Improving radiotherapy in immunosuppressive microenvironments by targeting complement receptor C5aR1.

An immunosuppressive microenvironment causes poor tumor T cell infiltration and is associated with reduced patient overall survival in colorectal cancer. How to improve treatment responses in these tumors is still a challenge. Using an integrated screening approach to identify cancer-specific vulnerabilities, we identified complement receptor C5aR1 as a druggable target, which when inhibited improved radiotherapy, even in tumors displaying immunosuppressive features and poor CD8+ T cell infiltration. While C5aR1 is well-known for its role in the immune compartment, we found that C5aR1 is also robustly expressed on malignant epithelial cells, highlighting potential tumor cell-specific functions. C5aR1 targeting resulted in increased NF-κB-dependent apoptosis specifically in tumors and not normal tissues, indicating that, in malignant cells, C5aR1 primarily regulated cell fate. Collectively, these data revealed that increased complement gene expression is part of the stress response mounted by irradiated tumors and that targeting C5aR1 could improve radiotherapy, even in tumors displaying immunosuppressive features.

Inhibition of C5AR1 impairs osteoclast mobilization and prevents bone loss.

Age-related and chemotherapy-induced bone loss depends on cellular senescence and the cell secretory phenotype. However, the factors secreted in the senescent microenvironment that contribute to bone loss remain elusive. Here, we report a central role for the inflammatory alternative complement system in skeletal bone loss. Through transcriptomic analysis of bone samples, we identified complement factor D, a rate-limiting factor of the alternative pathway of complement, which is among the most responsive factors to chemotherapy or estrogen deficiency. We show that osteoblasts and osteocytes are major inducers of complement activation, while monocytes and osteoclasts are their primary targets. Genetic deletion of C5ar1, the receptor of the anaphylatoxin C5a, or treatment with a C5AR1 inhibitor reduced monocyte chemotaxis and osteoclast differentiation. Moreover, genetic deficiency or inhibition of C5AR1 partially prevented bone loss and osteoclastogenesis upon chemotherapy or ovariectomy. Altogether, these lines of evidence support the idea that inhibition of alternative complement pathways may have some therapeutic benefit in osteopenic disorders.

C5aR1 signaling triggers lung immunopathology in COVID-19 through neutrophil extracellular traps.

Patients with severe COVID-19 develop acute respiratory distress syndrome (ARDS) that may progress to cytokine storm syndrome, organ dysfunction, and death. Considering that complement component 5a (C5a), through its cellular receptor C5aR1, has potent proinflammatory actions and plays immunopathological roles in inflammatory diseases, we investigated whether the C5a/C5aR1 pathway could be involved in COVID-19 pathophysiology. C5a/C5aR1 signaling increased locally in the lung, especially in neutrophils of critically ill patients with COVID-19 compared with patients with influenza infection, as well as in the lung tissue of K18-hACE2 Tg mice (Tg mice) infected with SARS-CoV-2. Genetic and pharmacological inhibition of C5aR1 signaling ameliorated lung immunopathology in Tg-infected mice. Mechanistically, we found that C5aR1 signaling drives neutrophil extracellular traps-dependent (NETs-dependent) immunopathology. These data confirm the immunopathological role of C5a/C5aR1 signaling in COVID-19 and indicate that antagonists of C5aR1 could be useful for COVID-19 treatment.

Platelets promote CRC by activating the C5a/C5aR1 axis via PSGL-1/JNK/STAT1 signaling in tumor-associated macrophages.

