C3aR Signaling Research Articles

Microglia mediate memory dysfunction via excitatory synaptic elimination in a fracture surgery mouse model

Cognitive impairment is a common issue among human patients undergoing surgery, yet the neural mechanism causing this impairment remains unidentified. Surgical procedures often lead to glial cell activation and neuronal hypoexcitability, both of which are known to contribute to postoperative cognitive dysfunction (POCD). However, the role of neuron-glia crosstalk in the pathology of POCD is still unclear. Through integrated transcriptomics and proteomics analyses, we found that the complement cascades and microglial phagocytotic signaling pathways are activated in a mouse model of POCD. Following surgery, there is a significant increase in the presence of complement C3, but not C1q, in conjunction with presynaptic elements. This triggers a reduction in excitatory synapses, a decline in excitatory synaptic transmission, and subsequent memory deficits in the mouse model. By genetically knockout out C3ar1 or inhibiting p-STAT3 signaling, we successfully prevented neuronal hypoexcitability and alleviated cognitive impairment in the mouse model. Therefore, targeting the C3aR and downstream p-STAT3 signaling pathways could serve as potential therapeutic approaches for mitigating POCD.

Revealing the signaling of complement receptors C3aR and C5aR1 by anaphylatoxins.

The complement receptors C3aR and C5aR1, whose signaling is selectively activated by anaphylatoxins C3a and C5a, are important regulators of both innate and adaptive immune responses. Dysregulations of C3aR and C5aR1 signaling lead to multiple inflammatory disorders, including sepsis, asthma and acute respiratory distress syndrome. The mechanism underlying endogenous anaphylatoxin recognition and activation of C3aR and C5aR1 remains elusive. Here we reported the structures of C3a-bound C3aR and C5a-bound C5aR1 as well as an apo-C3aR structure. These structures, combined with mutagenesis analysis, reveal a conserved recognition pattern of anaphylatoxins to the complement receptors that is different from chemokine receptors, unique pocket topologies of C3aR and C5aR1 that mediate ligand selectivity, and a common mechanism of receptor activation. These results provide crucial insights into the molecular understanding of C3aR and C5aR1 signaling and structural templates for rational drug design for treating inflammation disorders.

C3a Receptor Signaling Inhibits Neurodegeneration Induced by Neonatal Hypoxic-Ischemic Brain Injury.

Hypoxic-ischemic neonatal encephalopathy due to perinatal asphyxia is the leading cause of brain injury in newborns. Clinical data suggest that brain inflammation induced by perinatal insults can persist for years. We previously showed that signaling through the receptor for complement peptide C3a (C3aR) protects against cognitive impairment induced by experimental perinatal asphyxia. To investigate the long-term neuropathological effects of hypoxic-ischemic injury to the developing brain and the role of C3aR signaling therein, we subjected wildtype mice, C3aR deficient mice, and mice expressing biologically active C3a in the CNS to mild hypoxic-ischemic brain injury on postnatal day 9. We found that such injury triggers neurodegeneration and pronounced reactive gliosis in the ipsilesional hippocampus both of which persist long into adulthood. Transgenic expression of C3a in reactive astrocytes reduced hippocampal neurodegeneration and reactive gliosis. In contrast, neurodegeneration and microglial cell density increased in mice lacking C3aR. Intranasal administration of C3a for 3 days starting 1 h after induction of hypoxia-ischemia reduced neurodegeneration and reactive gliosis in the hippocampus of wildtype mice. We conclude that neonatal hypoxic-ischemic brain injury leads to long-lasting neurodegeneration. This neurodegeneration is substantially reduced by treatment with C3aR agonists, conceivably through modulation of reactive gliosis.

