Properties of C5a Receptor on Human Macrophages

Seow, Vernon (2014). Properties of C5a Receptor on Human Macrophages PhD Thesis, Institute for Molecular Bioscience, The University of Queensland. doi:10.14264/uql.2017.437

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Author Seow, Vernon
Thesis Title Properties of C5a Receptor on Human Macrophages
School, Centre or Institute Institute for Molecular Bioscience
Institution The University of Queensland
DOI 10.14264/uql.2017.437
Publication date 2014-01-01
Thesis type PhD Thesis
Supervisor David Fairlie
Jacky Suen
Total pages 302
Language eng
Subjects 1108 Medical Microbiology
1107 Immunology
Formatted 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 GMMΦ
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
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 C5ainduced
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

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
Keyword GPCR
C5a receptor
Human macrophages
Human monocytes
C5a receptor agonist
C5a receptor antagonist

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Created: Mon, 02 Jun 2014, 22:29:40 EST by Mr Zi-xing Seow on behalf of Learning and Research Services (UQ Library)