Rational design of antibody-like peptides for targeting the human complement fragment protein C5a.

Human complement fragment 5a (C5a) is one of the most potent glycoproteins generated downstream of C3a and C4a during late-stage activation of the complement signaling cascade. C5a recruits receptors like C5aR1 and C5aR2 and is established to play a critical role in complement-mediated inflammation. Thus, excessive C5a in the plasma due to aberrant activation of the complement contributes to the pathophysiology of several chronic inflammatory diseases. Therefore, restricting the excessive interaction of C5a with its receptors by neutralizing C5a has been one of the most effective therapeutic strategies for the management of inflammatory diseases. Indeed, antibodies targeting C5 (Eculizumab), the precursor of C5a, and C5a (Vilobelimab) have already been approved by the FDA. Still, small designer peptides that work like antibodies and can target and stop C5a from interacting with its receptors seem to be a possible therapeutic alternative to antibodies because they are smaller, cheaper to make, more specific to their target, and can get through membrane barriers. As a proof-of-principle, the current study describes the computational design and evaluation of a pair of peptides that are able to form stable high-affinity complexes with the epitope regions of C5a that are important for the recruitment of C5aR1 and C5aR2. The computational data further supports the potential of designer peptides for mimicking the function of antibodies targeting C5a. However, further experimental studies will be required to establish the structure-function relationship of the designer peptides and also to establish the hypothesis of antibody-like peptides targeting C5a.

Neutraligands of C5a can potentially occlude the interaction of C5a with the complement receptors C5aR1 and C5aR2.

The complement system is central to the rapid immune response witnessed in vertebrates and invertebrates, which plays a crucial role in physiology and pathophysiology. Complement activation fuels the proteolytic cascade, which produces several complement fragments that interacts with a distinct set of complement receptors. Among all the complement fragments, C5a is one of the most potent anaphylatoxins, which exerts solid pro-inflammatory responses in a myriad of tissues by binding to the complement receptors such as C5aR1 (CD88, C5aR) and C5aR2 (GPR77, C5L2), which are part of the rhodopsin subfamily of G-protein coupled receptors. In terms of signaling cascade, recruitment of C5aR1 or C5aR2 by C5a triggers the association of either G-proteins or ß-arrestins, providing a protective response under normal physiological conditions and a destructive response under pathophysiological conditions. As a result, both deficiency and unregulated activation of the complement lead to clinical conditions that require therapeutic intervention. Indeed, complement therapeutics targeting either the complement fragments or the complement receptors are being actively pursued by both industry and academia. In this context, the model structural complex of C5a-C5aR1 interactions, followed by a biophysical evaluation of the model complex, has been elaborated on earlier. In addition, through the drug repurposing strategy, we have shown that small molecule drugs such as raloxifene and prednisone may act as neutraligands of C5a by effectively binding to C5a and altering its biologically active molecular conformation. Very recently, structural models illustrating the intermolecular interaction of C5a with C5aR2 have also been elaborated by our group. In the current study, we provide the biophysical validation of the C5a-C5aR2 model complex by recruiting major synthetic peptide fragments of C5aR2 against C5a. In addition, the ability of the selected neutraligands to hinder the interaction of C5a with the peptide fragments derived from both C5aR1 and C5aR2 has also been explored. Overall, the computational and experimental data provided in the current study supports the idea that small molecule drugs targeting C5a can potentially neutralize C5a's ability to interact effectively with its cognate complement receptors, which can be beneficial in modulating the destructive signaling response of C5a under pathological conditions.

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.

Intracellular complement activation-An alarm raising mechanism?