Astrocytic Kir6.1 deletion aggravates neurodegeneration in the lipopolysaccharide-induced mouse model of Parkinson’s disease via astrocyte-neuron cross talk through complement C3-C3R signaling

Complement pathway over-activation has been implicated in a variety of neurological diseases. However, the signaling pathways governing astrocytic complement activation in Parkinson’s disease (PD) are poorly understood. Kir6.1, a pore-forming subunit of ATP-sensitive potassium (K-ATP) channel, is prominently expressed in astrocytes and exhibits anti-inflammatory effects. Therefore, we hypothesize that Kir6.1/K-ATP channel may regulate astrocytic complement activation in the pathogenesis of PD. In this study, astrocytic Kir6.1 knockout (KO) mice were used to examine the effect of astrocytic Kir6.1/K-ATP channel on astrocytic complement activation triggered by the lipopolysaccharide (LPS). Here, we found that astrocytic Kir6.1 KO mice showed more dopaminergic neuron loss and more astrocyte reactivity in substantia nigra compacta than controls. We also found that astrocytic Kir6.1 KO increased the expression of complement C3 in astrocytes in LPS-induced mouse model of PD. Mechanistically, astrocytic Kir6.1 KO promoted astroglial NF-κB activation to elicit extracellular release of C3, which in turn interacted with neuronal C3aR to induce neuron death. Blocking complement function by NF-κB inhibitor or C3aR antagonist rescued the aggravated neuron death induced by Kir6.1 KO. Collectively, our findings reveal that astrocytic Kir6.1/K-ATP channel prevents neurodegeneration in PD via astrocyte-neuron cross talk through NF-κB/C3/C3aR signaling and suggest that targeting astroglial Kir6.1/K-ATP channel-NF-κB-C3-neuronal C3aR signaling represents a novel therapeutic strategy for PD.

Complement C3a receptor-mediated vascular dysfunction: a complex interplay between aging and neurodegeneration.

Vascular dysfunction resulting in compromised blood-brain barrier (BBB) integrity is evident in aging and disease. Although the complement C3a/C3a receptor (C3a/C3aR) axis influences normal brain aging and disease progression, the mechanisms governing endothelial C3aR-mediated neurovascular inflammation and BBB permeability remain unexplored. In this issue of the JCI, Propson et al. investigated endothelial C3a/C3aR signaling in normal, aged, and neurodegenerative mouse models. Endothelial C3aR signaling modulated age-dependent increases in VCAM1, initiated peripheral lymphocyte infiltration, and enhanced microglial activity. Increased calcium release downstream of C3aR signaling disrupted the vascular endothelial cadherin (VE-cadherin) junctions, increased BBB permeability, and degraded vascular structure and function. Mice lacking C3aR (C3ar1-/-) and mice treated with a C3aR antagonist showed attenuated age-related microglial reactivity and neurodegeneration. These results confirm that complement-mediated signaling impacts vascular health and BBB function in normal aging and neurodegenerative disease, suggesting that complement inhibitors represent a therapeutic option for cerebral microvascular dysfunction.

Abstract 4117: Adipsin promotes tumor progression in ESR1 mutant breast cancer cells