Rationale: Platelets can influence the progression and prognosis of colorectal cancer (CRC) through multiple mechanisms, including crosstalk with tumor-associated macrophages (TAMs). However, the mechanisms underlying the crosstalk between platelets and TAMs remain unclear. The present study aimed to investigate the role of intratumoral platelets in regulating the function of TAMs and to identify the underlying mechanisms. Methods: The interaction of platelets with macrophages was assessed in the presence or absence of the indicated compounds in vivo. An azoxymethane/dextran sodium sulfate (AOM/DSS)-induced CRC mouse model was used to investigate the role of platelets in controlling CRC development. Multiplexed immunofluorescence staining, fluorescence-activated cell sorting (FACS), and RNA sequence analysis were used to examine the changes in TAMs. TAMs and bone marrow-derived macrophages (BMDMs) were treated with the indicated compounds or siRNA against specific targets, and the expression levels of signal transducer and activator of transcription 1 (STAT1), c-Jun N-terminal kinase (JNK), and P-selectin glycoprotein ligand-1 (PSGL-1) were measured by Western blotting. The mRNA expression levels of complement 5 (C5), complement 5a receptor 1 (C5ar1), Arginase 1 (Arg1) and Il10 were measured by real-time RT-PCR, and the complement 5a (C5a) concentration was measured by ELISA. The dual-luciferase reporter assay and ChIP assay were performed to examine the potential regulatory mechanisms of platelet induction of C5 transcription in TAMs. Results: In our study, we found that an increase in platelets exacerbated CRC development, while inhibiting platelet adhesion attenuated tumor growth. Platelets signal TAMs through P-selectin (CD62P) binding to PSGL-1 expressed on TAMs and activating the JNK/STAT1 pathway to induce the transcription of C5 and the release of C5a, shifting TAMs toward a protumor phenotype. Inhibiting the C5a/C5aR1 axis or PSGL-1 significantly reduced CRC growth. Conclusions: An increase in intratumoral platelets promoted CRC growth and metastasis by CD62P binding to PSGL-1 expressed on TAMs, leading to JNK/STAT1 signaling activation, which promoted C5 transcription and activated the C5a/C5aR1 axis in TAMs. Our study examined the mechanism of the crosstalk between platelets and TAMs to exacerbate CRC development and proposed a potential therapeutic strategy for CRC patients.

Overexpression of complement C5a indicates poor survival and therapeutic response in metastatic renal cell carcinoma.

INTRODUCTION: Complement C5a is an important component of the innate immune system. An increasing number of reports have revealed the relevance of C5a in tumor progression; however, its exact role in metastatic renal cell carcinoma (mRCC) remains unknown. METHODS: We evaluated C5a expression in tumor tissue microarrays of 231 mRCC patients and analyzed the relationship between C5a levels and clinical outcomes, and the expression of epithelial-mesenchymal transition (EMT)-related proteins, programmed cell death protein 1 (PD-1), and programmed cell death-ligand 1 (PD-L1). In-vitro functional experiments using exogenous C5a stimulation and C5a silencing in renal cell carcinoma cells were used to validate the tissue findings. RESULTS: High C5a expression was associated with poor therapeutic responses, poor overall and progression-free survival, and high expression of EMT-related proteins and PD-1/PD-L1 in mRCC patients. Exogenous C5a promoted proliferation, migration, and invasion of renal cell carcinoma cells, and induced the expression of EMT-related proteins and PD-1/PD-L1. Conversely, C5a silencing inhibited migration and invasion of renal cell carcinoma cells and decreased the expression of EMT-related proteins and PD-1/PD-L1. CONCLUSIONS: Our findings indicate that elevated C5a expression is associated with poor outcomes in patients with mRCC, and this effect may be partly attributed to the ability of C5a to promote EMT and PD-1/PD-L1 expression. C5a may be a potential novel target for the treatment of mRCC.

C5aR2 receptor: The genomic twin of the flamboyant C5aR1.

The complement fragment C5a is one of the most potent proinflammatory glycoproteins liberated by the activation of the biochemical cascade of the complement system. C5a is established to interact with a set of genomically related transmembrane receptors, like C5aR1 (CD88, C5aR) and C5aR2 (GPR77, C5L2) with comparable affinity. The C5aR1 is a classical G-protein-coupled receptor (GPCR), whereas C5aR2 is a nonclassical GPCR that tailors immune cell activity potentially through ß-arrestins rather than G-proteins. Currently, the exact function of the C5aR2 is actively debated in the context of C5aR1, even though both C5aR1 and C5aR2 are coexpressed on myriads of tissues. The functional relevance of C5aR2 appears to be context-dependent compared to the C5aR1, which has received enormous attention for its role in both acute and chronic inflammatory diseases. In addition, the structure of C5aR2 and its interaction specificity toward C5a is not structurally elucidated in the literature so far. The current study has attempted to close the gap by generating highly refined model structures of C5aR2, respectively in free (inactive), complexed to C-terminal peptide of C5a (meta-active) and the C5a (active), embedded to a model palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayer. The computational modeling and the 1.5-µs molecular dynamics data presented in the current study are expected to further enrich the understanding of C5a-C5aR2 interaction compared to C5a-C5aR1, which will surely help in elaborating the currently debated biological function of C5aR2 better in the foreseeable future.