It has become increasingly apparent that the complement system, being an ancient defense mechanism, is not operative only in the extracellular milieu but also intracellularly. In addition to the known synthetic machinery in the liver and by macrophages, many other cell types, including lymphocytes, adipocytes and epithelial cells produce selected complement components. Activation of e.g. C3 and C5 inside cells may have multiple effects ranging from direct antimicrobial defense to cell differentiation and possible influence on metabolism. Intracellular activation of C3 and C5 in T cells is involved in the maintenance of immunological tolerance and promotes differentiation of T helper cells into Th1-type cells that activate cell-mediated immune responses. Adipocytes are unique in producing many complement sensor proteins (like C1q) and Factor D (adipsin), the key enzyme in promoting alternative pathway amplification. The effects of complement activation products are mediated by intracellular and cell membrane receptors, like C3aR, C5aR1, C5aR2 and the complement regulator MCP/CD46, often jointly with other receptors like the T cell receptor, Toll-like receptors and those of the inflammasomes. These recent observations link complement activation to cellular metabolic processes, intracellular defense reactions and to diverse adaptive immune responses. The complement components may thus be viewed as intracellular alarm molecules involved in the cellular danger response.

The critical role of C5a as an initiator of neutrophil-mediated autoimmune inflammation of the joint and skin.

The deposition of IgG autoantibodies in peripheral tissues and the subsequent activation of the complement system, which leads to the accumulation of the anaphylatoxin C5a in these tissues, is a common hallmark of diverse autoimmune diseases, including rheumatoid arthritis (RA) and pemphigoid diseases (PDs). C5a is a potent chemoattractant for granulocytes and mice deficient in its precursor C5 or its receptor C5aR1 are resistant to granulocyte recruitment and, consequently, to tissue inflammation in several models of autoimmune diseases. However, the mechanism whereby C5a/C5aR regulates granulocyte recruitment in these diseases has remained elusive. Mechanistic studies over the past five years into the role of C5a/C5aR1 in the K/BxN serum arthritis mouse model have provided novel insights into the mechanisms C5a/C5aR1 engages to initiate granulocyte recruitment into the joint. It is now established that the critical actions of C5a/C5aR1 do not proceed in the joint itself, but on the luminal endothelial surface of the joint vasculature, where C5a/C5aR1 mediate the arrest of neutrophils on the endothelium by activating ß2 integrin. Then, C5a/C5aR1 induces the release of leukotriene B4 (LTB4) from the arrested neutrophils. The latter, subsequently, initiates by autocrine/paracrine actions via its receptor BLT1 the egress of neutrophils from the blood vessel lumen into the interstitial. Compelling evidence suggests that this C5a/C5aR1-LTB4/BLT1 axis driving granulocyte recruitment in arthritis may represent a more generalizable biological principle critically regulating effector cell recruitment in other IgG autoantibody-induced diseases, such as in pemphigoid diseases. Thus, dual inhibition of C5a and LTB4, as implemented in nature by the lipocalin coversin in the soft-tick Ornithodoros moubata, may constitute a most effective therapeutic principle for the treatment of IgG autoantibody-driven diseases.

Integrating complement into the molecular pathogenesis of Hidradenitis Suppurativa.

Complement inhibition has been identified as a potential therapeutic target for multiple inflammatory disorders including Hidradenitis Suppurativa (HS). It is currently unclear how complement integrates into our current model of molecular pathogenesis in HS and whether it represents a central component of pathogenesis, or a neutrophil-associated bystander. Levels of C5a in serum and tissue correlate with disease activity and degree of neutrophilic infiltrates in HS. C5a has been associated with Th17 immune axis activation in psoriasis, rheumatoid arthritis and Crohn's disease with strong similarities to TH17 activation in HS. Porphyromonas species (which are identified in the HS microbiome) are able to cleave inactive C5 into C5a implicating the cutaneous microbiome as an activator of complement. C3a and C5a are associated with activation of the NLRP3 inflammasome, implicated in the inflammatory drive in HS. Complement receptors are present upon dendritic cells, monocytes, fibroblasts and adipocytes, which may broaden the potential contribution of complement to multiple aspects of HS pathogenesis. Dysregulation of complement receptor pathways has been documented in obesity, insulin resistance and polycystic ovarian syndrome leading to the possibility that complement may explain the epidemiological associations between these conditions and HS. The therapeutic potential of complement inhibitors in HS may be related to the therapeutic target (complement receptor or complement subunit) and the presence of alternate receptors (such as C5aR2) or ligands (including C3a, PAMPs and DAMPs). Integrating complement into the known pathogenesis of HS may aid in explaining the contradictory results between Phase 2 studies of C5a antagonists. It also allows for the identification of existing knowledge gaps to target further clinical investigation and research.

