Receptor C5aR Research Articles

The Complement Receptors C3aR and C5aR Are a New Class of Immune Checkpoint Receptor in Cancer Immunotherapy.

Cancer immunotherapy has made remarkable clinical advances in recent years. Antibodies targeting the immune checkpoint receptors PD-1 and CTLA-4 and adoptive cell therapy (ACT) based on ex vivo expanded peripheral CTLs, tumor infiltrating lymphocytes (TILs), gene-engineered TCR- and chimeric antigen receptor (CAR)-T cells have all shown durable clinical efficacies in multiple types of cancers. However, these immunotherapeutic approaches only benefit a small fraction of cancer patients as various immune resistance mechanisms and limitations make their effective use a challenge in the majority of cancer patients. For example, adaptive resistance to therapeutic PD-1 blockade is associated with an upregulation of some additional immune checkpoint receptors. The efficacy of transferred tumor-specific T cells under the current clinical ACT protocol is often limited by their inefficient engraftment, poor persistence, and weak capability to attack tumor cells. Recent studies demonstrate that the complement receptor C3aR and C5aR function as a new class of immune checkpoint receptors. Complement signaling through C3aR and C5aR expressed on effector T lymphocytes prevent the production of the cytokine interleukin-10 (IL-10). Removing C3aR/C5aR-mediated transcriptional suppression of IL-10 expression results in endogenous IL-10 production by antitumor effector T cells, which drives T cell expansion and enhances T cell-mediated antitumor immunity. Importantly, preclinical, and clinical data suggest that a signaling axis consisting of complement/C3aR/C5aR/IL-10 critically regulates T cell mediated antitumor immunity and manipulation of the pathway ex vivo and in vivo is an effective strategy for cancer immunotherapy. Furthermore, a combination of treatment strategies targeting the complement/C3aR/C5aR/IL-10 pathway with other treatment modalities may improve cancer therapeutic efficacy.

C5a receptors C5aR1 and C5aR2 mediate opposing pathologies in a mouse model of melanoma.

The canonical complement component 5a (C5a) receptor (C5aR) 1 has well-described roles in tumorigenesis but the contribution of the second receptor, C5aR2, is unclear. The present study demonstrates that B16.F0 melanoma cells express mRNA for both C5aR1 and C5aR2 and signal through ERK and p38 MAPKs in response to C5a. Despite this, C5a had no impact on melanoma cell proliferation or migration in vitro. In vivo studies demonstrated that the growth of B16.F0 melanoma tumors was increased in C5aR2-/- mice but reduced in C5aR1-/- mice and wild-type mice treated with a C5aR1 antagonist. Analysis of tumor-infiltrating leukocyte populations showed no significant differences between wild-type and C5aR2-/- mice. Conversely, percentages of myeloid-derived suppressor cells, macrophages, and regulatory T lymphocytes were lower in tumors from C5aR1-/- mice, whereas total (CD3+) T lymphocytes and CD4+ subsets were higher. Analysis of cytokine and chemokine levels also showed plasma IFN-γ was higher and tumor C-C motif chemokine ligand 2 was lower in the absence of C5aR1. The results suggest that C5aR1 signaling supports melanoma growth by promoting infiltration of immunosuppressive leukocyte populations into the tumor microenvironment, whereas C5aR2 has a more restricted but beneficial role in limiting tumor growth. Overall, these data support the potential of C5aR1-inhibitory therapies for melanoma.-Nabizadeh, J. A., Manthey, H. D., Panagides, N., Steyn, F. J., Lee, J. D., Li, X. X., Akhir, F. N. M., Chen, W., Boyle, G. M., Taylor, S. M., Woodruff, T. M., Rolfe, B. E. C5a receptors C5aR1 and C5aR2 mediate opposing pathologies in a mouse model of melanoma.

