Complement in stem cells and development.

From its discovery in the late nineteenth century, as a 'complement' to the cellular immune response, the complement system has been widely affirmed as a powerful controller of innate and adaptive immune responses. In recent decades however, new roles for complement have been discovered, with multiple complement proteins now known to function in a broad array of non-immune systems. This includes during development, where complement exerts control over stem cell populations from fertilization and implantation throughout embryogenesis and beyond post-natal development. It is involved in processes as diverse as cell localisation, tissue morphogenesis, and the growth and refinement of the brain. Such physiological actions of complement have also been described in adult stem cell populations, with roles in proliferation, differentiation, survival, and regeneration. With such a broad range of complement functions now described, it is likely that current research only describes a fraction of the full reach of complement proteins. Here, we review how complement control of physiological cell processes has been harnessed in stem cell populations throughout both development and in adult physiology.

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.

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.

Complement: The Emerging Architect of the Developing Brain.

Complement activation products have long been associated with roles in the innate immune system, linking the humoral and cellular responses. However, among their recently described non-inflammatory roles, complement proteins also have multiple emerging novel functions in brain development. Within this context, separate proteins and pathways of complement have carved out physiological niches in the formation, development, and refinement of neurons. They demonstrate actions that are both reminiscent of peripheral immune actions and removed from them. We review here three key roles for complement proteins in the developing brain: progenitor proliferation, neuronal migration, and synaptic pruning.

Complement C3a receptor modulates embryonic neural progenitor cell proliferation and cognitive performance.

The complement system of innate immunity is emerging as a novel player in neurodevelopmental processes. The receptor for C3a, C3aR, shares a close evolutionary and functional relationship with C5a receptors. Whilst the C5a receptor, C5aR1, has been demonstrated to promote embryonic neural stem cell proliferation, little is known about the role of C3aR in this process. Here we show that C3aR is expressed in a similar manner to C5aR1 in mice, at the apical pole of the embryonic ventricular zone, though it has an opposing function. Using in utero delivery of C3aR agonist and antagonist compounds to the embryonic ventricle, we demonstrate that C3aR functions to decrease proliferation of apical neural progenitor cells (NPC). Intriguingly, C3aR-/- animals also have altered NPC proliferation, but demonstrate an opposing phenotype to animals subjected to pharmacological blockade of C3aR. Finally, despite a grossly normal development of C3aR-/- animals, cognitive behavioural testing of adult mice showed subtle deficits in recall memory. These data demonstrate that in addition to C5a, C3a also has a critical role in the normal development of the mammalian brain.

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.

De novo peptide design with C3a receptor agonist and antagonist activities: theoretical predictions and experimental validation.

Targeting the complement component 3a receptor (C3aR) with selective agonists or antagonists is believed to be a viable therapeutic option for several diseases such as stroke, heart attack, reperfusion injuries, and rheumatoid arthritis. We designed a number of agonists, partial agonists, and antagonists of C3aR using our two-stage de novo protein design framework. Of the peptides tested using a degranulation assay in C3aR-transfected rat basophilic leukemia cells, two were prominent agonists (EC(50) values of 25.3 and 66.2 nM) and two others were partial agonists (IC(50) values of 15.4 and 26.1 nM). Further testing of these lead compounds in a calcium flux assay in U937 cells yielded similar results although with reduced potencies compared to transfected cells. The partial agonists also displayed full antagonist activity when tested in a C3aR inhibition assay. In addition, the electrostatic potential profile was shown to potentially discriminate between full agonists and partial agonists.