Distinct neutrophil C5a receptor inflammatory events in cows initiated by chemoattractant C5a and lipopolysaccharide around parturition and in mid lactation.

In neutrophils, toll-like receptor and complement component 5a (C5a) signaling are critical pathways regulating innate immunity. In cows, not much is known about the second C5a receptor, complement component 5a receptor 2 (C5AR2). It is an interesting player in sepsis treatment because it is considered to have an anti-inflammatory effect during normal inflammation. Periparturient cows are prone to severe infections, and the objectives of this study were to investigate the expression and functionality of C5AR2 during peripartum. We investigated the effect of 2 major inflammatory stimuli, C5a and lipopolysaccharide (LPS), on the expression of a selected number of genes (C5AR1, C5AR2, TLR4, ITGAM, COX2, and CXCL8) and functions linked to these receptors. Overall, TLR4, ITGAM, and C5AR2, all of which are involved in early inflammation, showed a lower expression in periparturient cows. However, an overall lower expression seems not to be the only explanation for the increased risk of sepsis in periparturient cows. Normally, in response to inflammation and as seen in the mid-lactation group, the expression of these genes increases after stimulation with LPS. However, in periparturient cows, stimulation with LPS led to a decrease in expression of these receptors, indicating a different response of neutrophils in response to LPS during this period. A decrease in ITGAM (coding for CD11b) expression complicates correct neutrophil localization and phagocytosis. Its downregulation upon stimulation might be detrimental for adequate eradication of the pathogen and might increase the risk of an imbalanced inflammation; C5AR2 seems to play a central role in this altered response. In addition, myeloperoxidase (MPO) activity in periparturient cows is lower in response to C5a stimulation. It has been suggested that MPO plays an important role in neutrophil shutdown and, thereby, timely resolution of inflammation. A decreased MPO activity might thus prolong the inflammatory reaction of the neutrophils. This finding was supported by the increased viability of the neutrophils obtained from periparturient cows. Even after stimulation, we found a lower caspase-3 activity in this group, indicating that they might be activated for a longer time compared with the neutrophils from mid-lactation cows. Accordingly, these alterations might contribute to a temporal mismatch in inflammatory responses, as often seen in severe periparturient infections.

A pathogenic role of complement in arterial hypertension and hypertensive end organ damage.

The self-amplifying cascade of messenger and effector molecules of the complement system serves as a powerful danger-sensing system that protects the host from a hostile microbial environment, while maintaining proper tissue and organ function through effective clearance of altered or dying cells. As an important effector arm of innate immunity, it also plays important roles in the regulation of adaptive immunity. Innate and adaptive immune responses have been identified as crucial players in the pathogenesis of arterial hypertension and hypertensive end organ damage. In line with this view, complement activation may drive the pathology of hypertension and hypertensive injury through its impact on innate and adaptive immune responses. It is well known that complement activation can cause tissue inflammation and injury and complement-inhibitory drugs are effective treatments for several inflammatory diseases. In addition to these proinflammatory properties, complement cleavage fragments of C3 and C5 can exert anti-inflammatory effects that dampen the inflammatory response to injury. Recent experimental data strongly support a role for complement in arterial hypertension. The remarkably similar clinical and histopathological features of malignant nephrosclerosis and atypical hemolytic uremic syndrome, which is driven by complement activation, suggest a role for complement also in the development of malignant nephrosclerosis. Herein, we will review canonical and noncanonical pathways of complement activation as the framework to understand the multiple roles of complement in arterial hypertension and hypertensive end organ damage.

