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An investigation into the molecular mechanisms of complement terminal pathway induced inflammation.

Walters, David John 2018. An investigation into the molecular mechanisms of complement terminal pathway induced inflammation. PhD Thesis, Cardiff University.
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Abstract

The complement system is a bastion of the innate immune system which mediates opsonic, inflammatory and lytic responses to pathogenic activating stimuli, culminating in the formation of the membrane attack complex (MAC) (Sarma and Ward 2011). MAC deposition on non-nucleated or bacterial cell membranes results in chemiosmotic flux; subsequently inducing cell swelling and rupture (Morgan 2000). However, sublytic MAC formation may occur on nucleated self-cells, inducing the release of inflammatory cytokines, including IL-1β (Tegla et al 2011; Ricklin, Reis and Lambris 2016). IL-1β proteolytic maturation and secretion requires activation of inflammasomes, with the NLRP3 inflammasome being demonstrated as the central mediator of MAC induced IL-1β secretion in a range of cell types (Triantafilou et al 2013; Laudisi et al 2013; Suresh et al 2016). However, the cellular mechanisms linking MAC deposition and NLRP3 activation are poorly defined, with a range of activation mechanisms proposed. In the work described in this thesis I investigated the mechanisms linking sublytic MAC to NLRP3 activation in monocytic and synovial cell lines to delineate conserved cell signalling pathways involved in NLRP3 activation, as well as investigating the broader interactions of NLRP3 with the complement system. Firstly, classical and reactive lysis systems of complement activation were established and validated to generate sublytic membrane attack on cells for study of the interaction with NLRP3. Within these experiments, interesting observations surrounding the role of CD59 in the regulation of C3b, as well as the role of C5 depletion or inhibition on C3b deposition were made. LPS primed THP-1 monocytes were used as a model for NLRP3 activation by sublytic MAC. In concordance with the published literature and studies by our group, both primary and secondary Ca2+flux and ROS production had roles in MAC mediated IL-1β secretion (Triantafilou et al 2013). However, extracellular KCl was demonstrated to be a non-specific inhibitor of IL-1β release as the addition of NaCl to the stimulation media demonstrated inhibition of NLRP3 activation in the same manner as KCl in this system. To further explore pharmacological manipulation of sublytic MAC mediated activation, inhibitors of a range of known MAC activated kinases were used to identify signalling pathways linking sublytic MAC and NLRP3 activation. Only an AKT inhibitor, Perifosine, xvii induced statistically significant changes in MAC mediated IL-1β release; however, this was shown to be through the activation of cell death and stress pathways such as JNK rather than attributable to its effects on AKT signalling. Subsequent simultaneous inhibition of JNK and ERK signalling significantly inhibited both IL-1β secretion and Caspase-1 activation in response to sublytic MAC. MAC-induced activation of NLRP3 and the induction of IL-1β release was also subsequently demonstrated using purified components in the reactive lysis system; cells exposed to sublytic MAC via reactive lysis were subjected to RNA-Seq analysis, providing preliminary evidence of transcriptomic changes with implications for inflammatory cell signalling in response to MAC. Finally, NLRP3 expression was knocked out using the CRISPR/CAS9 system in SW 982 synoviocytes, to investigate interactions between NLRP3 expression and MAC mediated responses. NLRP3 -/- cells were protected from MAC mediated cell death, a consequence of increased CD59 expression on the cell surface. Collectively, this thesis improves understanding of the mechanisms linking complement and NLR inflammasomes, highlighting novel cell signalling pathways linking the two systems and providing evidence that NLRP3 modulates MAC sensitivity by regulating CD59 localisation within the cell.

Item Type: Thesis (PhD)
Date Type: Completion
Status: Unpublished
Schools: Medicine
Date of First Compliant Deposit: 22 May 2019
Last Modified: 22 May 2019 13:51
URI: http://orca-mwe.cf.ac.uk/id/eprint/122784

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