Abstract:
The NLRP3 inflammasome comprises a sensor NLRP3, an adaptor ASC and an effector caspase-1 which together form a multiprotein complex that enables maturation of the inflammatory cytokines IL-1β and IL-18 and executes an inflammatory type of cell death called pyroptosis. NLRP3 is strictly regulated during its priming and activation phases, and several post-translational modifiers of NLRP3 contribute to its activation and regulation. Previous studies have identified Bruton tyrosine kinase (BTK) as a post- translational activator of NLRP3 that interacts and phosphorylates multiple tyrosine residues in the central NLRP3 NACHT linker domain. In this study we have aimed at analysing BTK’s activation and interaction with NLRP3, and the downstream effects of BTK-mediated phosphorylation on NLRP3 oligomerization. We observed that BTK was phosphorylated, a common indication of BTK activation, during the early TLR priming phase using immunoblotting analysis. We further hypothesized that Syk, a tyrosine kinase known to phosphorylate BTK in B cells to be upstream of BTK activation. Indeed, pharmacological Syk inhibition abolished BTK phosphorylation in primary mouse macrophages. Furthermore, confocal microscopy in primary mouse macrophages revealed that within 20 mins of TLR4 stimulation, BTK was recruited transiently to the plasma membrane, in good agreement with findings that the BTK Pleckstrin homology (PH) domain is known to interact with phospho inositol (PIP3) in the plasma membrane in B cells. However, Xid BTK with a point mutant in its PH domain (R28C), did not show plasma membrane localization upon TLR priming in mouse macrophages. As Xid BTK macrophages did not show plasma membrane recruitment, we speculated if the Xid mutation would affect the interaction of NLRP3 and BTK. Proximity ligation assays of the NLRP3 and Xid BTK interaction indicated that the interaction was indeed reduced compared to the WT BTK, possibly due to reduced BTK activation or aberrant localization in this phenotype. Using co-immunoprecipitation, we further established that BTK interacts with proline residues P199 and P202 in the NLRP3 NACHT linker which, when substituted to alanine or glycine, showed reduced interaction with BTK. In the publicly available cryo-EM structures of NLRP3, the Pro sites were in proximity to the BTK-modified Tyr residues in the NACHT linker. Finally, analysing the downstream effect of BTK on NLRP3 oligomerization, we observed a significant decrease in NLRP3 specks in Btk KO vs WT murine macrophages using immunofluorescence staining which corresponded to a decrease in IL-1β. Altogether, we have endeavoured to address the mechanisms of BTK activation and interaction with NLRP3 in the inflammasome cascade. Although the interaction sites of BTK and NLRP3 and the role of Syk in NLRP3 inflammasome requires further investigation, our results highlight the requirement for precise targeting of specific interaction interfaces of BTK and NLRP3. Additionally, the involvement of Syk raises the possibility that Syk inhibitors could be potential and attractive alternate targets for inflammasome inhibition in NLRP3 related diseases.
Inflammasomes