Role of the Anaphylatoxin Receptor C5aR2 in Angiotensin II-Induced Hypertension and Hypertensive End-Organ Damage.

BACKROUND: Complement activation may facilitate hypertension through its effects on immune responses. The anaphylatoxin C5a, a major inflammatory effector, binds to the C5a receptors 1 and 2 (C5aR1, C5aR2). We have recently shown that C5aR1-/- mice have reduced hypertensive renal injury. The role of C5aR2 in hypertension is unknown. METHODS: For examination of C5aR2 expression on infiltrating and resident renal cells a tandem dye Tomato-C5aR2 knock-in reporter mouse was used. Human C5aR2 expression was analyzed in a single-cell RNAseq data set from the kidneys of hypertensive patients. Finally, we examined the effect of angiotensin II-induced hypertension in C5aR2-deficient mice. RESULTS: Flow cytometric analysis of leukocytes isolated from kidneys of the reporter mice showed that dendritic cells are the major C5aR2-expressing population (34%) followed by monocyte/macrophages (30%) and neutrophils (14%). Using confocal microscopy C5aR2 was not detected in resident renal or cardiac cells. In the human kidney, C5aR2 was also mainly found in monocytes, macrophages, and dendritic cells with a significantly higher expression in hypertension (P < 0.05). Unilateral nephrectomy was performed followed by infusion of Ang II (0.75 ng/g/min) and a high salt diet in wildtype (n = 18) and C5aR2-deficient mice (n = 14). Blood pressure, renal injury (albuminuria, glomerular filtration rate, glomerular and tubulointerstitial injury, inflammation), and cardiac injury (cardiac fibrosis, heart weight, gene expression) did not differ between hypertensive wildtype and C5aR2-/- mice. CONCLUSIONS: In summary, C5aR2 is mainly expressed in myeloid cells in the kidney in mice and humans but its deficiency has no effect on Ang II-induced hypertensive injury.

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

Pregnancy is a fascinating immunological phenomenon because it allows allogeneic fetal and placental tissues to survive inside the mother. As a component of innate immunity with high inflammatory potential, the complement system must be tightly regulated during pregnancy. Dysregulation of the complement system plays a role in pregnancy complications including pre-eclampsia and intrauterine growth restriction. Complement components are also used as biomarkers for pregnancy complications. However, the mechanisms of detrimental role of complement in pregnancy is poorly understood. C5a is the most potent anaphylatoxin and generates multiple immune reactions via two transmembrane receptors, C5aR1 and C5aR2. C5aR1 is pro-inflammatory, but the role of C5aR2 remains largely elusive. Interestingly, murine NK cells have been shown to express C5aR2 without the usual co-expression of C5aR1. Furthermore, C5aR2 appears to regulate IFN-γ production by NK cells in vitro. As IFN-γ produced by uterine NK cells is one of the major factors for the successful development of a vital pregnancy, we investigated the role anaphylatoxin C5a and its receptors in the establishment of pregnancy and the regulation of uterine NK cells by examinations of murine C5ar2-/- pregnancies and human placental samples. C5ar2-/- mice have significantly reduced numbers of implantation sites and a maternal C5aR2 deficiency results in increased IL-12, IL-18 and IFN-γ mRNA expression as well as reduced uNK cell infiltration at the maternal-fetal interface. Human decidual leukocytes have similar C5a receptor expression patterns showing clinical relevance. In conclusion, this study identifies C5aR2 as a key contributor to dNK infiltration and pregnancy success.

Conformational variants of the ternary complex of C5a, C5aR1, and G-protein.