Abstract ESR1 point mutations at ESR1 ligand-binding domain are frequently identified in metastatic tumors, cfDNA (Cell-free DNA), ctDNA (Circulating tumor DNA) and ptDNA (plasma tumor DNA) derived from ESR1 positive breast cancer patients treated SERMs, SERDs, and AIs. ESR1 mutations enhance ESR1 transcriptional activity in the absence of estrogen and induce estradiol independent growth as well as increase resistance to SERM or SERD in ESR1 mutant overexpressed cells. ESR1 mutations are found more frequently in cfDNA and tumor DNA from patients with metastatic disease compared to patients with primary tumor, Although SERM, SERD, AI, and CDK4/6 inhibitor therapies have demonstrated preclinical and clinical benefits for breast cancer with ESR1 mutations, the development of resistance remains a significant challenge and the detailed mechanisms and potential therapeutic targets in metastatic breast cancer with ESR1 point mutations is yet to be revealed. Tumor and organ microenvironments are crucial for cancer progression and metastasis. Crosstalk between multiple non-malignant cell types in the microenvironments such as blood and lymphatic endothelial cells and cancer cells promotes tumor growth and metastasis. Possibly the anti-angiogenic and anti-lymphangiogenic therapies can be combined with each other for improved outcomes for patients. In this study, we identified the secretion of Adipsin from two different genome-edited MCF-7 harboring Y537S and D538G ESR1 cells compared to wild-type cells using the Human Cytokine Array Q440 to quantitatively detect 440 human inflammatory factors, growth factors, chemokines, receptors, and cytokines simultaneously. We validated that the expression of Adipsin was highly upregulated in MCF-7-Y537S and D538G ESR1 mutant cells by real-time qRT-PCR and ELISA. Interestingly, we observed that when cells were cultured in estrogen deprivation, the mRNA expression of the Adipsin was significantly increased in MCF-7-ESR1 mutant cells while the treatment of tamoxifen and fulvestrant abrogated the upregulation of Adipsin. These results suggest that Adipsin is an ER target gene. Since the cleavage of factor B by Adipsin results in the conversion of C3 to C3a, which binds to its cell surface receptor (C3aR), our data showed that the C3a production was significantly increased in ESR1 mutant cells compared to WT by ELISA. In addition, we found that C3aR expression was increased in ESR1 mutant cells. To elucidate that the C3aR signaling pathway promotes the proliferation of MCF-7-ESR1 mutant cells, we examined the ESR1 mutant cell viability using SB290157, C3aR inhibitor and found that ESR1 mutant cell growth was decreased in the treatment of SB290157. Furthermore, we found that apoptosis was significantly induced in ESR1 mutant cells compared to WT by Annexin V assay. These findings implicate Adipsin signaling as a critical event in breast cancer metastasis with ESR1 mutations. Further, these studies suggest that Adipsin acts as a key regulator orchestrating breast cancer with ESR1 mutations. Therefore, we have provided evidence that supports the hypothesis that functional inhibition of the Adipsin signaling pathway has the potential to circumvent breast cancer metastasis. Citation Format: Marie K. Malone, Kideok Jin. Adipsin promotes tumor progression in ESR1 mutant breast cancer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4117.

C3a-C3aR signaling promotes breast cancer lung metastasis via modulating carcinoma associated fibroblasts

BackgroundMounting evidence suggests that complement components promote tumor progression via modulating immune suppression, angiogenesis, or tumor cell proliferation. However, the role of C3a-C3aR signaling in regulating lung metastasis of breast cancer remains unknown.MethodsWe performed various ex-vivo and in-vivo assays. Genetic and pharmacological C3aR blockade models were applied to investigate the role of C3a-C3aR in metastasis of breast cancer.ResultsC3a-C3aR signaling in CAFs facilitates the metastasis of breast cancer. Mechanically, C3a-C3aR signaling augments pro-metastatic cytokine secretion and extracellular matrix components expression of CAFs via the activation of PI3K-AKT signaling. Genetic or pharmacological blockade of C3aR signaling effectively inhibited lung metastasis of breast cancer in mouse models.ConclusionsC3a-C3aR signaling in CAFs facilitates the metastasis of breast cancer. Targeting C3aR signaling is a potential anti-metastasis strategy for breast cancer therapy.

Complement component 3a receptor deficiency attenuates chronic stress-induced monocyte infiltration and depressive-like behavior

