Deciphering new immune-evasive mechanisms relevant to mycobacterial infection

Abstract

NLRP3 inflammasome activation is critical for inflammation and plays a central role in a wide range of both infectious (including Mycobacterium tuberculosis) and inflammatory diseases. However, dysregulation of inflammasome activation can have serious consequences. As a result, NLRP3 inflammasome activation is highly regulated at several levels, including via post-translational modifications. The ubiquitin system provides a flexible and specific mechanism to target and regulate protein function across many pathways, including the NLRP3 inflammasome activation. The de-ubiquitinase (DUB) USP7 has been widely studied in cancer, but its role in regulating inflammatory processes is still unclear. USP7 is known to play a role in NF-kB regulation and, together with USP47, can promote NLRP3 activation. However, how each of these two DUBs differentially contribute to NLRP3 activation, whether at the priming or activation level, is unknown. The first aim of this work was to dissect the contribution of USP7 to NLRP3 inflammasome activation. For this, we compared the effect of the selective USP7 inhibitors GNE-6776 and FT671 to P22077, a dual USP7 and USP47 inhibitor, on inflammasome activation in THP1 and primary human monocyte-derived macrophage (MDM) cells. We found that USP7 contributed to NF-kB activation and inflammasome priming, but that the extent of its contribution is dependent on cell type and the presence of LPS priming. The second aim of this project was to investigate whether USP7 can regulate NLRP3 inflammasome activation during mycobacterial infection. The Mycobacterium bovis vaccine strain BCG was used for these experiments, where we found that USP7 contributes to inflammasome activation and especially to IL-18 secretion. Finally, the third aim was to explore the role of sirtuin 1, a NAD+-dependent histone deacetylase known to be involved in anti-Mtb responses and a previously identified substrate of USP7. We confirmed the interaction between USP7 and SIRT1 in THP1 cells and found that SIRT1 was able to contribute to nigericin-induced inflammasome activation in PMA-differentiated THP1. Taken together, these findings suggest that USP7 contributes to NF-kB activation as well as NLRP3 inflammasome activation induced by nigericin and BCG; USP7 inhibition alone however does not affect inflammasome activation to the same extent as the combined inhibition of USP7 and USP47. We also identified a potential pro-inflammatory role for SIRT1 as a contributor to inflammasome activation.

Date of Award	1 Aug 2023
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Eileithyia Swanton (Supervisor), David Brough (Supervisor) & Gloria Lopez-Castejon (Supervisor)