Involvement of complement peptides C3a and C5a in osteoarthritis pathology.

Osteoarthritis (OA) affects more than 500 million people worldwide and is among the five diseases in Germany causing the highest suffering of the patients and cost for the society. The quality of life of OA patients is severely compromised, and adequate therapy is lacking owing to a knowledge gap that acts as a major barrier to finding safe and effective solutions. Chronic, low-grade inflammation plays a central role in OA pathogenesis and is associated with both OA pain and disease progression. Innate immune pathways, such as the complement- and pattern-recognition receptor pathways, are pivotal to the inflammation in OA and key components of the innate immune system implicated in OA include DAMP-TLR signaling, the complement system, carboxypeptidase B (CPB), and mononuclear cells. Anaphylatoxins C3a and C5a are small polypeptides (77 and 74 amino acids, respectively) which are released by proteolytic cleavage of the complement components C3 and C5. The alternative complement pathway seems to play a crucial role in OA pathogenesis as these complement components, mostly C3 and its activation peptide C3a, were detected at high levels in osteoarthritic cartilage, synovial membrane, and cultured chondrocytes. Targeting the complement system by using anti-complement drugs as a therapeutic option bears the risk of major side effects such as increasing the risk of infection, interfering with cell regeneration and metabolism, and suppressing the clearance of immune complexes. Despite those adverse effects, several synthetic complement peptide antagonists show promising effects in ameliorating inflammatory cell responses also in joint tissues.

Unexpected Off-Target Activities for Recombinant C5a in Human Macrophages.

The anaphylatoxin C5a is core effector of complement activation. C5a exerts potent proinflammatory and immunomodulatory actions through interacting with its C5a receptors, C5aR1 and C5aR2, modulating multiple signaling and functional activities of immune cells. Native C5a contains a large N-linked glycosylation site at Asn64, which accounts for up to 25% of its m.w. To date, the vast majority of published studies examining C5a are performed using Escherichia coli-generated recombinant C5a, which is readily available from numerous commercial suppliers, but lacks this glycosylation moiety. However, a plasma-purified "native" form of C5a is also commercially available. The different size and glycosylation of these two C5a versions could have functional implications. Therefore, the current study aimed to compare recombinant human C5a to purified plasma-derived human C5a in driving the signaling and functional activities of human primary macrophages. We found that both versions of C5a displayed similar potencies at triggering C5aR1- and C5aR2-mediated cell signaling, but elicited distinct functional responses in primary human monocyte-derived macrophages. Multiple commercial sources of recombinant C5a, but not the plasma-purified or a synthetic C5a version, induced human monocyte-derived macrophages to produce IL-6 and IL-10 in a C5a receptor-independent manner, which was driven through Syk and NF-κB signaling and apparently not due to endotoxin contamination. Our results, therefore, offer caution against the sole use of recombinant human C5a, particularly in functional/cytokine assays conducted in human primary immune cells, and suggest studies using recombinant human C5a should be paired with C5aR1 inhibitors or purified/synthetic human C5a to confirm relevant findings.

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.

Complement C5a promotes antigen cross-presentation by Peyer's patch monocyte-derived dendritic cells and drives a protective CD8+ T cell response.

