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.

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.

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.

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.

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.

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.

1,2-Diacetylbenzene impaired hippocampal memory by activating proinflammatory cytokines and upregulating the prolactin pathway: An in vivo and in vitro study.

Memory loss is the most common occurrence of dementia in the elderly population. Evidence shows 1,2-Diacetylbenzene (DAB) can exacerbate cerebral dysfunction. The molecular mechanisms involved in DAB actions in the hippocampus have not been well elucidated to date. qPCR, western blot, Morris water maze, and RNAseq analysis were used to identify the association between inflammation and hyperphosphorylated tau in male DAB-treated mice (1 or 5 mg/kg/day), rats (3 mg/kg/day), in vitro BV2 microglial cells (1 or 5 µM), and the hippocampal transcriptome of male DAB-treated rats. We found that DAB induces memory deficits by activating pro-inflammatory cytokines as well as down-regulating memory and learning genes. Several genes involved in learning, memory, and behavior induced by DAB (e.g., PRL, Pit-1, PRLR, Ttr, Notch2, Ntsr1, C5ar2, Cd74) were not changed or downregulated in young rats, but upregulated in old rats. Detoxification pathways were upregulated in young rats treated with DAB, whereas prolactin (PRL) signaling pathways were upregulated in old DAB-treated rats. Further work is needed to gain a better understanding of the roles of PRL during aging.

Signaling Through FcγRIIA and the C5a-C5aR Pathway Mediate Platelet Hyperactivation in COVID-19.

Patients with COVID-19 present with a wide variety of clinical manifestations. Thromboembolic events constitute a significant cause of morbidity and mortality in patients infected with SARS-CoV-2. Severe COVID-19 has been associated with hyperinflammation and pre-existing cardiovascular disease. Platelets are important mediators and sensors of inflammation and are directly affected by cardiovascular stressors. In this report, we found that platelets from severely ill, hospitalized COVID-19 patients exhibited higher basal levels of activation measured by P-selectin surface expression and had poor functional reserve upon in vitro stimulation. To investigate this question in more detail, we developed an assay to assess the capacity of plasma from COVID-19 patients to activate platelets from healthy donors. Platelet activation was a common feature of plasma from COVID-19 patients and correlated with key measures of clinical outcome including kidney and liver injury, and APACHEIII scores. Further, we identified ferritin as a pivotal clinical marker associated with platelet hyperactivation. The COVID-19 plasma-mediated effect on control platelets was highest for patients that subsequently developed inpatient thrombotic events. Proteomic analysis of plasma from COVID-19 patients identified key mediators of inflammation and cardiovascular disease that positively correlated with in vitro platelet activation. Mechanistically, blocking the signaling of the FcγRIIa-Syk and C5a-C5aR pathways on platelets, using antibody-mediated neutralization, IgG depletion or the Syk inhibitor fostamatinib, reversed this hyperactivity driven by COVID-19 plasma and prevented platelet aggregation in endothelial microfluidic chamber conditions. These data identified these potentially actionable pathways as central for platelet activation and/or vascular complications and clinical outcomes in COVID-19 patients. In conclusion, we reveal a key role of platelet-mediated immunothrombosis in COVID-19 and identify distinct, clinically relevant, targetable signaling pathways that mediate this effect.

Effect of a C5a receptor antagonist on macrophage function in an intestinal transplant rat model.

BACKGROUND: C5a promotes alloreactivity via the C5a receptor 1 (C5aR1) on immune cells, but this has not been confirmed in the case of small intestine transplantation immunity. In the present study, we examined the effect of C5aR1 antagonist (PMX53) on macrophage function in small intestinal transplantation. METHODS: The model was created by heterotopic intestinal transplantation using donor Dark Agouti and recipient Lewis rats. PMX53 was administered starting on the day of operation until postoperative day 7. The graft survivals were compared, and HE staining of grafts, lymphocyte mixed reaction test (MLR, mixed culture of T cells from lymph nodes and spleen cells from donors), and changes in macrophage and T cell accumulation in grafts on day 6 after transplantation were evaluated. In addition, the effect of PMX53 on macrophage differentiation and activation was assessed using macrophages derived from bone marrow (BMDM). RESULTS: Graft survival was significantly prolonged in the therapeutic group compared to the untreated group. Histological evaluation showed that PMX53 inhibited the shortening of the graft villus, and the stimulation index of MLR was significantly lower in the therapeutic group compared to the untreated group. In the therapeutic group, the accumulation of macrophages in intestinal graft and monocyte in blood were reduced, compared with the untreated group. PMX53 decreased the differentiation in BMDM and the mRNA expression of IL-1ß and TNF-α in activated BMDM. CONCLUSION: Inhibition of C5a/C5aR1 signaling appears to regulate macrophage differentiation and suppress rejection in small intestine transplantation immunity.

Vascular Endothelial Cells Produce Coagulation Factors That Control Their Growth via Joint Protease-Activated Receptor and C5a Receptor 1 (CD88) Signaling.

As per the classical view of the coagulation system, it functions solely in plasma to maintain hemostasis. An experimental approach modeling vascular reconstitution was used to show that vascular endothelial cells (ECs) endogenously synthesize coagulation factors during angiogenesis. Intracellular thrombin generated from this synthesis promotes the mitotic function of vascular endothelial cell growth factor A (VEGF-A). The thrombin concurrently cleaves C5a from EC-synthesized complement component C5 and unmasks the tethered ligand for EC-expressed protease-activated receptor 4 (PAR4). The two ligands jointly trigger EC C5a receptor-1 (C5ar1) and PAR4 signaling, which together promote VEGF receptor 2 growth signaling. C5ar1 is functionally associated with PAR4, enabling C5a or thrombin to elicit Gαi and/or Gαq signaling. EC coagulation factor and EC complement component synthesis concurrently down-regulate with contact inhibition. The connection of these processes with VEGF receptor 2 signaling provides new insights into mechanisms underlying angiogenesis. Knowledge of endogenous coagulation factor/complement component synthesis and joint PAR4/C5ar1 signaling could be applied to other cell types.