Chimeric protein probes for C5a receptors through fusion of the anaphylatoxin C5a core region with a small-molecule antagonist

Blockade of the interaction of anaphylatoxin C5a with its receptor C5aR1 has been actively studied as a potential treatment for many inflammatory diseases; but current C5a antagonists exhibit inadequate potency and poor species cross-reactivity, and novel biochemical tools are needed to investigate whether the core region of C5a contains important interaction epitopes that can explain these limitations. Herein, we report the development of chimeric protein C5a probes containing both the complete core region of rat or human C5a, and the small-molecule antagonist PMX53-1. These probes were chemically synthesized through hydrazide-based native chemical ligation of a linear peptide hydrazide with the requisite cyclopeptidic antagonist, both of which were made by solid-phase synthesis. Quasi-racemic X-ray crystallography established that attachment of PMX53-1 did not affect the structure of the core region of C5a. Subsequent C5aR1 activity assays demonstrated the probes can provide valuable insights into the development of C5a antagonists; for example, they exhibited significantly better binding affinity and much improved species cross-reactivity than PMX53-1, supporting the notion that the effect of some epitopes outside the C-terminus of C5a should be taken into consideration when designing better C5a antagonists. Surprisingly, the core region of C5a was found to partially agonize C5aR1, suggesting the presence of more than one agonistic interaction in the binding of C5a to C5aR1. This study exemplifies the value of chemical protein synthesis in developing novel receptor probes for drug discovery research.

SAT-302 INHIBITION OF COMPLEMENT C5A/C5A RECEPTOR 1 DECREASES RENAL INJURY IN DIABETIC KIDNEY DISEASE VIA METABOLIC REPROGRAMMING

The complement system plays a central role in the activation of innate immunity, inflammation and tissue remodelling. The complement activation fragment C5a is a potent pro-inflammatory effector molecule. We have shown complement activation in patients with diabetes including renal deposition of the canonical C5a receptor C5aR1 and C5a/C5aR1 activation in experimental diabetes. This study aimed to determine whether genetic deletion or pharmacological inhibition of C5aR1 could confer renoprotection in diabetes. Streptozotocin (STZ)-induced diabetic C57BL/6 mice were treated with the highly selective C5aR1 antagonist, PMX53 (2mg/kg/day) in drinking water for 24 weeks (n=10). C5aR1 deficient mice (C5ar1-/-) and their wild type littermates were rendered diabetic with STZ and followed for 24 weeks (n=10). Kidney injury was assessed by urinary albumin excretion and glomerulosclerosis (GSI). Immunohistochemistry for Collagen IV, FoxP3+ regulatory T cells and F4/80+ macrophages was performed. Transcriptomics of renal cortex was performed by RNA-sequencing using the Illumina platform and pathway analyses using Gene Set Enrichment Analysis. Lipidomics of renal cortex was performed using liquid chromatography electrospray inonization tandem mass spectrometry. Diabetic C5ar1-/- mice showed protection against renal injury with decreased albumin excretion. Treatment of diabetic mice with PMX53 led to a reduction in albuminuria, inhibition of glomerular injury and resolution of inflammation via a decrease in F4/80+ macrophages and activation of FoxP3+ regulatory T cells. Transcriptomic analyses showed that the diabetes gene signature was reversed by PMX53. Pathways that were reduced by PMX53 in diabetic mice were related to mitochondrial function and lipid metabolism. The top differential gene downregulated in the diabetic kidney was acyl-CoA dehydrogenase 10 (Acad10), which participates in fatty acid β-oxidation in mitochondria. Blockade of C5aR1 signalling in diabetic mice restored Acad10 expression. Lipidomics analyses revealed a dysregulation of acylcarnitine profile in the diabetic kidney, as well as pathological cardiolipin remodeling, both of which are important for mitochondrial function and metabolism. Treatment of PMX53 restored acylcarnitine profile and cardiolipin remodeling in diabetic kidney. Our findings demonstrate that genetic or pharmacological disruption of C5aR1 is renoprotective in diabetes via restoration of pathways involved in mitochondrial fatty acid oxidation and by suppressing the pathogenic inflammatory response. This study indicates that targeting the C5a/C5aR1 pathway may provide a substantial therapeutic benefit for this devastating complication of diabetes.

