outcome after stroke. Hypoxic-ischemic encephalopathy due to birth asphyxia is the leading cause of neurological disability resulting from birth complications. Despite major progress in neonatal care, approximately 50% of survivors develop complications such as mental retardation, cerebral palsy or epilepsy. The C3a receptor (C3aR) is expressed by many cell types including neurons and glia. While there is a body of evidence for its deleterious effects in the acute phase after ischemic injury to the adult brain, C3aR signaling contributes to better outcome in the post-acute and chronic phase after ischemic stroke in adults and in the ischemic immature brain. Here we discuss recent insights into the novel roles of C3aR signaling in the ischemic brain with focus on the therapeutic opportunities of modulating C3aR activity to improve the outcome after ischemic stroke and birth asphyxia.

The role and mechanisms of Microglia in Neuromyelitis Optica Spectrum Disorders.

Neuromyelitis optica spectrum disorder (NMOSD) is an autoimmune neurological disease that can cause blindness and disability. As the major mediators in the central nervous system, microglia plays key roles in immunological regulation in neuroinflammatory diseases, including NMOSD. Microglia can be activated by interleukin (IL)-6 and type I interferons (IFN-Is) during NMOSD, leading to signal transducer and activator of transcription (STAT) activation. Moreover, complement C3a secreted from activated astrocytes may induce the secretion of complement C1q, inflammatory cytokines and progranulin (PGRN) by microglia, facilitating injury to microglia, neurons, astrocytes and oligodendrocytes in an autocrine or paracrine manner. These processes involving activated microglia ultimately promote the pathological course of NMOSD. In this review, recent research progress on the roles of microglia in NMOSD pathogenesis is summarized, and the mechanisms of microglial activation and microglial-mediated inflammation, and the potential research prospects associated with microglial activation are also discussed.

Elevated Expression Levels of Lung Complement Anaphylatoxin, Neutrophil Chemoattractant Chemokine IL-8, and RANTES in MERS-CoV-Infected Patients: Predictive Biomarkers for Disease Severity and Mortality.

The complement system, a network of highly-regulated proteins, represents a vital part of the innate immune response. Over-activation of the complement system plays an important role in inflammation, tissue damage, and infectious disease severity. The prevalence of MERS-CoV in Saudi Arabia remains significant and cases are still being reported. The role of complement in Middle East Respiratory Syndrome coronavirus (MERS-CoV) pathogenesis and complement-modulating treatment strategies has received limited attention, and studies involving MERS-CoV-infected patients have not been reported. This study offers the first insight into the pulmonary expression profile including seven complement proteins, complement regulatory factors, IL-8, and RANTES in MERS-CoV infected patients without underlying chronic medical conditions. Our results significantly indicate high expression levels of complement anaphylatoxins (C3a and C5a), IL-8, and RANTES in the lungs of MERS-CoV-infected patients. The upregulation of lung complement anaphylatoxins, C5a, and C3a was positively correlated with IL-8, RANTES, and the fatality rate. Our results also showed upregulation of the positive regulatory complement factor P, suggesting positive regulation of the complement during MERS-CoV infection. High levels of lung C5a, C3a, factor P, IL-8, and RANTES may contribute to the immunopathology, disease severity, ARDS development, and a higher fatality rate in MERS-CoV-infected patients. These findings highlight the potential prognostic utility of C5a, C3a, IL-8, and RANTES as biomarkers for MERS-CoV disease severity and mortality. To further explore the prediction of functional partners (proteins) of highly expressed proteins (C5a, C3a, factor P, IL-8, and RANTES), the computational protein-protein interaction (PPI) network was constructed, and six proteins (hub nodes) were identified.

Dynamic regulation of B cell complement signaling is integral to germinal center responses.

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.

Quantification of Porcine Complement Activation Fragment C3a by a Neoepitope-Based Enzyme-Linked Immunosorbent Assay.

Enzyme-linked immunosorbent assay (ELISA) enables fast and simple quantification of analytes in the pico- to nanogram range in complex samples. Here, we describe an ELISA for the detection of porcine C3a as a marker for complement activation. Antibody specificity is critical for a robust assay. This assay is based on a pair of antibodies specific for the porcine C3a molecule and thus does not react with native C3.