The complement fragment C5a is closely associated with adaptive immune induction in the mucosa. However, the mechanisms that control CD8+ T cell responses by C5a have not been extensively explored. This study reveals that C5/C5a in the Peyer's patch (PP) subepithelial dome increases upon oral Listeria infection. We hypothesize that C5aR+ PP cells play an important role in the induction of antigen-specific T cell immunity. Using single-cell RNA sequencing, we identify C5aR- and lysozyme-expressing dendritic cells (C5aR+ LysoDCs) in PP and examine their role in CD8+ T cell immune induction. Stimulation of C5aR+ LysoDCs by C5a increases reactive oxygen species levels, leading to efficient antigen cross-presentation, which elicits an antigen-specific CD8+ T cell response. In C5-deficient mice, oral co-administration of C5a and Listeria enhances Listeria-specific cytotoxic T cell levels. Collectively, these findings suggest a role of the complement system in intestinal T cell immunity.

The significance of metalloproteinase 3 (MMP-3), chemokine CXC ligand 13 (CXCL-13) and complement component C5a in different stages of ANCA associated vasculitis.

The aim of the study was to evaluate the significance of metalloproteinase 3 (MMP-3), chemokine CXC ligand 13 (CXCL-13) and complement component 5a (C5a) in different stages of ANCA associated vasculitis (AAV). 89 adults were included into the study. 28 patients with active AAV (Birmingham Vasculitis Activity Score, BVAS > 3) formed the Active Group. 24 individuals who were in remission after 6 months of induction therapy formed the Short R Group, while 34 patients with longitudinal remission formed the Long R Group. 28 patients without autoimmune diseases similar in terms of age, gender and stage of kidney disease formed the Control Group. Receiver operating characteristic curve analysis (ROC) was used to evaluate MMP-3, CXCL-13 and C5a as markers of the different phases of vasculitis. In ROC analysis, MMP-3, CXCL-13 and C5a presented a good ability in distinguishing active vasculitis (Active Group) from the Control Group (AUC > 0.8), whereas only CXCL-13 displayed potential ability in distinguishing active vasculitis (Active Group) from long term remission (Long R Group, AUC = 0.683). MMP-3 significantly and positively correlated with serum creatinine concentration (r = 0.51, p = 0.011; r = 0.44, p = 0.009; r = -0.66, p < 0.001) and negatively with eGFR (r = -0.5, p = 0.012; r = -0.35, p = 0.039; r = -0.63, p < 0.001) in the Short R, Long R and Control Groups. MMP-3, CXCL-13, C5a can be potential markers in differentiating an active phase of vasculitis from other pathologies. However they can be treated as complementary to the well-known markers. CXCL-13 seems to be a potential marker in distinguishing active vasculitis from long term remission. MMP-3 level can be related to kidney function expressed by eGFR, therefore its elevation should be interpreted with caution in patients with kidney failure.

Chromosome 3 cluster rs11385942 variant links complement activation with severe COVID-19.

BACKGROUND: Genetic variation at a multigene cluster at chromosome 3p21.31 and the ABO blood group have been associated with the risk of developing severe COVID-19, but the mechanism remains unclear. Complement activation has been associated with COVID-19 severity. OBJECTIVE: The aim of this study was to examine whether chromosome 3p21.31 and the ABO variants are linked to the activation of the complement cascade in COVID-19 patients. METHODS: We considered 72 unrelated European hospitalized patients with genetic data and evaluation of circulating C5a and soluble terminal complement complex C5b-9 (SC5b-9). Twenty-six (36.1%) patients carried the rs11385942 G>GA variant and 44 (66.1%) non-O blood group associated with increased risk of severe COVID-19. RESULTS: C5a and SC5-b9 plasma levels were higher in rs11385949 GA carriers than in non-carriers (P = 0.041 and P = 0.012, respectively), while C5a levels were higher in non-O group than in O group patients (P = 0.019). The association between rs11385949 and SC5b-9 remained significant after adjustment for ABO and disease severity (P = 0.004) and further correction for C5a (P = 0.018). There was a direct relationship between upper airways viral load and SC5b-9 in carriers of the rs11385949 risk allele (P = 0.032), which was not observed in non-carriers. CONCLUSIONS: The rs11385949 G>GA variant, tagging the chromosome 3 gene cluster variation and predisposing to severe COVID-19, is associated with enhanced complement activation, both with C5a and terminal complement complex, while non-O blood group with C5a levels. These findings provide a link between genetic susceptibility to more severe COVID-19 and complement activation.

