Properties of the C5a Receptor on Human Macrophages Vernon Seow Bachelor of Science (Honours Class 1, Microbiology) A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2014 Institute for Molecular Bioscience I Abstract Macrophages (MΦ) are important to host defence and inflammation but plasticity in their gene expression profiles contributes to their functional heterogeneity. C5a is one of the most potent proinflammatory agents generated upon complement activation and binds to a specific receptor C5aR. Overproduction of C5a can contribute to numerous immune and inflammatory conditions. The objective of this thesis was to focus on human macrophage populations differentiated from primary 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, briefly surveying C5a, C5aR and some important analogues of C5a that had been developed to study roles for C5a in vivo. This chapter discusses new insights into C5aR-related biological functions and diseases, macrophage polarization and the usefulness of mouse models for human biology in relation to how closely human and mouse macrophages reproduce functional responses. Chapter Two addresses macrophage polarization. Macrophage heterogeneity was previously achieved by differentiating monocytes with either GM-CSF or M-CSF (generating GM- MΦ or M-MΦ), but was mostly studied on murine rather than human cells. This chapter describes a comparison of gene expression in populations of human macrophages (GM-MΦ versus M-MΦ), both in the basal state as well as in response to stimulation by LPS, using a combination of real-time PCR and cDNA microarray analysis, cellular migration, and functional responses. About 1000 genes are differently regulated between unstimulated GM-MΦ versus M-MΦ. Although evidence is presented in this chapter that human GM-MΦ and M-MΦ have distinct pro- and anti-inflammatory responses in human monocyte-derived macrophages, their activation by LPS supported more of a continuum without clear functional distinctions between each population. Chapter Three investigates the effects of C5aR activation on GM-MΦ versus M-MΦ using cDNA microarray. Previous reports only described gene expression in murine, rather than human, macrophages. Of ~40 genes substantially regulated by C5a in GM-MΦ and M-MΦ, 60% were common to both macrophage types. Furthermore, three different functional assays showed that C5a was equipotent on GM-MΦ and M-MΦ. These similarities for C5a-mediated functions could be due to GM-MΦ and M-MΦ having similar C5aR transcriptional and translational expression. A separate microarray experiment was conducted to overview the temporal gene expression profile induced by C5a on M-MΦ, enabling identification of rate-limiting genes as therapeutic targets in C5a-mediated inflammatory disorders. Chapter Four reports a comparative study of three known antagonists (3D53, W54011, JJ47) of C5a action via C5aR on M-MΦ. They were assessed for their relative advantages and II disadvantages as antagonists of C5aR. Traditionally, drug discovery has focused on optimizing ligand affinity for a specific receptor, enhancing functional potency, and improving drug-like properties for optimal pharmacokinetics and oral bioavailability. However, these optimization processes do not always improve in vivo efficacy. This chapter highlights some important considerations, often neglected in drug development – namely residence time, insurmountable or non-competitive antagonism for drug-receptor interactions. The non-competitive antagonist 3D53 showed superior antagonist activity compared to competitive antagonists W54011 and JJ47 despite inferior oral bioavailability, and this was because 3D53 remained bound to C5aR on macrophages for over 16h whereas compounds W54011 and JJ47 were no longer bound to C5aR after 2h. As a result, 3D53 retained high potency (IC50 20nM) in the face of competition from increasing concentrations of C5a (0.1-300nM), whereas W54011 and JJ47 were ineffective antagonists against concentrations of C5a above 30nM. The benefits of longer residence time were also demonstrated in 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 male Wistar rats. Chapter Five reports crosstalk between TLR4 and C5aR in human macrophages. C5aR is shown to differentially modulate LPS-induced inflammatory responses in primary human monocytes versus macrophages. While C5a enhanced secretion of LPS-induced IL6 and TNF from human monocytes, C5a inhibited these responses in GM-MΦ and M-MΦ. LPS amplified C5a- induced Gαi/c-Raf/MEK/ERK signalling in macrophages but not in monocytes. This synergy was independent of IL10, PI3K, p38, JNK, GM-CSF and M-CSF. C5a-mediated suppression of IL6 and TNF did not compromise but instead enhanced the clearance of Salmonella Typhimurium from macrophages. These findings implicated C5aR as a regulatory switch that modulates TLR4 signalling via the Gαi/c-Raf/MEK/ERK signalling axis in human macrophages but not in human monocytes. Chapter Six summarises all key findings in chapters 2-5, which represent important new knowledge in the field of complement C5a-C5aR and their effects on primary human monocytes and macrophages. Advances made in this thesis are specifically highlighted, discussed in relation to their novelty and significance and differences from the literature, and possible future research directions that build on the findings in this thesis are also outlined in relation to the fields of immunology and pharmacology. III Declaration by author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the General Award Rules of The University of Queensland, immediately made available for research and study in accordance with the Copyright Act 1968. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis. IV Publications during candidature Peer-reviewed papers J. Lim, A. Iyer, L. Liu, J. Y. Suen, R. J. Lohman, V. Seow, M. K. Yau, L. Brown, and D. P. Fairlie. 2013. Diet-induced obesity, adipose inflammation, and metabolic dysfunction correlating with PAR2 expression are attenuated by PAR2 antagonism. FASEB J. 27: 4757-4767. D. M. Hohenhaus, K. Schaale, K. A. Le Cao, V. Seow, A. Iyer, D. P. Fairlie, and M. J. Sweet. 2013. An mRNA atlas of G protein-coupled receptor expression during primary human monocyte/macrophage differentiation and lipopolysaccharide-mediated activation identifies targetable candidate regulators of inflammation. Immunobiology 218: 1345-1353. V. Seow, J. Lim, A. Iyer, J. Y. Suen, J. K. Ariffin, D. M. Hohenhaus, M. J. Sweet, and D. P. Fairlie. 2013. Inflammatory responses induced by lipopolysaccharide are amplified in primary human monocytes but suppressed in macrophages by complement protein c5a. J. Immunol. 191: 4308- 4316. Vesey, D. A., J. Y. Suen, V. Seow, R. J. Lohman, L. Liu, G. C. Gobe, D. W. Johnson, and D. P. Fairlie. 2013. PAR2-induced inflammatory responses in human kidney tubular epithelial cells. Am. J. Physiol. Renal Physiol. 304: F737-750. Lim, J., A. Iyer, J. Y. Suen, V. Seow, R. C. Reid, L. Brown, and D. P. Fairlie. 2013. C5aR and C3aR antagonists each inhibit diet-induced obesity, metabolic dysfunction, and adipocyte and macrophage signaling. FASEB J. 27: 822-831. V Publications included in this thesis Publication citation – incorporated as Chapter 5. V. Seow, J. Lim, A. Iyer, J. Y. Suen, J. K. Ariffin, D. M. Hohenhaus, M. J. Sweet, and D. P. Fairlie. 2013. Inflammatory responses induced by lipopolysaccharide are amplified in primary human monocytes but suppressed in macrophages by complement protein c5a. J. Immunol. 191: 4308- 4316. Contributor Statement of contribution Vernon Seow (Candidate) Designed experiments (80%) Wrote the paper (80%) Drs. Junxian Lim, Abishek Iyer, Jacky Y. Suen Designed experiments (15%) Wrote and edited paper (20%) Daniel M. Hohenhaus, Juliana K. Ariffin Designed experiments (5%) Drs. David P. Fairlie and Matthew J. Sweet Reviewed paper (100%) Publication citation – incorporated as Chapter 6 Lim, J., A. Iyer, J. Y. Suen, V. Seow, R. C. Reid, L. Brown, and D. P. Fairlie. 2013. C5aR and C3aR