C5aR2 Deficiency Lessens C5aR1 Distribution and Expression in Neutrophils and Macrophages.

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.

Emerging role of C5aR2: novel insights into the regulation of uterine immune cells during pregnancy.

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.

Reduced decay-accelerating factor expression promotes complement-mediated cystogenesis in murine ADPKD.

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.

C5aR1 inhibition reprograms tumor associated macrophages and reverses PARP inhibitor resistance in breast cancer.

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.

C5a-C5aR1 axis controls mitochondrial fission to promote podocyte injury in lupus nephritis.

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.

The C5aR1 complement receptor: A novel immunomodulator of insulin action in skeletal muscle.

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.

C5aR2 Regulates STING-Mediated Interferon Beta Production in Human Macrophages.

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.

C5a/C5aR1 axis as a key driver promotes epithelial-to-mesenchymal transition in airway epithelial cells in silica nanoparticles-induced pulmonary fibrosis.

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.

Disabled C3ar1/C5ar1 Signaling in Foxp3+ T Regulatory Cells Leads to TSDR Demethylation and Long-Term Stability.

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.

The complement receptor C5aR2 regulates neutrophil activation and function contributing to neutrophil-driven epidermolysis bullosa acquisita.

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.

Ras-Related Protein Rab5a Regulates Complement C5a Receptor Trafficking, Chemotaxis, and Chemokine Secretion in Human Macrophages.

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.

Loss of C3a and C5a receptors promotes adipocyte browning and attenuates diet-induced obesity via activating inosine/A2aR pathway.

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.

TLQP-21 is a low potency partial C3aR activator on human primary macrophages.

TLQP-21 is a 21-amino acid neuropeptide derived from the VGF precursor protein. TLQP-21 is expressed in the nervous system and neuroendocrine glands, and demonstrates pleiotropic roles including regulating metabolism, nociception and microglial functions. Several possible receptors for TLQP-21 have been identified, with complement C3a receptor (C3aR) being the most commonly reported. However, few studies have characterised the activity of TLQP-21 in immune cells, which represent the major cell type expressing C3aR. In this study, we therefore aimed to define the activity of both human and mouse TLQP-21 on cell signalling in primary human and mouse macrophages. We first confirmed that TLQP-21 induced ERK signalling in CHO cells overexpressing human C3aR, and did not activate human C5aR1 or C5aR2. TLQP-21 mediated ERK signalling was also observed in primary human macrophages. However, the potency for human TLQP-21 was 135,000-fold lower relative to C3a, and only reached 45% at the highest dose tested (10 µM). Unlike in humans, mouse TLQP-21 potently triggered ERK signalling in murine macrophages, reaching near full activation, but at ~10-fold reduced potency compared to C3a. We further confirmed the C3aR dependency of the TLQP-21 activities. Our results reveal significant discrepancy in TLQP-21 C3aR activity between human and murine receptors, with mouse TLQP-21 being consistently more potent than the human counterpart in both systems. Considering the supraphysiological concentrations of hTLQP-21 needed to only partially activate macrophages, it is likely that the actions of TLQP-21, at least in these immune cells, may not be mediated by C3aR in humans.

C5L2 modulates BDNF production in human dental pulp stem cells via p38α pathway.

Tissue injury affects nerve fibers and triggers an immune response, leading to inflammation. The complement system gets activated during inflammatory conditions and has been reported to be involved in the regeneration process. We have demonstrated that the C5a receptor (C5aR) has crucial roles in regeneration and healing processes including nerve sprouting and hard tissue formation. Another C5a-like 2 receptor (C5AR2; C5L2) has been cloned which is still considered controversial due to limited studies. We previously established that C5L2 regulates brain-derived neurotrophic factor (BDNF) secretion in pulp fibroblasts. However, there is no study available on human dental pulp stem cells (DPSCs), especially in the inflammatory context. Stem cell therapy is an emerging technique to treat and prevent several diseases. DPSCs are a great option to be considered due to their great ability to differentiate into a variety of cells and secrete nerve regeneration factors. Here, we demonstrated that C5L2 modulates BDNF secretion in DPSCs. Our results stated that C5L2 silencing through siRNA could increase BDNF production, which could accelerate the nerve regeneration process. Moreover, stimulation with lipopolysaccharide (LPS) enhanced BDNF production in C5L2 silenced DPSCs. Finally, we quantified BDNF secretion in supernatant and cell lysates using ELISA. Our results showed enhanced BDNF production in C5L2 silenced DPSCs and hampered by the p38MAPKα inhibitor. Taken together, our data reveal that C5L2 modulates BDNF production in DPSCs via the p38MAPKα pathway.

Mild Hypothermia Alleviates Complement C5a-Induced Neuronal Autophagy During Brain Ischemia-Reperfusion Injury After Cardiac Arrest.

