Liam G. Coulthard

C5L2: a controversial receptor of complement anaphylatoxin, C5a

C5a is the paramount proinflammatory mediator of the complement cascade, and has been previously thought to act only through a single, G‐protein‐coupled, C5a receptor (C5aR; also termed CD88). In 2000, a second C5a receptor, C5L2 (previously known as GPR77), was discovered; yet, despite 12 yr of intensive research, its biological, or pathophysiological, function is both enigmatic and controversial. Unlike C5aR, this receptor does not couple to G proteins, and early studies promoted the hypothesis that C5L2 functions as a decoy receptor. However, recent data have provided other evidence for more complicated and conflicting interactions between C5L2 and other inflammatory mediators. C5L2 has been recently demonstrated to physically interact with both C5aR and β‐arrestin to negatively regulate C5aR signaling toward an anti‐inflammatory manner, and to reduce pathology, in several disease models in vivo. In direct contrast, other groups have demonstrated that C5L2 stimulation caused release of HMGB1 both in vitro and in vivo, and enhanced pathology in sepsis models, suggesting a clear proinflammatory signaling role. These astoundingly contradictory data challenge our precepts and complicate the foundational bases for the possible targeting of C5L2 as a therapeutic option in inflammatory disease. C5L2 may be the great masquerader in complement biology; its function dependent on the cell type, species, and disease context. Because of these unusual and unforeseen complexities, we present the current state of knowledge on C5L2 structure, expression and, most controversially, its putative functions.—Li, R., Coulthard, L.G., Wu, M. C. L., Taylor, S. M., Woodruff, T. M. C5L2: a controversial receptor of complement anaphylatoxin, C5a. FASEB J. 27, 855–864 (2013). www.fasebj.org

Is the Complement Activation Product C3a a Proinflammatory Molecule? Re-evaluating the Evidence and the Myth

The complement activation product C3a is often described as a proinflammatory mediator, alongside its downstream cousin, C5a. However, emerging studies show that C3a has several anti-inflammatory facets in vivo. For example, in the acute inflammatory response, C3a acts in direct opposition to C5a, through preventing the accumulation of neutrophils in inflamed tissues by independently regulating their mobilization. This acute, protective, and opposing activity of C3a to C5a is also illustrated in models of septicemia. In this article, we reinvestigate the discovery and original classification of C3a as a proinflammatory mediator and highlight the emerging studies demonstrating anti-inflammatory effects for C3a in the immune response. It is our hope that this review illuminates these apparently contradictory roles for C3a and challenges the general dogma surrounding C3a, which, historically, has ubiquitously been described as a proinflammatory mediator. In light of this, we urge investigators to use “inflammatory modulator” as the descriptor for C3a.

Elevated complement factor C5a in maternal and umbilical cord plasma in preeclampsia

Preeclampsia is a leading cause of morbidity and mortality worldwide, encompassing significant short- and long-term health sequelae. Recently, there has been accumulating evidence for a role of the complement system in the pathogenesis of numerous complications of pregnancy, including preeclampsia. The present cross-sectional study compared the plasma concentrations of complement factors C3a and C5a between normotensive pregnancies and pregnancies complicated with either preeclampsia or gestational hypertension alone. We found that maternal plasma C5a concentration was significantly higher in preeclamptic pregnancy than in pregnancy affected by gestational hypertension alone or normotensive pregnancy. Umbilical cord plasma C5a concentrations were also higher in pregnancies complicated by preeclampsia compared to gestational hypertension or normotensive pregnancy. Maternal and cord plasma C5a concentrations were significantly correlated, suggesting that C5a can freely diffuse between maternal and fetal circulation. There were no significant differences in C3a concentrations in maternal or cord plasma between any groups. These results support the hypothesis that C5a may play a role in preeclampsia, but not in gestational hypertension.

C5a Receptor Signaling Prevents Folate Deficiency–Induced Neural Tube Defects in Mice

The complement system is involved in a range of diverse developmental processes, including cell survival, growth, differentiation, and regeneration. However, little is known about the role of complement in embryogenesis. In this study, we demonstrate a novel role for the canonical complement 5a receptor (C5aR) in the development of the mammalian neural tube under conditions of maternal dietary folic acid deficiency. Specifically, we found C5aR and C5 to be expressed throughout the period of neurulation in wild-type mice and localized the expression to the cephalic regions of the developing neural tube. C5aR was also found to be expressed in the neuroepithelium of early human embryos. Ablation of the C5ar1 gene or the administration of a specific C5aR peptide antagonist to folic acid–deficient pregnant mice resulted in a high prevalence of severe anterior neural tube defect-associated congenital malformations. These findings provide a new and compelling insight into the role of the complement system during mammalian embryonic development.

