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Title: Regulation of endogenous interleukin-36 activity
Author: Jaafar, Ali
ISNI:       0000 0004 8509 1289
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2019
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IL-36 is a pro-inflammatory cytokine that consists of three agonists IL-36α, IL36β and IL-36γ and one antagonist, IL-36Ra. In addition to their receptor (IL-36R), IL36 cytokines require IL-1RAcP as a co-receptor for their signalling. IL-36 cytokines and their receptor are members of the IL-1 family of proteins. IL-36 has been shown to play important roles in activation of NF-κB and MAPK pathways. Activated IL-36 induces expression of inflammatory mediators such as CXCL8 and recruits inflammatory cells such as neutrophils into the site of infection. Uncontrolled signalling of IL-36 cytokines can lead to excessive inflammation and potentially chronic inflammatory diseases such as psoriasis. Previous studies that were used to assess the biological activity of recombinant, fully active IL-36 cytokines have either used exogenous transfected IL-36R or endogenous IL-36R for 24 h. Here in this thesis I used HT-29 cells, which express endogenous IL-36R. They have been stably transfected with an NF-κB reporter gene and cloned. I confirmed by gene editing, that IL1LR2 is the only gene that encodes IL-36R in HT-29 cells. Our results showed that the EC₅₀ values of the active forms of n⁵-IL-36β and n¹⁸-IL-36γ, which I prepared, and previously prepared n⁶-IL-36α in our laboratory was considerably greater than previously had been reported. The duration of the response of the NF-κB reporter gene to n⁶-IL-36α and n⁵-IL-36β is between 3 and 9 h. Our data also showed that replacing of n⁶ amino acid lysine of IL-36α with either glycine or serine leads to increase EC₅₀ of the IL-36α protein. Finally, limited in vitro digestion of n¹-IL-36α by chymotrypsin leads to cleavage of n¹-IL-36α in four different sites. Reporter gene data showed that the mixed digested protein has similar activity to n⁶-IL-36α. Proteolytic processing of endogenous IL-36 proteins is still largely unexplored, but an in vitro study showed that specific truncation of their N-termini activates them. Another study showed that serine proteases enzymes derived from neutrophils can cleave proIL-36 proteins but not necessarily at the same sites suggested by the previous in vitro study. The last section of this thesis focuses on expression of endogenous IL-36 cytokines at the level of mRNA and protein and my attempts to induce proteolytic processing of endogenous IL-36γ. RT-PCR and RT-qPCR data presented here showed that the expression of IL-36β and IL-36γ mRNAs but not IL-36α is induced in response to IL-1α, TNF, PMA, flagellin, TNF and PMA or TNF and flagellin in both human epithelial cell lines (HaCaT and A-431). IL-36γ is the most abundant IL-36 mRNA that is regulated by these stimuli. Furthermore, IL-36α was not found to induce expression either of itself or other IL-36 agonists in HaCaT cells. Our research within this thesis shows that the synergistic action of PMA or flagellin with TNF in HaCaT and A-431 or Staphylococcus aureus in HaCAT induces endogenous IL-36γ protein. However, processing of endogenous IL-36γ protein was not induced in response to these stimuli. The apoptotic stimuli, cycloheximide and staurosporine but not calcium ionophore induced a mobility change of endogenous IL-36γ suggesting a processing at a site before n¹⁸ of IL-36γ. The same effect was not seen in primary keratinocytes. The significance of this processing has not yet been investigated.
Supervisor: Nicklin, Martin Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available