The complement receptor C5aR2 promotes protein kinase R expression and contributes to NLRP3 inflammasome activation and HMGB1 release from macrophages

The NLR family pyrin domain-containing 3 (NLRP3) inflammasome is a multimeric protein complex that mediates maturation of the cytokines IL-1β and IL-18 as well as release of the proinflammatory protein high-mobility group box 1 (HMGB1) and contributes to several inflammatory diseases, including sepsis, gout, and type 2 diabetes. In this context, the well-studied active complement fragment C5a and its receptor C5aR1 or C5aR2 orchestrate the inflammatory responses in many diseases. Although a C5a-C5aR interaction in NLRP3-associated diseases has been suggested, little is known about the details of C5a-C5aR cross-talk with the NLRP3 inflammasome in macrophages. In this study, using mice and murine macrophages and cytokines, immunoblotting, siRNA, and quantitative real-time PCR assays, we demonstrate that C5aR2 deficiency restricts activation of the NLRP3 inflammasome and release of HMGB1 both in vitro and in vivo Mechanistically, we found that C5aR2 promotes NLRP3 activation by amplifying dsRNA-dependent PKR expression, which is an important NLRP3-activating factor. We also observed that elevation of PKR expression because of the C5a-C5aR2 interaction depends on the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase pathway and type I IFN signaling. In conclusion, these findings reveal that C5aR2 contributes to NLRP3 inflammasome activation and HMGB1 release from macrophages.

Anaphylatoxin receptor C3aR on platelets participates in platelet function and links innate immunity with thrombosis

Abstract Background Beyond the superficial function of blood coagulation, platelets play an important role in the genesis of vascular inflammatory processes. Interestingly, platelets express the complement receptor C3aR. Here, we examine the role of the complement receptor C3aR on platelets for platelet function and in diseases. Methods We detected the expression of C3aR on murine and human platelets using western blot and flow cytometry. We examined proteins, that are in close connection to the C3aR receptor on platelets by mass spectrometry to elucidate the intracellular signaling. We examined the role of the platelet C3aR for platelet function using different platelet function tests. To investigate the relevance of our findings in vivo, we made use of mice that are deficient for C3aR. After performing bleeding time and intravital microscopy experiments, we induced myocardial infarction and stroke in knockout animals. Results We found that C3aR on platelets plays an important role in platelet function and thrombus formation. We discovered that the complement activation fragment C3a regulates bleeding time after tail injury and thrombosis using C3aR−/− mice or C3−/− mice with re-injection of C3a. After induction of a myocardial infarction or stroke in mice, C3aR−/− mice showed a better outcome in comparison to WT mice. When analyzing the intracellular signaling, we discovered that those effects were PI3 kinase and Rap-1 dependent. Conclusions Taken together, we identified the C3a receptor on platelets as an important player in platelet function and thrombus formation. Linking the systems of innate immunology and hemostasis, the C3a receptor on platelets could be a possible therapeutic target in diseases featuring thromboinflammation.

Aristolochic acid I aggravates renal injury by activating the C3a/C3aR complement system

Previous studies have reported that the complement system is unconventionally activated in many kinds of glomerulonephritis. Multiple complement components participate in the pathogenic process by triggering immune response or other intracellular signaling pathways. Here, we have investigated the role of C3a and its receptor C3aR in aristolochic acid nephropathy (AAN), which, is featured with progressive interstitial fibrosis. Over release of C3a and increased expression of C3aR parallels to the up-regulation of α-SMA and TGF-β1 in AAN, which appeared to promote epithelial-mesenchymal-transition (EMT). To identify the role of complement activation in AAN, we used an inhibitor of C3aR (C3aRA) to block the coupling of C3a to its receptor. Our results confirmed from decreased EMT, the protective effect of C3aRA in cell apoptosis and inflammatory response induced by aristolochic acid I. These results showed that C3a and its receptor C3aR played pathogenic roles in AAN, and renal tubular epithelial cells were potentially pivotal targets of complement activation that could cause pro-fibrotic effects.