Brief report: complement C5a promotes human embryonic stem cell pluripotency in the absence of FGF2.

The complement activation product, C5a, is a pivotal member of the innate immune response; however, a diverse number of nonimmune functions are now being ascribed to C5a signaling, including roles during embryonic development. Here, we identify the expression of the C5a precursor protein, C5, as well as the C5a receptors, C5aR and C5L2, in both human embryonic stem cells and human‐induced pluripotent stem cells. We show that administration of a physiologically relevant dose of purified human C5a (1 nM) stimulates activation of ERK1/2 and AKT signaling pathways, and is able to promote maintenance of the pluripotent state in the absence of FGF2. C5a also reduced cell loss following dissociation of human pluripotent stem cells. Our results reveal that complement C5a signaling supports human stem cell pluripotency and survival, and thus may play a key role in shaping early human embryonic development. Stem Cells 2014;32:3278—3284

Complement in the fundamental processes of the cell.

Once regarded solely as an activator of innate immunity, it is now clear that the complement system acts in an assortment of cells and tissues, with immunity only one facet of a diverse array of functions under the influence of the complement proteins. Throughout development, complement activity has now been demonstrated from early sperm-egg interactions in fertilisation, to regulation of epiboly and organogenesis, and later in refinement of cerebral synapses. Complement has also been shown to regulate homeostasis of adult tissues, controlling cell processes such as migration, survival, repair, and regeneration. Given the continuing emergence of such novel actions of complement, the existing research likely represents only a fraction of the myriad of functions of this complex family of proteins. This review is focussed on outlining the current knowledge of complement family members in the regulation of cell processes in non-immune systems. It is hoped this will spur research directed towards revealing more about the role of complement in these fundamental cell processes.

New concepts on the therapeutic control of complement anaphylatoxin receptors

The complement system is a pivotal driver of innate immunity, coordinating the host response to protect against pathogens. At the heart of the complement response lie the active fragments, C3a and C5a, acting through their specific receptors, C3aR, C5aR1, and C5aR2, to direct the cellular response to inflammation. Their potent function however, places them at risk of damaging the host, with aberrant C3a and C5a signaling activity linked to a wide range of disorders of inflammatory, autoimmune, and neurodegenerative etiologies. As such, the therapeutic control of these receptors represents an attractive drug target, though, the realization of this clinical potential remains limited. With the success of eculizumab, and the progression of a number of novel C5a-C5aR1 targeted drugs to phase II and III clinical trials, there is great promise for complement therapeutics in future clinical practice. In contrast, the toolbox of drugs available to modulate C3aR and C5aR2 signaling remains limited, however, the emergence of new selective ligands and molecular tools, and an increased understanding of the function of these receptors in disease, has highlighted their unique potential for clinical applications. This review provides an update on the growing arsenal of drugs now available to target C5, and C5a and C3a receptor signaling, and discusses their utility in both clinical and pre-clinical development.

Complement C5aR1 Signaling Promotes Polarization and Proliferation of Embryonic Neural Progenitor Cells through PKCζ.