The emerging structural complexity of G-quadruplex RNAs.

G-quadruplexes (G4s) are four-stranded nucleic acid structures that arise from the stacking of G-quartets, cyclic arrangements of four guanines engaged in Hoogsteen base-pairing. Until recently, most RNA G4 structures were thought to conform to a sequence pattern in which guanines stacking within the G4 would also be contiguous in sequence (e.g., four successive guanine trinucleotide tracts separated by loop nucleotides). Such a sequence restriction, and the stereochemical constraints inherent to RNA (arising, in particular, from the presence of the 2'-OH), dictate relatively simple RNA G4 structures. Recent crystallographic and solution NMR structure determinations of a number of in vitro selected RNA aptamers have revealed RNA G4 structures of unprecedented complexity. Structures of the Sc1 aptamer that binds an RGG peptide from the Fragile-X mental retardation protein, various fluorescence turn-on aptamers (Corn, Mango, and Spinach), and the spiegelmer that binds the complement protein C5a, in particular, reveal complexity hitherto unsuspected in RNA G4s, including nucleotides in syn conformation, locally inverted strand polarity, and nucleotide quartets that are not all-G. Common to these new structures, the sequences folding into G4s do not conform to the requirement that guanine stacks arise from consecutive (contiguous in sequence) nucleotides. This review highlights how emancipation from this constraint drastically expands the structural possibilities of RNA G-quadruplexes.

The evolutionary analysis of complement component C5 and the gene co-expression network and putative interaction between C5a and C5a anaphylatoxin receptor (C5AR/CD88) in human and two Cyprinid fish.

The complement system is a complex network of soluble and membrane-associated serum proteins that regulate immune response. Activation of the complement C5 generates C5a and C5b which generate chemoattractive effect on myeloid cells and initiate the membrane attack complex (MAC) assembly. However, the study of evolutionary process and systematic function of C5 are still limited. In this study, we performed an evolutionary analysis of C5. Phylogeny analysis indicated that C5 sequences underwent complete divergence in fish and non-fish vertebrate. It was found that codon usage bias improved and provided evolution evidence of C5 in species. Notably, the codon usage bias of grass carp was evolutionarily closer to the zebrafish genome compared with humans and stickleback. This suggested that the zebrafish cell line may provide an alternative environment for heterologous protein expression of grass carp. Sequence comparison showed a higher similarity between human and mouse, grass carp, and zebrafish. Moreover, selective pressure analysis revealed that the C5 genes in fish and non-fish vertebrates exhibited different evolutionary patterns. To study the function of C5, gene co-expression networks of human and zebrafish were built which revealed the complexity of C5 function networks in different species. The protein structure simulation of C5 indicated that grass carp and zebrafish are more similar than to human, however, differences between species in C5a proteins are extremely smaller. Spatial conformations of C5a-C5AR (CD88) protein complex were constructed, which showed that possible interaction may exist between C5a and CD88 proteins. Furthermore, the protein docking sites/residues were measured and calculated according to the minimum distance for all atoms from C5a and CD88 proteins. In summary, this study provides insights into the evolutionary history, function and potential regulatory mechanism of C5 in fish immune responses.

Circulating FH Protects Kidneys From Tubular Injury During Systemic Hemolysis.