The complement component fragment 5a (C5a) binds and activates two complement receptors like C5aR1 and C5aR2, which play a significant role in orchestrating the proinflammatory function of C5a in tissues through the recruitment of heterotrimeric G-proteins and ß-arrestins. Dysregulation of the complement induces excessive production of C5a, which triggers aberrant activation of the C5a-C5aR1-G-protein and C5a-C5aR2-ß-arrestin signalling axes in tissues, contributing to the pathology of numerous immune-inflammatory diseases. Thus, understanding the interaction of C5a with C5aR1 and C5aR2, as well as the interaction of G-protein and ß-arrestins, respectively, with C5a-C5aR1 and C5a-C5aR2, holds tremendous therapeutic value. In the absence of structural data, we have previously elaborated the binary complexes of C5a-C5aR1 and C5a-C5aR2, as well as the ternary complex of C5a-C5aR2-ß-arrestin1, in highly refined model structures. While our ternary model complex of C5a-C5aR1-G-protein was in progress, two cryo-electron microscopy-based ternary structural complexes of C5aR1 were made available by others. However, it is observed that the interaction of the crucial NT-peptide of C5aR1 with C5a, including the portion of the G⍺i-subunit that harbors the switch-I region, is not fully resolved in both complexes. The current study addresses the issues and provides two highly refined alternative model ternary complexes of C5a-C5aR1-G-protein. The study highlights the conformational heterogeneity in C5aR1 by comparing the two conformational variants of the model ternary complex in the context of C5a-C5aR2-ß-arrestin1 for further devising methods and molecules targeting both surface and intracellular C5aR1/C5aR2 for effectively mitigating the proinflammatory role of C5a in various disease settings.Communicated by Ramaswamy H. Sarma.

The anaphylatoxin C5a: Structure, function, signaling, physiology, disease, and therapeutics.

The complement system is one of the oldest known tightly regulated host defense systems evolved for efficiently functioning cell-based immune systems and antibodies. Essentially, the complement system acts as a pivot between the innate and adaptive arms of the immune system. The complement system collectively represents a cocktail of ∼50 cell-bound/soluble glycoproteins directly involved in controlling infection and inflammation. Activation of the complement cascade generates complement fragments like C3a, C4a, and C5a as anaphylatoxins. C5a is the most potent proinflammatory anaphylatoxin, which is involved in inflammatory signaling in a myriad of tissues. This review provides a comprehensive overview of human C5a in the context of its structure and signaling under several pathophysiological conditions, including the current and future therapeutic applications targeting C5a.

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.

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.

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.

An Integrated Analysis of C5AR2 Related to Malignant Properties and Immune Infiltration of Breast Cancer.

BACKGROUND: C5AR2 (GPR77, C5L2) is the second receptor for C5a that is a potent protein generated by complement activation. C5AR2 can mediate its own signaling events and exert significant immunomodulatory effects through those events. However, research of C5AR2 in cancer is limited, and its function remains unclear in breast cancer. METHODS: The expression of C5AR2 and its correlations with prognosis, immune infiltration, tumor mutation burden (TMB), and microsatellite instability (MSI) in more than thirty types of cancers were described through GTEx, TCGA, PrognoScan, TIMER2.0, CCLE, HPA, and TISIDB database. C5AR2 showed strong relationships to those immune marker sets in breast cancer. Otherwise, CCK8 assay and Transwell assay were conducted to illustrate the role of C5AR2 in migration, invasion, and proliferation of breast cancer cells. RESULTS: Generally, C5AR2 expression differed across most cancerous and noncancerous tissues, and high C5AR2 expression significantly related to poor prognosis in BRCA, GBM, KICH, LAML, LGG, LIHC, PAAD, and STAD. Moreover, C5AR2 expression levels were dramatically correlated with recognized immune infiltration, especially the polarization of macrophages in breast cancer. Gene set enrichment analysis confirmed that C5AR2 participates in regulating multiple signaling pathways involved in tumorigenesis as well as tumor immunity. C5AR2 overexpression facilitated the functions such as migration, invasion, and proliferation in breast cancer cells, which is consistent with bioinformatics analysis. CONCLUSIONS: C5AR2 is involved in immune infiltration and malignant characteristics of breast cancer, which may be a prospective biomarker for breast cancer.

Anaphylatoxin receptor promiscuity for commonly used complement C5a peptide agonists.

