Inflammation is a highly conserved process utilised by an organism to protect itself against damage or infection. However, inflammation can also be extremely damaging and is implicated in many diseases. Indeed, targeting inflammation can be of great therapeutic benefit. Inflammation is initiated and propagated by pro-inflammatory cytokines such as interleukin-1 (IL-1). The two major pro-inflammatory members of the IL-1 family are IL-1a and IL-1b, both of which signal through the IL-1 type 1 receptor following secretion via unconventional mechanisms. IL-1 signalling is stringently regulated. IL-1b, which is relatively well researched, is translated as an inactive pro form which must be cleaved by a multi protein complex called an inflammasome. The mechanisms governing the formation of inflammasomes and subsequent IL-1b processing are not completely understood. IL-1a is poorly researched in comparison to IL-1b and undergoes subcellular transport to the nucleus of the cell as a result of the presence of a nuclear localisation sequence (NLS); the biological significance of this is poorly understood. In order to effectively target IL-1 signalling in disease it is crucial to understand how this signalling is regulated. The aim of this thesis was to improve the understanding of IL-1 regulation by focusing on formation of the inflammasome and importance of the IL-1a NLS. I contributed to the discovery that the fenamate class of nonsteroidal anti-inflammatory drugs (NSAIDs) prevents formation of the NLRP3 inflammasome via reversible blockade of the VRAC Cl- channel and may be effective therapeutics in Alzheimer's disease (AD). Building on this discovery, I was also involved in the design and development of novel NLRP3 inhibiting compounds using the Ca2+ blocker 2APB as a starting point. After multiple rounds of structure-activity relationships and phenotypic screening NBC6 was developed, a molecule 100-fold more potent than 2APB and with no effect on Ca2+-signalling. Finally, CRSIPR/Cas9 was used to neutralise the NLS on IL-1a which resulted in an observed loss of IL-1a transcription. Further analysis revealed that this procedure had inadvertently disrupted the secondary structure of the crucial regulatory long-noncoding RNA AS-IL1a, the most likely explanation for the loss of IL-1a expression. Together, these data greatly improve our understanding of the cellular and molecular mechanisms underpinning the regulation of IL-1. Not only do these studies pave the way for novel therapeutics to treat inflammatory disease but they also open new avenues of research for the rapidly accelerating field of inflammation.
|Date of Award||31 Dec 2018|
- The University of Manchester
|Supervisor||Douglas Kell (Supervisor), Hervé Boutin (Supervisor), David Brough (Supervisor) & James Galea (Supervisor)|