Intravascular hemolysis of any cause can induce acute kidney injury (AKI). Hemolysis-derived product heme activates the innate immune complement system and contributes to renal damage. Therefore, we explored the role of the master complement regulator Factor H (FH) in the kidney's resistance to hemolysis-mediated AKI. Acute systemic hemolysis was induced in mice lacking liver expression of FH (hepatoFH-/-, ~20% residual FH) and in WT controls, by phenylhydrazine injection. The impaired complement regulation in hepatoFH-/- mice resulted in a delayed but aggravated phenotype of hemolysis-related kidney injuries. Plasma urea as well as markers for tubular (NGAL, Kim-1) and vascular aggression peaked at day 1 in WT mice and normalized at day 2, while they increased more in hepatoFH-/- compared to the WT and still persisted at day 4. These were accompanied by exacerbated tubular dilatation and the appearance of tubular casts in the kidneys of hemolytic hepatoFH-/- mice. Complement activation in hemolytic mice occurred in the circulation and C3b/iC3b was deposited in glomeruli in both strains. Both genotypes presented with positive staining of FH in the glomeruli, but hepatoFH-/- mice had reduced staining in the tubular compartment. Despite the clear phenotype of tubular injury, no complement activation was detected in the tubulointerstitium of the phenylhydrazin-injected mice irrespective of the genotype. Nevertheless, phenylhydrazin triggered overexpression of C5aR1 in tubules, predominantly in hepatoFH-/- mice. Moreover, C5b-9 was deposited only in the glomeruli of the hemolytic hepatoFH-/- mice. Therefore, we hypothesize that C5a, generated in the glomeruli, could be filtered into the tubulointerstitium to activate C5aR1 expressed by tubular cells injured by hemolysis-derived products and will aggravate the tissue injury. Plasma-derived FH is critical for the tubular protection, since pre-treatment of the hemolytic hepatoFH-/- mice with purified FH attenuated the tubular injury. Worsening of acute tubular necrosis in the hepatoFH-/- mice was trigger-dependent, as it was also observed in LPS-induced septic AKI model but not in chemotherapy-induced AKI upon cisplatin injection. In conclusion, plasma FH plays a key role in protecting the kidneys, especially the tubules, against hemolysis-mediated injury. Thus, FH-based molecules might be explored as promising therapeutic agents in a context of AKI.

Crosstalk between GBM cells and mesenchymal stemlike cells promotes the invasiveness of GBM through the C5a/p38/ZEB1 axis.

BACKGROUND: Mesenchymal stemlike cells (MSLCs) have been detected in many types of cancer including brain tumors and have received attention as stromal cells in the tumor microenvironment. However, the cellular mechanisms underlying their participation in cancer progression remain largely unexplored. The aim of this study was to determine whether MSLCs have a tumorigenic role in brain tumors. METHODS: To figure out molecular and cellular mechanisms in glioma invasion, we have cultured glioma with MSLCs in a co-culture system. RESULTS: Here, we show that MSLCs in human glioblastoma (GBM) secrete complement component C5a, which is known for its role as a complement factor. MSLC-secreted C5a increases expression of zinc finger E-box-binding homeobox 1 (ZEB1) via activation of p38 mitogen-activated protein kinase (MAPK) in GBM cells, thereby enhancing the invasion of GBM cells into parenchymal brain tissue. CONCLUSION: Our results reveal a mechanism by which MSLCs undergo crosstalk with GBM cells through the C5a/p38 MAPK/ZEB1 signaling loop and act as a booster in GBM progression. KEY POINTS: 1. MSLCs activate p38 MAPK-ZEB1 signaling in GBM cells through C5a in a paracrine manner, thereby boosting the invasiveness of GBM cells in the tumor microenvironment.2. Neutralizing of C5a could be a potential therapeutic target for GBM by inhibition of mesenchymal phenotype.

The C5a/C5aR2 axis promotes renal inflammation and tissue damage.

C5a is a potent inflammatory mediator that binds C5aR1 and C5aR2. Although pathogenic roles of the C5a/C5aR1 axis in inflammatory disorders are well documented, the roles for the C5a/C5aR2 axis in inflammatory disorders and underlying mechanisms remain unclear. Here, we show that the C5a/C5aR2 axis contributes to renal inflammation and tissue damage in a mouse model of acute pyelonephritis. Compared with WT littermates, C5ar2-/- mice had significantly reduced renal inflammation, tubular damage, and renal bacterial load following bladder inoculation with uropathogenic E. coli. The decrease in inflammatory responses in the kidney of C5ar2-/- mice was correlated with reduced intrarenal levels of high mobility group box-1 protein (HMGB1), NLRP3 inflammasome components, cleaved caspase-1, and IL-1ß. In vitro, C5a stimulation of macrophages from C5ar1-/- mice (lacking C5aR1 but expressing C5aR2) led to significant upregulation of HMGB1 release, NLRP3/cleaved caspase-1 inflammasome activation, and IL-1ß secretion. Furthermore, blockade of HMGB1 significantly reduced C5a-mediated upregulation of NLRP3/cleaved caspase-1 inflammasome activation and IL-1ß secretion in the macrophages, implying a HMGB1-dependent upregulation of NLRP3/cleaved caspase-1 inflammasome activation in macrophages. Our findings demonstrate a pathogenic role for the C5a/C5aR2 axis in renal injury following renal infection and suggest that the C5a/C5aR2 axis contributes to renal inflammation and tissue damage through upregulation of HMGB1 and NLRP3/cleaved caspase-1 inflammasome.