Major depressive disorder (MDD) is one of the most common and debilitating neuropsychiatric illnesses. Accumulating evidence suggests a potential role of the immune system in the pathophysiology of MDD. The complement system represents one of the major effector mechanisms of the innate immune system, and plays a critical role in inflammation. However, the role of complement components in MDD is not well understood. Here, we found significant increase in component 3 (C3) expression in the prefrontal cortex (PFC) of depressed suicide subjects. We tested the role of altered C3 expression in mouse model of depression and found that increased C3 expression in PFC as a result of chronic stress causes depressive-like behavior. Conversely, mice lacking C3 were resilient to stress-induced depressive-like behavior. Moreover, selective overexpression of C3 in PFC was sufficient to cause depressive-like behavior in mice. We found that C3a (activated product of C3) receptor, C3aR+ monocytes were infiltrated into PFC following chronic stress. However, C3aR knockout mice displayed significantly reduced monocyte recruitment into PFC and reduced levels of the proinflammatory cytokine IL-1β in PFC after chronic stress. In addition, C3aR knockout mice did not exhibit chronic stress-induced behavior despair. Similarly, chronic stress-induced increases in C3aR+ monocytes and IL-1β in PFC, and depressive-like behavior were attenuated by myeloid cell depletion. These postmortem and preclinical studies identify C3aR signaling as a key factor in MDD pathophysiology.

The Complement C3a Receptor Contributes to Melanoma Tumorigenesis by Inhibiting Neutrophil and CD4+ T Cell Responses.

The complement peptide C3a is a key component of the innate immune system and a major fragment produced following complement activation. We used a murine model of melanoma (B16-F0) to identify a hitherto unknown role for C3a-C3aR signaling in promoting tumor growth. The results show that the development and growth of B16-F0 melanomas is retarded in mice lacking C3aR, whereas growth of established melanomas can be arrested by C3aR antagonism. Flow cytometric analysis showed alterations in tumor-infiltrating leukocytes in the absence of C3aR. Specifically, neutrophils and CD4(+) T lymphocyte subpopulations were increased, whereas macrophages were reduced. The central role of neutrophils was confirmed by depletion experiments that reversed the tumor inhibitory effects observed in C3aR-deficient mice and returned tumor-infiltrating CD4(+) T cells to control levels. Analysis of the tumor microenvironment showed upregulation of inflammatory genes that may contribute to the enhanced antitumor response observed in C3aR-deficient mice. C3aR deficiency/inhibition was also protective in murine models of BRAF(V600E) mutant melanoma and colon and breast cancer, suggesting a tumor-promoting role for C3aR signaling in a range of tumor types. We propose that C3aR activation alters the tumor inflammatory milieu, thereby promoting tumor growth. Therapeutic inhibition of C3aR may therefore be an effective means to trigger an antitumor response in melanoma and other cancers.

Complement Component 3C3 and C3a Receptor Are Required in Chitin-Dependent Allergic Sensitization to Aspergillus fumigatus but Dispensable in Chitin-Induced Innate Allergic Inflammation

ABSTRACTLevels of the anaphylatoxin C3a are increased in patients with asthma compared with those in nonasthmatics and increase further still during asthma exacerbations. However, the role of C3a during sensitization to allergen is poorly understood. Sensitization to fungal allergens, such as Aspergillus fumigatus, is a strong risk factor for the development of asthma. Exposure to chitin, a structural polysaccharide of the fungal cell wall, induces innate allergic inflammation and may promote sensitization to fungal allergens. Here, we found that coincubation of chitin with serum or intratracheal administration of chitin in mice resulted in the generation of C3a. We established a model of chitin-dependent sensitization to soluble Aspergillus antigens to test the contribution of complement to these events. C3−/− and C3aR−/− mice were protected from chitin-dependent sensitization to Aspergillus and had reduced lung eosinophilia and type 2 cytokines and serum IgE. In contrast, complement-deficient mice were not protected against chitin-induced innate allergic inflammation. In sensitized mice, plasmacytoid dendritic cells from complement-deficient animals acquired a tolerogenic profile associated with enhanced regulatory T cell responses and suppressed Th2 and Th17 responses specific for Aspergillus. Thus, chitin induces the generation of C3a in the lung, and chitin-dependent allergic sensitization to Aspergillus requires C3aR signaling, which suppresses regulatory dendritic cells and T cells and induces allergy-promoting T cells.

