Understanding the mechanisms that drive NLRP3-dependent inflammation

  • Bali Lee

Student thesis: Phd

Abstract

Inflammation, largely driven by the innate immune system, is a powerful first line of defence against infectious agents. It is characterised by immune cell recruitment, pain and loss of function. The non-specific nature of inflammation enables it to respond rapidly and robustly to a perceived threat. However, when dysregulated it can cause damage to previously healthy tissues. The NLRP3 inflammasome is an innate immune sensor capable of recognising and responding to a vast array of stimuli. These include the bacterial toxin, nigericin, intracellular signals of danger like ATP, and foreign particulate matter like asbestos and silica. Activation of the NLRP3 inflammasome results in pyroptosis, an inflammatory form of cell death, and release of the pro-inflammatory cytokine IL-1ß. IL-1ß is a master regulator of inflammation it can travel within the circulatory system encouraging the differentiation and proliferation of local immune cells. Aberrant or dysregulated NLRP3 activation is associated with numerous non-communicable diseases including, Alzheimer’s, atherosclerosis and diabetes. More recently, aberrant NLRP3 activation was implicated in hyper inflammation in response to COVID-19. Thus, the NLRP3 inflammasome presents an attractive therapeutic target. Activation of the NLRP3 inflammasome is tightly regulated, due to its potent effects, and involves a two-step process. The first step, priming, is relatively well characterised and involves upregulation of the NLRP3 and interleukin-1 protein. The second, activation step, has proven slightly more elusive. As despite numerous studies investigating NLRP3, a common unifying signal that results in NLRP3 activation and oligomerisation remains to be identified. The subcellular localisation of both inactive and active NLRP3 is also controversial with multiple contradictory reports present in the literature. Thus, the primary aim of this thesis was to gain further insight into the mechanisms driving NLRP3-dependent inflammation, with the hope that this could aid in the development of better therapeutics for the diseases that NLRP3 inflammasome activation is implicated in. Research published right at the start of this PhD suggested that the NLRP3 protein senses and responds to fragmentation of the trans-Golgi network. The work conducted in this thesis demonstrates that NLRP3 is actually responding to disrupted endocytic traffic and that this is a common effect across a broad range of different stimuli. NLRP3 sensing of disrupted endocytic traffic results in its recruitment to endolysosomal vesicles, which we hypothesise serve as platform for the subsequent oligomerisation of NLRP3. NLRP3 also responds in this way to monensin, a known disruptor of endocytic traffic that is incapable of activating NLRP3, indicating that although this step may be essential it is not sufficient to cause NLRP3 activation. Following from this, we observed that at homeostasis a proportion of NLRP3 is present on TGN membranes. Chemical disruption of the Golgi structure did not activate the NLRP3 nor did it cause NLRP3 to dissociate from the TGN. Interestingly, NLRP3 genetically targeted to various cell organelles was found to still oligomerise ASC. This raises the intriguing possibility that NLRP3 is negatively regulated on the TGN and that its concentration on any membrane is otherwise sufficient to induce its activation. Finally, we identified nylon and polyethylene microplastics as novel activators of the NLRP3 inflammasome in vitro, with nylon microplastics also found to cause disruptions in endocytic traffic and inflammation in vivo. In summary, the findings of this thesis provide new insights into the mechanisms governing NLRP3 inflammasome activation and identify nylon and polyethylene microplastics as novel NLRP3 stimuli.
Date of Award31 Dec 2022
Original languageEnglish
Awarding Institution
  • The University of Manchester
SupervisorStuart Allan (Supervisor), Catherine Lawrence (Supervisor), David Brough (Supervisor), Hiren Patel (Supervisor), Adrian Parry-Jones (Supervisor) & Jack Rivers-Auty (Supervisor)

Keywords

  • Polyethylene
  • Microplastics
  • IL-1
  • Inflammasome
  • Endosome
  • Trafficking
  • Inflammation
  • NLRP3

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