The complement system is an essential component of innate immunity. Its activation generates the effector cleavage proteins, anaphylatoxins C3a and C5a, that exert activity by interacting with three structurally related seven-transmembrane receptors. C3a activates C3aR, whilst C5a interacts with both C5aR1 and C5aR2 with equal potency. Of the three receptors, C5aR1 in particular is considered the most functionally potent inflammatory driver and has been the major target for pharmacological development. Multiple peptidic C5a agonists have been developed to target C5aR1, with the full agonists EP54 (YSFKPMPLaR) and EP67 (YSFKDMP(MeL)aR), and the partial agonist C028 (C5apep, NMe-FKPdChaChadR) being the most commonly utilised in research. Recent studies have indicated that small complement peptide ligands may lack selectivity amongst the three anaphylatoxin receptors, however this has not been uniformly confirmed for these commonly used C5a agonists. In the present study, we therefore characterised the pharmacological activity of EP54, EP67, and C5apep at human C5aR1, C5aR2 and C3aR, by conducting signalling assays in transfected cell lines, and in human primary macrophages. Our results revealed that none of the compounds tested were selective for human C5aR1. Both EP54 and EP67 were potent, full C3aR agonists, and EP54 and C5apep potently and partially activated human C5aR2. Therefore, we caution against the usage of these ligands, particularly EP54 and EP67, as C5a surrogates in C5a/ C5aR research.

The role of complement in arterial hypertension and hypertensive end organ damage.

Increasing evidence indicates that hypertension and hypertensive end organ damage are not only mediated by haemodynamic injury but that inflammation also plays an important role. The complement system protects the host from a hostile microbial environment and maintains tissue and cell integrity through the elimination of altered or dead cells. As an important effector arm of innate immunity, it plays also central roles in the regulation of adaptive immunity. Thus, complement activation may drive the pathology of hypertension through its effects on innate and adaptive immune responses, aside from direct effects on the vasculature. Recent experimental data strongly support a role for complement in all stages of arterial hypertension. The remarkably similar clinical and histopathological features of malignant nephrosclerosis and atypical haemolytic uraemic syndrome suggest also a role for complement in the development of malignant nephrosclerosis. Here, we review the role of complement in hypertension and hypertensive end organ damage. LINKED ARTICLES: This article is part of a themed issue on Canonical and non-canonical functions of the complement system in health and disease. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v178.14/issuetoc.

The "C3aR Antagonist" SB290157 is a Partial C5aR2 Agonist.

Innate immune complement activation generates the C3 and C5 protein cleavage products C3a and C5a, defined classically as anaphylatoxins. C3a activates C3aR, while C5a activates two receptors (C5aR1 and C5aR2) to exert their immunomodulatory activities. The non-peptide compound, SB290157, was originally reported in 2001 as the first C3aR antagonist. In 2005, the first report on the non-selective nature of SB290157 was published, where the compound exerted clear agonistic, not antagonistic, activity in variety of cells. Other studies also documented the non-selective activities of this drug in vivo. These findings severely hamper data interpretation regarding C3aR when using this compound. Unfortunately, given the dearth of C3aR inhibitors, SB290157 still remains widely used to explore C3aR biology (>70 publications to date). Given these issues, in the present study we aimed to further explore SB290157's pharmacological selectivity by screening the drug against three human anaphylatoxin receptors, C3aR, C5aR1 and C5aR2, using cell models. We identified that SB290157 exerts partial agonist activity at C5aR2 by mediating ß-arrestin recruitment at higher compound doses. This translated to a functional outcome in both human and mouse primary macrophages, where SB290157 significantly dampened C5a-induced ERK signaling. We also confirmed that SB290157 acts as a potent agonist at human C3aR in transfected cells, but as an antagonist in primary human macrophages. Our results therefore provide even more caution against using SB290157 as a research tool to explore C3aR function. Given the reported immunomodulatory and anti-inflammatory activities of C5aR2 agonism, any function observed with SB290157 could be due to these off-target activities.

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

The 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.