Regulation of C3a Receptor Signaling in Human Mast Cells by G Protein Coupled Receptor Kinases

BackgroundThe complement component C3a activates human mast cells via its cell surface G protein coupled receptor (GPCR) C3aR. For most GPCRs, agonist-induced receptor phosphorylation leads to receptor desensitization, internalization as well as activation of downstream signaling pathways such as ERK1/2 phosphorylation. Previous studies in transfected COS cells overexpressing G protein coupled receptor kinases (GRKs) demonstrated that GRK2, GRK3, GRK5 and GRK6 participate in agonist-induced C3aR phosphorylation. However, the roles of these GRKs on the regulation of C3aR signaling and mediator release in human mast cells remain unknown.Methodology/Principal FindingsWe utilized lentivirus short hairpin (sh)RNA to stably knockdown the expression of GRK2, GRK3, GRK5 and GRK6 in human mast cell lines, HMC-1 and LAD2, that endogenously express C3aR. Silencing GRK2 or GRK3 expression caused a more sustained Ca2+ mobilization, attenuated C3aR desensitization, and enhanced degranulation as well as ERK1/2 phosphorylation when compared to shRNA control cells. By contrast, GRK5 or GRK6 knockdown had no effect on C3aR desensitization, but caused a significant decrease in C3a-induced mast cell degranulation. Interestingly, GRK5 or GRK6 knockdown rendered mast cells more responsive to C3a for ERK1/2 phosphorylation.Conclusion/SignificanceThis study demonstrates that GRK2 and GRK3 are involved in C3aR desensitization. Furthermore, it reveals the novel finding that GRK5 and GRK6 promote C3a-induced mast cell degranulation but inhibit ERK1/2 phosphorylation via C3aR desensitization-independent mechanisms. These findings thus reveal a new level of complexity for C3aR regulation by GRKs in human mast cells.

C5aR but not C3aR signaling in mDCs differentially regulates maladaptive immunity and inflammation in house dust mite-mediated experimental allergic asthma

C5aR but not C3aR signaling in mDCs differentially regulates maladaptive immunity and inflammation in house dust mite-mediated experimental allergic asthma

Opposing roles for C5aR- and C3aR signaling in the development of maladaptive immunity in allergic asthma

Opposing roles for C5aR- and C3aR signaling in the development of maladaptive immunity in allergic asthma

Anaphylatoxins play a key role in the development of experimental lupus cerebritis (100.4)

Abstract Complement activation generates the C3a and C5a anaphylatoxins, which act through specific G-protein coupled receptors (R). Anaphylatoxins correlate with flares in patients with systemic lupus erythematosus. C3aR and C5aR mRNA and protein expression were significantly increased in brains of the lupus mouse model, MRL/lpr mice compared to those of the non-diseased congenic MRL/+ strain. To examine the role of C3aR and C5aR signaling in brain, specific antagonists, N2-[(2,2-diphenylethoxy)acetyl]-L-arginine and acetyl-Phe-(Orn-Pro-D-cyclohexylalanine)-Trp-Arg, respectively, were administered to MRL/lpr mice. Littermate controls were given vehicle alone. Control MRL/lpr mice had increased infiltration of blood cells into brain and an increase in a number of relevant cytokines and cell-bound accessory molecules (γ-IFN, TNF-α, IL-1β, MIP-2, MCP-1, RANTES, TNFR1, and ICAM-1) which were reduced by inhibition of C3aR and C5aR. In addition, the number of apoptotic cells that were increased in MRL/lpr mice compared to MRL/+ mice, was significantly reduced by inhibition of C3aR and C5aR, with a corresponding increase in Iκβα and phosphorylated Akt, and decrease in Bcl-xL and phosphorylated BAD. Thus, pharmacological blockade of anaphylatoxin receptors reduces pathological features in the brain in experimental lupus and represents a potentially viable treatment for human lupus cerebritis.