049 Dual inhibition of the complement factor C5 and leukotriene B4by the bifunctional soft tick-derived lipocalin Coversin synergizes in suppressing disease in a murine model of bullous pemphigoid-like epidermolysis bullosa acquisita

The treatment of autoimmune diseases still relies on immunosuppression and is associated with severe side-effects. The development of drugs abrogating pathogenic pathways more specifically is, therefore, most desirable. In nature, such specificity is exemplified, e.g., by the soft-tick-derived lipocalin Coversin, which locally suppresses immune responses by simultaneously inhibiting the complement factor C5 and leukotriene B4 (LTB4). C5a, a proteolytic fragment of C5, and LTB4have been highlighted as critical drivers of skin inflammation in pemphigoid diseases (PDs), a group of autoimmune blistering skin diseases. Here, we demonstrate that both Coversin and its mutated form L-Coversin, which only inhibits LTB4, dose-dependently attenuate disease in a model of bullous pemphigoid-like epidermolysis bullosa acquisita (BP-like EBA).Coversin, however, reduces disease more effectively than L-Coversin, indicating that inhibition of C5 and that of LTB4 synergize in their suppressing effects in that model. Further supporting a similar therapeutic potential of Coversin and L-Coversin in humans, we show that C5a and LTB4are both present in the blister fluid of BP patients in quantities inducing the recruitment of granulocytes and that their receptors C5aR1 and BLT1, respectively, are abundant specifically in lesional skin. Collectively, our results highlight Coversin and L-Coversin as potential therapeutics for PDs.

Complementing the Cancer-Immunity Cycle.

Reactivation of cytotoxic CD8+ T-cell responses has set a new direction for cancer immunotherapy. Neutralizing antibodies targeting immune checkpoint programmed cell death protein 1 (PD-1) or its ligand (PD-L1) have been particularly successful for tumor types with limited therapeutic options such as melanoma and lung cancer. However, reactivation of T cells is only one step toward tumor elimination, and a substantial fraction of patients fails to respond to these therapies. In this context, combination therapies targeting more than one of the steps of the cancer-immune cycle may provide significant benefits. To find the best combinations, it is of upmost importance to understand the interplay between cancer cells and all the components of the immune response. This review focuses on the elements of the complement system that come into play in the cancer-immunity cycle. The complement system, an essential part of innate immunity, has emerged as a major regulator of cancer immunity. Complement effectors such as C1q, anaphylatoxins C3a and C5a, and their receptors C3aR and C5aR1, have been associated with tolerogenic cell death and inhibition of antitumor T-cell responses through the recruitment and/or activation of immunosuppressive cell subpopulations such as myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs), or M2 tumor-associated macrophages (TAMs). Evidence is provided to support the idea that complement blocks many of the effector routes associated with the cancer-immunity cycle, providing the rationale for new therapeutic combinations aimed to enhance the antitumor efficacy of anti-PD-1/PD-L1 checkpoint inhibitors.

The Novel C5aR Antagonist DF3016A Protects Neurons Against Ischemic Neuroinflammatory Injury

The central nervous system (CNS) constitutively expresses complement (C) membrane receptors and complement proteins, including the component C5a. This is a crucial terminal effector of the C cascade, mostly involved in pain and neuroinflammatory conditions. Aberrant activation of C5a protein and its receptor C5aR has been reported to play a critical role in neurodegenerative diseases, with important clinical consequences. Here we have investigated the effects of DF3016A, a novel selective C5aR antagonist, able to penetrate the blood-brain barrier (BBB), on cortical neurons exposed to oxygen-glucose deprivation-reoxygenation (OGD/R), a neuroinflammation-related process. We demonstrated that a mild ischemic insult induces an early upregulation of C5aR associated with the over-production of pro-inflammatory cytokines and the over-expression of the transcriptional regulatory factor miR-181. Furthermore, we report the first experimental evidence of the effect of DF3016A, modulating complement component C5a, on neurons in a model of injury. Interestingly, DF3016A protects neuronal viability by restoring intracellular calcium levels, thus opposing the increase in pro-inflammatory cytokine levels and miR-181 expression. Based on our results, we suggest that DF3016A is a novel C5aR antagonist promoting protective effects against OGD/R-induced damage that could be a new therapeutic approach to controlling CNS neuroinflammatory conditions.

Correction to: Functional Relevance of the Anaphylatoxin Receptor C3aR for Platelet Function and Arterial Thrombus Formation Marks an Intersection Point Between Innate Immunity and Thrombosis.

Correction to: Functional Relevance of the Anaphylatoxin Receptor C3aR for Platelet Function and Arterial Thrombus Formation Marks an Intersection Point Between Innate Immunity and Thrombosis.