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.

Basophils activation of patients with chronic spontaneous urticaria in response to C5a despite failure to respond to IgE-mediated stimuli.

Urticaria is characterized by the occurrence of wheals and flares in response to vasoactive mediators, such as histamine. Various studies have suggested the involvement of basophils in the pathogenesis of chronic spontaneous urticaria (CSU). However, histamine release from peripheral basophils in response to stimuli acting on the high affinity IgE receptor (FcϵRI) is impaired in many patients with CSU (non/low responders). We previously demonstrated that tissue factor (TF)s expressed on vascular endothelial cells in response to a combination of various stimuli, such as that of histamine and lipopolysaccharide (LPS), activates the extrinsic coagulation pathway and produces anaphylatoxin, complement 5a (C5a), which then activates basophils and mast cells via the C5a receptor (C5aR). We have revealed that histamine release was induced in response to C5a and formyl-l-methionyl-l-leucyl-l-phenylalanine (fMLP), regardless of the response to anti-IgE antibody, the reduced numbers of basophils and severity of urticaria. Moreover, we found that spontaneous release of histamine ex vivo from basophils of patients with CSU is higher than that from healthy individuals. These results suggest that basophils and the complement system, which could be activated by coagulation factors, may play a critical role in the pathogenesis of CSU, especially in cases refractory to treatment involving the IgE/FcϵRI pathway.

Pharmacological activation of the C5a receptor leads to stimulation of the ß-adrenergic receptor and alleviates cognitive impairment in a murine model of familial Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disease of the brain causing either familial or sporadic dementia. We have previously administered the modified C5a receptor agonist (EP67) for a short period to a transgenic mouse model of AD (5XFAD) and have observed not only reduction in ß-amyloid deposition and gliosis but also improvement in cognitive impairment. Inquiring, however, on the effects of EP67 in an already heavily burdened animal, thus representing a more realistic scenario, we treated 6-month-old 5XFAD mice for a period of 14 weeks. We recorded a significant decrease in both fibrillar and pre-fibrillar ß-amyloid as well as remarkable amelioration of cognitive impairment. Following proteomic analysis and pathway association, we postulate that these events are triggered through the upregulation of ß-adrenergic and GABAergic signaling. In summary, our results reveal how inflammatory responses can be employed in inducing tangible phenotype improvements even in advanced stages of AD.

C5aR1 antagonism alters microglial polarization and mitigates disease progression in a mouse model of Alzheimer's disease.

Multiple studies have recognized the involvement of the complement cascade during Alzheimer's disease pathogenesis. However, the specific role of C5a-C5aR1 signaling in the progression of this neurodegenerative disease is still not clear. Furthermore, its potential as a therapeutic target to treat AD still remains to be elucidated. Canonically, generation of the anaphylatoxin C5a as the result of complement activation and interaction with its receptor C5aR1 triggers a potent inflammatory response. Previously, genetic ablation of C5aR1 in a mouse model of Alzheimer's disease exerted a protective effect by preventing cognitive deficits. Here, using PMX205, a potent, specific C5aR1 antagonist, in the Tg2576 mouse model of Alzheimer's disease we show a striking reduction in dystrophic neurites in parallel with the reduced amyloid load, rescue of the excessive pre-synaptic loss associated with AD cognitive impairment and the polarization of microglial gene expression towards a DAM-like phenotype that are consistent with the neuroprotective effects seen. These data support the beneficial effect of a pharmacological inhibition of C5aR1 as a promising therapeutic approach to treat Alzheimer's disease. Supportive of the safety of this treatment is the recent FDA-approval of another other C5a receptor 1 antagonist, Avacopan, as a treatment for autoimmune inflammatory diseases.

Modulation of C5a-C5aR1 signaling alters the dynamics of AD progression.

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.

C5a receptor antagonism coming of age for vascular pathology.

The Food and Drug Administration recently approved the new drug avacopan for a relatively rare disease, anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis. Avacopan is an antagonist of receptor-1 for anaphylatoxin C5a (C5aR1) that is the first one to meet all expectations of an orally bioavailable drug. Pharmacological effects of C5a on vascular tissue are reviewed; they are essentially indirect, via resident or infiltrating leukocytes, and largely mediated by vasoconstrictor prostanoids that are potentially thrombogenic. The in vivo acute neutropenic effect of C5a and various responses of isolated neutrophils to the peptide have been exploited in the preclinical development of avacopan, but not the prominent hemodynamic responses. Possible clinical risks and extension of therapeutic C5aR1 blockade are discussed. Therapeutic intervention on the blood-derived peptide C5a and on its G protein coupled receptor for specific forms of vascular injury contrasts with other current research approaches in vascular pathology, such as investigating the roles of cytokines and intracellular signaling.