Complement C5a Induces Renal Injury in Diabetic Kidney Disease by Disrupting Mitochondrial Metabolic Agility.

The sequelae of diabetes include microvascular complications such as diabetic kidney disease (DKD), which involves glucose-mediated renal injury associated with a disruption in mitochondrial metabolic agility, inflammation, and fibrosis. We explored the role of the innate immune complement component C5a, a potent mediator of inflammation, in the pathogenesis of DKD in clinical and experimental diabetes. Marked systemic elevation in C5a activity was demonstrated in patients with diabetes; conventional renoprotective agents did not therapeutically target this elevation. C5a and its receptor (C5aR1) were upregulated early in the disease process and prior to manifest kidney injury in several diverse rodent models of diabetes. Genetic deletion of C5aR1 in mice conferred protection against diabetes-induced renal injury. Transcriptomic profiling of kidney revealed diabetes-induced downregulation of pathways involved in mitochondrial fatty acid metabolism. Interrogation of the lipidomics signature revealed abnormal cardiolipin remodeling in diabetic kidneys, a cardinal sign of disrupted mitochondrial architecture and bioenergetics. In vivo delivery of an orally active inhibitor of C5aR1 (PMX53) reversed the phenotypic changes and normalized the renal mitochondrial fatty acid profile, cardiolipin remodeling, and citric acid cycle intermediates. In vitro exposure of human renal proximal tubular epithelial cells to C5a led to altered mitochondrial respiratory function and reactive oxygen species generation. These experiments provide evidence for a pivotal role of the C5a/C5aR1 axis in propagating renal injury in the development of DKD by disrupting mitochondrial agility, thereby establishing a new immunometabolic signaling pathway in DKD.

High Concentration of C5a-Induced Mitochondria-Dependent Apoptosis in Murine Kidney Endothelial Cells.

Patients with a relapse of idiopathic nephrotic syndrome have significantly increased levels of serum complement component 5a (C5a), and proteinuria has been noted in mice treated with C5a via changes in permeability of kidney endothelial cells (KECs) in established animal models. However, the apoptosis of KECs treated with high concentrations of C5a has also been observed. As mitochondrial damage is known to be important in cell apoptosis, the aim of this study was to examine the association between C5a-induced mouse KEC apoptosis and mitochondrial damage. Mouse KECs were isolated and treated with different concentrations of C5a. Cell viability assays showed that a high-concentration mouse recombinant protein C5a (rmC5a) treatment reduced mouse KEC growth. Cell cycle phase analysis, including apoptosis (sub-G1 phase) showed an increased percentage of the subG1 phase with a high-concentration rmC5a treatment. Cytochrome c and caspase 3/9 activities were significantly induced in the mouse KECs after a high-dose rmC5a (50 ng/mL) treatment, and this was rescued by pretreatment with the C5a receptor (C5aR) inhibitor (W-54011) and N-acetylcysteine (NAC). Reactive oxygen species (ROS) formation was detected in C5a-treated mouse KECs; however, W-54011 or NAC pretreatment inhibited high-dose rmC5a-induced ROS formation and also reduced cytochrome c release, apoptotic cell formation, and apoptotic DNA fragmentation. These factors determined the apoptosis of mouse KECs treated with high-dose C5a through C5aR and subsequently led to apoptosis via ROS regeneration and cytochrome c release. The results showed that high concentrations of C5a induced mouse KEC apoptosis via a C5aR/ROS/mitochondria-dependent pathway. These findings may shed light on the potential mechanism of glomerular sclerosis, a process in idiopathic nephrotic syndrome causing renal function impairment.