Distinct roles of the anaphylatoxin receptors C3aR, C5aR1 and C5aR2 in experimental meningococcal infections

ABSTRACTThe complement system is pivotal in the defense against invasive disease caused by Neisseria meningitidis (Nme, meningococcus), particularly via the membrane attack complex. Complement activation liberates the anaphylatoxins C3a and C5a, which activate three distinct G-protein coupled receptors, C3aR, C5aR1 and C5aR2 (anaphylatoxin receptors, ATRs). We recently discovered that C5aR1 exacerbates the course of the disease, revealing a downside of complement in Nme sepsis. Here, we compared the roles of all three ATRs during mouse nasal colonization, intraperitoneal infection and human whole blood infection with Nme. Deficiency of complement or ATRs did not alter nasal colonization, but significantly affected invasive disease: Compared to WT mice, the disease was aggravated in C3ar−/- mice, whereas C5ar1−/- and C5ar2−/- mice showed increased resistance to meningococcal sepsis. Surprisingly, deletion of either of the ATRs resulted in lower cytokine/chemokine responses, irrespective of the different susceptibilities of the mice. This was similar in ex vivo human whole blood infection using ATR inhibitors. Neutrophil responses to Nme were reduced in C5ar1−/- mouse blood. Upon stimulation with C5a plus Nme, mouse macrophages displayed reduced phosphorylation of ERK1/2, when C5aR1 or C5aR2 were ablated or inhibited, suggesting that both C5a-receptors prime an initial macrophage response to Nme. Finally, in vivo blockade of C5aR1 alone (PMX205) or along with C5aR2 (A8Δ71−73) resulted in ameliorated disease, whereas neither antagonizing C3aR (SB290157) nor its activation with a “super-agonist” peptide (WWGKKYRASKLGLAR) demonstrated a benefit. Thus, C5aR1 and C5aR2 augment disease pathology and are interesting targets for treatment, whereas C3aR is protective in experimental meningococcal sepsis.

Toll-like receptor 2 confers partial neuroprotection during prion disease.

Neuroinflammation and neurodegeneration are common during prion infection, but the mechanisms that underlie these pathological features are not well understood. Several components of innate immunity, such as Toll-like receptor (TLR) 4 and Complement C1q, have been shown to influence prion disease. To identify additional components of innate immunity that might impact prion disease within the central nervous system (CNS), we screened RNA from brains of pre-clinical and clinical 22L-infected mice for alterations in genes associated with innate immunity. Transcription of several genes encoding damage-associated molecular pattern (DAMP) proteins and receptors were increased in the brains of prion-infected mice. To investigate the role of some of these proteins in prion disease of the CNS, we infected mice deficient in DAMP receptor genes Tlr2, C3ar1, and C5ar1 with 22L scrapie. Elimination of TLR2 accelerated disease by a median of 10 days, while lack of C3aR1 or C5aR1 had no effect on disease tempo. Histopathologically, all knockout mouse strains tested were similar to infected control mice in gliosis, vacuolation, and PrPSc deposition. Analysis of proinflammatory markers in the brains of infected knockout mice indicated only a few alterations in gene expression suggesting that C5aR1 and TLR2 signaling did not act synergistically in the brains of prion-infected mice. These results indicate that signaling through TLR2 confers partial neuroprotection during prion infection.

Asparaginyl Endopeptidase (Legumain) Supports Human Th1 Induction via Cathepsin L-Mediated Intracellular C3 Activation.