The complement system, typically associated with innate immunity, is emerging as a key controller of nonimmune systems including in development, with recent studies linking complement mutations with neurodevelopmental disease. A key effector of the complement response is the activation fragment C5a, which, through its receptor C5aR1, is a potent driver of inflammation. Surprisingly, C5aR1 is also expressed during early mammalian embryogenesis; however, no clearly defined function is ascribed to C5aR1 in development. Here we demonstrate polarized expression of C5aR1 on the apical surface of mouse embryonic neural progenitor cells in vivo and on human embryonic stem cell-derived neural progenitors. We also show that signaling of endogenous C5a during mouse embryogenesis drives proliferation of neural progenitor cells within the ventricular zone and is required for normal brain histogenesis. C5aR1 signaling in neural progenitors was dependent on atypical protein kinase C ζ, a mediator of stem cell polarity, with C5aR1 inhibition reducing proliferation and symmetric division of apical neural progenitors in human and mouse models. C5aR1 signaling was shown to promote the maintenance of cell polarity, with exogenous C5a increasing the retention of polarized rosette architecture in human neural progenitors after physical or chemical disruption. Transient inhibition of C5aR1 during neurogenesis in developing mice led to behavioral abnormalities in both sexes and MRI-detected brain microstructural alterations, in studied males, demonstrating a requirement of C5aR1 signaling for appropriate brain development. This study thus identifies a functional role for C5a–C5aR1 signaling in mammalian neurogenesis and provides mechanistic insight into recently identified complement gene mutations and brain disorders. SIGNIFICANCE STATEMENT The complement system, traditionally known as a controller of innate immunity, now stands as a multifaceted signaling family with a broad range of physiological actions. These include roles in the brain, where complement activation is associated with diseases, including epilepsy and schizophrenia. This study has explored complement regulation of neurogenesis, identifying a novel relationship between the complement activation peptide C5a and the neural progenitor proliferation underpinning formation of the mammalian brain. C5a was identified as a regulator of cell polarity, with inhibition of C5a receptors during embryogenesis leading to abnormal brain development and behavioral deficits. This work demonstrates mechanisms through which dysregulation of complement causes developmental disease and highlights the potential risk of complement inhibition for therapeutic purposes in pregnancy.

Comparative efficacy of a secretory phospholipase A2 inhibitor with conventional anti-inflammatory agents in a rat model of antigen-induced arthritis

IntroductionPreviously, secretory phospholipase A2 (sPLA2) inhibition has been used as an adjunct to conventional rheumatoid arthritis therapy in human clinical trials without significant improvement of arthritic pathology. In this study, we compared the efficacy of a potent and orally active group IIa secretory phospholipase A2 inhibitor (sPLA2I) to conventional anti-arthritic agents; infliximab, leflunomide and prednisolone, in a rat model of antigen-induced arthritis.MethodsInitially, to establish efficacy and dose-response, rats were orally dosed with the sPLA2I (1 and 5 mg/kg) two days prior to arthritis induction, and then daily throughout the 14-day study period. In the second trial, rats were orally dosed with the sPLA2I (5 and 10 mg/kg/day) beginning two days after the induction of arthritis, at the peak of joint swelling. Separate groups of rats were also dosed with the tumour necrosis factor-alpha (TNF-α) inhibitor infliximab (single 3 mg/kg i.v. injection), leflunomide (10 mg/kg/day, oral) or prednisolone (1 mg/kg/day, oral) at this same time point and used as comparative treatments.ResultsIn the pathology prevention trial, both 1 and 5 mg/kg dose groups of sPLA2I demonstrated a significant reduction in joint swelling and gait disturbances; however, only the higher 5 mg/kg dose resulted in significantly reduced histopathology scores. In the post-induction trial, rats dosed with sPLA2I showed a significant improvement in joint swelling and gait scoring, whereas none of the conventional therapeutics achieved a significant decrease in both of these two disease markers. Histopathological scoring at the end-point of the study demonstrated significantly reduced median scores in rats treated with 10 mg/kg sPLA2I and leflunomide.ConclusionsThe results from this study suggest a pathogenic role for sPLA2 enzymes in this model of arthritis in rats, and the potential clinical utility of sPLA2 inhibition as a safer, and more effective, alternative to conventional anti-arthritic therapeutics.

Co-ordinated expression of innate immune molecules during mouse neurulation.

The innate immune system is the first line of defence against pathogens and infection. Recently, it has become apparent that many innate immune factors have roles outside of immunity and there is growing evidence that these factors play important functional roles during the development of a range of model organisms. Several studies have documented developmental expression of individual factors of the toll-like receptor and complement systems, and we recently demonstrated a key role for complement C5a receptor (C5aR1) signalling in neural tube closure in mice. Despite these emerging studies, a comprehensive expression analysis of these molecules in embryonic development is lacking. In the current study, we therefore, examined the expression of key innate immune factors in the early development period of neurulation (7.5-10.5dpc) in mice. We found that complement factor genes were differentially expressed during this period of murine development. Interestingly, the expression patterns we identified preclude activation of the classical and alternative pathways and formation of the membrane attack complex. Additionally, several other classes of innate immune molecules were expressed during the period of neurulation, including toll-like receptors (TLR-2, -3, -4 and -9), receptor for advanced glycation end-products (RAGE), and their signalling adapters (TRAF-4, TRAF-6, TAK-1 and MyD88). Taken together, this study highlights a number of innate immune factors as potential novel players in early embryonic development.