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Complement-mediated microglial priming: an in vitro study

Wheat, Richard 2016. Complement-mediated microglial priming: an in vitro study. PhD Thesis, Cardiff University.
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Abstract

The concept of microglial priming has developed through in vivo studies and is operationally defined as an exaggerated microglial production of soluble mediators (NO and cytokines e.g. IL-1β, TNF-α, IL-6) following a pro-inflammatory activation event (e.g. LPS-treatment). In practice microglial priming predisposes the brain to degeneration through the promotion of inflammatory mechanisms. In vivo studies of Crry (a major murine cell-surface C3-regulator) KO mice previously identified a novel role for C in the induction of the primed microglial phenotype, implicating iC3b ligation of microglial CR3. The purpose of this study was to further investigate C-dependent microglial priming and its mechanism(s) through study of microglia in isolation in vitro. Experiments using purified fluid-phase human iC3b failed to demonstrate any phenotypic effects of ligand exposure. Given the results of previous investigations concerning CR3 ligands, combined with the results of binding studies and sequence comparisons, it appears likely that, while still able to engage the cell-borne CR3, fluid-phase iC3b is incapable of exerting significant effects on the microglial phenotype. Studies using Zymsoan and C-fixing mAb-sensitised TC plastic as a means to generate ligands to investigate the consequences of microglial CR3 engagement by iC3b were confounded by the stimulatory effects of the C-activating agents (i.e. zymsoan or mAb) which prevented attempts to dissect the effects of the isolated interaction. Nonetheless, specific effects were attributable to the C3-derived CR3 ligands generated, which dramatically and significantly reduced the pro-inflammatory responses evoked by the C-activating agents. Investigations using C3-activation fragments immobilised on native (i.e. non-sensitised) TC plastic demonstrated phenotypic effects of microglial iC3b-CR3 ligation consistent with the previously reported mechanism of C-dependent microglial priming. Experiments using cultured Crry KO microglia demonstrated increased sensitivity to autologous C activation. Phenotyping experiments, however, failed to show any consequence of Crry expression status, even when the intrinsic sensitivity of Crry KO cells to C3 activation and deposition was effected, thus mimicking the in vivo scenario (including the potential for iC3b ligation of CR3). Data gathered from the several systems designed to ligate CR3 of microglial cells with C3-derived ligands highlight the broad range of potential cellular responses mediated by CR3 and emphasise the importance of context for the consequence of this interaction. In so doing, these data also further evidence that under certain circumstances, iC3b-CR3 binding can induce a primed microglial phenotype.

Item Type: Thesis (PhD)
Status: Unpublished
Schools: Medicine
Subjects: Q Science > QR Microbiology > QR180 Immunology
Date of First Compliant Deposit: 4 April 2017
Last Modified: 05 Jun 2017 05:53
URI: http://orca-mwe.cf.ac.uk/id/eprint/99615

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