Autocrine activation of the complement receptors C3aR and CD46 by complement activation components C3a and C3b produced through C3 cleavage by the protease cathepsin L (CTSL) during T cell stimulation is a requirement for IFN-γ production and Th1 induction in human CD4+ T cells. Thus, lack of autocrine CD46 activation, such as in CD46-deficient patients, is associated with defective Th1 responses and recurrent infections. We have identified LGMN [the gene coding for legumain, also known as asparaginyl endopeptidase (AEP)] as one of the key genes induced by CD46 co-stimulation during human CD4+ T cell activation. AEP processes and activates a range of proteins, among those α1-thymosin and CTSL, which both drive intrinsically Th1 activity—but has so far not been described to be functionally active in human T cells. Here we found that pharmacological inhibition of AEP during activation of human CD4+ T cells reduced CTSL activation and the CTSL-mediated generation of intracellular C3a. This translated into a specific reduction of IFN-γ production without affecting cell proliferation or survival. In line with these findings, CD4+ T cells isolated from Lgmn−/− mice also displayed a specific defect in IFN-γ secretion and Th1 induction. Furthermore, we did not observe a role for AEP-driven autocrine α1-thymosin activation in T cell-derived IFN-γ production. These data suggest that AEP is an “upstream” activator of the CTSL-C3-IFN-γ axis in human CD4+ T cells and hence an important supporter of human Th1 induction.

Functional Relevance of the Anaphylatoxin Receptor C3aR for Platelet Function and Arterial Thrombus Formation Marks an Intersection Point Between Innate Immunity and Thrombosis.

Platelets have distinct roles in the vascular system in that they are the major mediator of thrombosis, critical for restoration of tissue integrity, and players in vascular inflammatory conditions. In close spatiotemporal proximity, the complement system acts as the first line of defense against invading microorganisms and is a key mediator of inflammation. Whereas the fluid phase cross-talk between the complement and coagulation systems is well appreciated, the understanding of the pathophysiological implications of such interactions is still scant. We analyzed coexpression of the anaphylatoxin receptor C3aR with activated glycoprotein IIb/IIIa on platelets of 501 patients with coronary artery disease using flow cytometry; detected C3aR expression in human or murine specimen by polymerase chain reaction, immunofluorescence, Western blotting, or flow cytometry; and examined the importance of platelet C3aR by various in vitro platelet function tests, in vivo bleeding time, and intravital microscopy. The pathophysiological relevance of C3aR was scrutinized with the use of disease models of myocardial infarction and stroke. To approach underlying molecular mechanisms, we identified the platelet small GTPase Rap1b using nanoscale liquid chromatography coupled to tandem mass spectrometry. We found a strong positive correlation of platelet complement C3aR expression with activated glycoprotein IIb/IIIa in patients with coronary artery disease and coexpression of C3aR with glycoprotein IIb/IIIa in thrombi obtained from patients with myocardial infarction. Our results demonstrate that the C3a/C3aR axis on platelets regulates distinct steps of thrombus formation such as platelet adhesion, spreading, and Ca2+ influx. Using C3aR-/- mice or C3-/- mice with reinjection of C3a, we uncovered that the complement activation fragment C3a regulates bleeding time after tail injury and thrombosis. Notably, C3aR-/- mice were less prone to experimental stroke and myocardial infarction. Furthermore, reconstitution of C3aR-/- mice with C3aR+/+ platelets and platelet depletion experiments demonstrated that the observed effects on thrombosis, myocardial infarction, and stroke were specifically caused by platelet C3aR. Mechanistically, C3aR-mediated signaling regulates the activation of Rap1b and thereby bleeding arrest after injury and in vivo thrombus formation. Overall, our findings uncover a novel function of the anaphylatoxin C3a for platelet function and thrombus formation, highlighting a detrimental role of imbalanced complement activation in cardiovascular diseases.

C5aR1 interacts with TLR2 in osteoblasts and stimulates the osteoclast-inducing chemokine CXCL10.

The anaphylatoxin C5a is generated upon activation of the complement system, a crucial arm of innate immunity. C5a mediates proinflammatory actions via the C5a receptor C5aR1 and thereby promotes host defence, but also modulates tissue homeostasis. There is evidence that the C5a/C5aR1 axis is critically involved both in physiological bone turnover and in inflammatory conditions affecting bone, including osteoarthritis, periodontitis, and bone fractures. C5a induces the migration and secretion of proinflammatory cytokines of osteoblasts. However, the underlying mechanisms remain elusive. Therefore, in this study we aimed to determine C5a‐mediated downstream signalling in osteoblasts. Using a whole‐genome microarray approach, we demonstrate that C5a activates mitogen‐activated protein kinases (MAPKs) and regulates the expression of genes involved in pathways related to insulin, transforming growth factor‐β and the activator protein‐1 transcription factor. Interestingly, using coimmunoprecipitation, we found an interaction between C5aR1 and Toll‐like receptor 2 (TLR2) in osteoblasts. The C5aR1‐ and TLR2‐signalling pathways converge on the activation of p38 MAPK and the generation of C‐X‐C motif chemokine 10, which functions, among others, as an osteoclastogenic factor. In conclusion, C5a‐stimulated osteoblasts might modulate osteoclast activity and contribute to immunomodulation in inflammatory bone disorders.

