Properties of C5a Receptor on Human Macrophages

Abstract

Macrophages (MP) are important to host defence and inflammation but plasticity in theirgene expression profiles contributes to their functional heterogeneity. C5a is one of the most potentproinflammatory agents generated upon complement activation and binds to a specific receptorC5aR. Overproduction of C5a can contribute to numerous immune and inflammatory conditions.The objective of this thesis was to focus on human macrophage populations differentiated fromprimary human monocytes, and examine effects of C5a, C5aR agonists and antagonists, and TLR4-C5aR crosstalk on inflammatory profiles of human monocytes and macrophages.Chapter One summarizes roles for C5a activation on human macrophages, brieflysurveying C5a, C5aR and some important analogues of C5a that had been developed to study rolesfor C5a in vivo. This chapter discusses new insights into C5aR-related biological functions anddiseases, macrophage polarization and the usefulness of mouse models for human biology inrelation to how closely human and mouse macrophages reproduce functional responses.Chapter Two addresses macrophage polarization. Macrophage heterogeneity waspreviously achieved by differentiating monocytes with either GM-CSF or M-CSF (generating GMMPor M-MP), but was mostly studied on murine rather than human cells. This chapter describes acomparison of gene expression in populations of human macrophages (GM-MP versus M-MP),both in the basal state as well as in response to stimulation by LPS, using a combination of real-timePCR and cDNA microarray analysis, cellular migration, and functional responses. About 1000genes are differently regulated between unstimulated GM-MP versus M-MP. Although evidence ispresented in this chapter that human GM-MP and M-MP have distinct pro- and anti-inflammatoryresponses in human monocyte-derived macrophages, their activation by LPS supported more of acontinuum without clear functional distinctions between each population.Chapter Three investigates the effects of C5aR activation on GM-MP versus M-MP usingcDNA microarray. Previous reports only described gene expression in murine, rather than human,macrophages. Of ~40 genes substantially regulated by C5a in GM-MP and M-MP, 60% werecommon to both macrophage types. Furthermore, three different functional assays showed that C5awas equipotent on GM-MP and M-MP. These similarities for C5a-mediated functions could be dueto GM-MP and M-MP having similar C5aR transcriptional and translational expression. A separatemicroarray experiment was conducted to overview the temporal gene expression profile induced byC5a on M-MP, enabling identification of rate-limiting genes as therapeutic targets in C5a-mediatedinflammatory disorders.Chapter Four reports a comparative study of three known antagonists (3D53, W54011,JJ47) of C5a action via C5aR on M-MP. They were assessed for their relative advantages and disadvantages as antagonists of C5aR. Traditionally, drug discovery has focused on optimizingligand affinity for a specific receptor, enhancing functional potency, and improving drug-likeproperties for optimal pharmacokinetics and oral bioavailability. However, these optimizationprocesses do not always improve in vivo efficacy. This chapter highlights some importantconsiderations, often neglected in drug development n namely residence time, insurmountable ornon-competitive antagonism for drug-receptor interactions. The non-competitive antagonist 3D53showed superior antagonist activity compared to competitive antagonists W54011 and JJ47 despiteinferior oral bioavailability, and this was because 3D53 remained bound to C5aR on macrophagesfor over 16h whereas compounds W54011 and JJ47 were no longer bound to C5aR after 2h. As aresult, 3D53 retained high potency (IC50 20nM) in the face of competition from increasingconcentrations of C5a (0.1-300nM), whereas W54011 and JJ47 were ineffective antagonists againstconcentrations of C5a above 30nM. The benefits of longer residence time were also demonstratedin vivo in rats. Orally administered 3D53 was an effective anti-inflammatory agent for 16h,compared to less than 2h for W54011 and JJ47, in inhibiting C5aR-mediated paw oedema in maleWistar rats.Chapter Five reports crosstalk between TLR4 and C5aR in human macrophages. C5aR isshown to differentially modulate LPS-induced inflammatory responses in primary humanmonocytes versus macrophages. While C5a enhanced secretion of LPS-induced IL6 and TNF fromhuman monocytes, C5a inhibited these responses in GM-MP and M-MP. LPS amplified C5ainducedGai/c-Raf/MEK/ERK signalling in macrophages but not in monocytes. This synergy wasindependent of IL10, PI3K, p38, JNK, GM-CSF and M-CSF. C5a-mediated suppression of IL6 andTNF did not compromise but instead enhanced the clearance of Salmonella Typhimurium frommacrophages. These findings implicated C5aR as a regulatory switch that modulates TLR4signalling via the Gai/c-Raf/MEK/ERK signalling axis in human macrophages but not in humanmonocytes.Chapter Six summarises all key findings in chapters 2-5, which represent important newknowledge in the field of complement C5a-C5aR and their effects on primary human monocytesand macrophages. Advances made in this thesis are specifically highlighted, discussed in relation totheir novelty and significance and differences from the literature, and possible future researchdirections that build on the findings in this thesis are also outlined in relation to the fields ofimmunology and pharmacology