Anaphylatoxin Receptors C3aR and C5aR1 Are Important Factors That Influence the Impact of Ethanol on the Adipose Secretome

Background and aims: Chronic ethanol exposure results in inflammation in adipose tissue; this response is associated with activation of complement as well as the development of alcohol-related liver disease (ALD). Adipose communicates with other organs, including liver, via the release of soluble mediators, such as adipokines and cytokines, characterized as the “adipose secretome.” Here we investigated the role of the anaphaylatoxin receptors C3aR and C5aR1 in the development of adipose tissue inflammation and regulation of the adipose secretome in murine ALD (mALD).Methods: Wild-type C57BL/6 (WT), C3aR−/−, and C5aR1−/− mice were fed Lieber-DeCarli ethanol diet for 25 days (6% v/v, 32% kcal) or isocaloric control diets; indicators of inflammation and injury were assessed in gonadal adipose tissue. The adipose secretome was characterized in isolated adipocytes and stromal vascular cells.Results: Ethanol feeding increased the expression of adipokines, chemokines and leukocyte markers in gonadal adipose tissue from WT mice; C3aR−/− were partially protected while C5aR1−/− mice were completely protected. In contrast, induction of CYP2E1 and accumulation of TUNEL-positive cells in adipose in response to ethanol feeding was independent of genotype. Bone marrow chimeras, generated with WT and C5aR1−/− mice, revealed C5aR1 expression on non-myeloid cells, likely to be adipocytes, contributed to ethanol-induced adipose inflammation. Chronic ethanol feeding regulated both the quantity and distribution of adipokines secreted from adipocytes in a C5aR1-dependent mechanism. In WT mice, chronic ethanol feeding induced a predominant release of pro-inflammatory adipokines from adipocytes, while the adipose secretome from C5aR1−/− mice was characterized by an anti-inflammatory/protective profile. Further, the cargo of adipocyte-derived extracellular vesicles (EVs) was distinct from the soluble secretome; in WT EVs, ethanol increased the abundance of pro-inflammatory mediators while EV cargo from C5aR1−/− adipocytes contained a greater diversity and more robust expression of adipokines.Conclusions: C3aR and C5aR1 are potent regulators of ethanol-induced adipose inflammation in mALD. C5aR1 modulated the impact of chronic ethanol on the content of the adipose secretome, as well as influencing the cargo of an extensive array of adipokines from adipocyte-derived EVs. Taken together, our data demonstrate that C5aR1 contributes to ethanol-mediated changes in the adipose secretome, likely contributing to intra-organ injury in ALD.

The distinct roles of the anaphylatoxin receptors C5aR1, C5aR2 and C3aR during experimental invasive meningococcal disease

In this study, the experimental surface tensions were measured for aqueous solutions of cetyltrimethyl ammonium bromide, 1-propanol, 2-propanol, and 1-butanol with a pendant drop apparatus. The temperature and pressure of all experiments were 298.15 K and 1 bar, respectively. Subsequently, a model based on the equality of the chemical potential of components at the interface and the bulk liquid was used. The results of this part showed that the surface tensions were reproduced well. The average absolute deviation percent of surface tension was 1.11. Then the surface tensions of (cetyltrimethylammonium bromide + alcohols) aqueous mixtures were measured at different concentrations. Moreover, the critical micelle concentrations of the applied systems were determined. The present model was used for aqueous mixtures of (cetyltrimethylammonium bromide + alcohols). The average absolute deviation percent of surface tension was 2.72, so the model successfully predicted the surface tension for aqueous solutions of (cetyltrimethylammonium bromide + alcohols). Furthermore, the results of the model proved that the presence of alcohols decreased the surface coverage of cetyltrimethylammonium bromide and increased the values of the critical micelle concentration.

RA10.02: A NOVEL ROLE FOR THE COMPLEMENT CASCADE IN CHEMORADIATION THERAPY RESISTANT OESOPHAGEAL ADENOCARCINOMA

Abstract Background Resistance to chemoradiation therapy (CRT) in oesophageal adenocarcinoma (OAC) is an important clinical challenge. Elucidating underlying mechanisms of treatment resistance is crucial to improving survival rates. Methods OAC tumour biopsies were collected prior to neoadjuvant CRT. Tumour conditioned media was generated by culturing human explants for 24 h. Response to treatment was determined pathologically using the Mandard Grading System. C3, CFB and MCP-1 mRNA expression was assessed by qPCR. Secreted C3, C3a and MCP-1 were measured by ELISA. An isogenic model of radioresistant OAC was established by chronically irradiating OE33 cells with clinically-relevant doses of 2 Gy X-ray radiation. C5aR and C3aR expression was determined by flow cytometry. Results We demonstrate, for the first time, that the central factor of the complement cascade, complement component 3 (C3), is expressed in OAC tumours and is increased in pre-treatment OAC biopsies from patients (n = 13) who have a subsequent poor response to neoadjuvant CRT (P < 0.05). In addition, C3 is secreted from ex vivo human OAC tumour explants (n = 13) and correlates with levels of the monocyte chemoattractant MCP-1 (P < 0.008) and complement factor B (CFB) a component of the alternative pathway of complement activation (P < 0.0001). In vitro, radioresistant OE33 R cells had increased mRNA levels of C3 (P < 0.01), CFB (P < 0.05) and MCP-1 (P < 0.05) compared to radiosensitive OE33 P cells. OEE3 R cells expressed increased levels of secreted C3 and the anaphylatoxin C3a, compared to radiosensitive OE33P cells (both P < 0.001). Furthermore, we demonstrate for the first time that the anaphylatoxin receptor C5aR is expressed by OAC cells, suggesting that OAC cells can respond to complement anaphylatoxins. Conclusion This study highlights, for the first time, a novel role for the complement cascade in the resistance of OAC to CRT and highlights C3 as a novel predictive marker of response to CRT in OAC. Disclosure All authors have declared no conflicts of interest.

Myocardial expression of the anaphylatoxin receptor C3aR is associated with cardiac inflammation and prognosis in patients with non-ischaemic heart failure.

AimThe aim of this study is to analyse the prognostic value of complement anaphylatoxin receptors in patients with non‐ischaemic cardiomyopathy undergoing endomyocardial biopsy.Methods and resultsIn 102 patients (72.5% male patients, median age 54 years) with non‐ischaemic cardiomyopathy, myocardial expression of C3aR was assessed among other parameters. The primary study endpoint was a composite of death, heart transplantation, heart failure‐related re‐hospitalization, and deterioration of left ventricular ejection fraction within a mean follow‐up of 11.9 months. The number of cells, which stained positive for C3aR, was significantly increased in patients with inflammatory compared with non‐inflammatory cardiomyopathy (1.75 ± 0.31 cells in inflammatory cardiomyopathy vs. 0.94 ± 0.26 in non‐inflammatory cardiomyopathy, P = 0.049). Subsequently, positive expression for C3aR was judged based on a semi‐quantitative scoring system. Significantly, more patients with positive MHCII and CD68 expression showed an increased number of C3aR‐positive cells. C3aR expression based on this score was more pronounced in patients with human herpesvirus 6 viral genome detection. Kaplan–Meier curves illustrate that the C3aR‐negative group reached the primary endpoint significantly more often (mean follow‐up 11.9 months, log rank 5.963, P = 0.015). Lack of C3aR expression was a strong independent predictor for the primary endpoint in Cox regression analysis [hazard ratio 0.46 (0.26–0.82, P = 0.009)].ConclusionsC3aR‐positive cells are found more often in patients with inflammatory cardiomyopathy. The relevance of C3aR‐positive cells in patients with non‐ischaemic cardiomyopathy should be further evaluated as potential predictors or modulators of adverse cardiac remodelling, the substrate of progressive heart failure.