TBK1 kinase acts like a parking brake to prevent premature activation of NLRP3 inflammasome

Activation of the NLRP3 inflammasome can be beneficial during infection and vaccination. Nonetheless, when NLRP3 activity is uncontrolled and chronic, it becomes detrimental and contributes to inflammation-driven pathology in diseases such as Alzheimer's disease, Parkinson's disease and atherosclerosis. In healthy individuals therefore, there must exist a licencing mechanism that prevents unwanted NLRP3 inflammasome responses. Here, we characterized one such mechanism. Using pharmacological and genetic approaches we showed that TBK1 limits the responses downstream of the NLRP3 inflammasome activation and it works against the PP2A phosphatase ON switch to balance the NLRP3 activity. Several viruses such as coronavirus SARS-CoV-2 are known to inhibit TBK1 activity as a part of their immune evasion strategy. We propose that TBK1 kinase acts like a parking brake that limits NLRP3 pathway activity, and that viruses that inhibit TBK1 boost the NLRP3-driven inflammation by removing an important break from this pathway. We now propose to characterise the mechanism behind our findings and its relevance to the current pandemic coronavirus disease.

The team leading the proposal is made of PI (Jelena Bezbradica Mirkovic who is expert in macrophage biology, innate signaling and inflammasomes), highly productive inflammasome expert postdoc (Dr Benjamin Demarco) who will join the team in May 2021; the proteomics collaborators at Oxford (Prof Benedikt Kessler and Dr Roman Fischer), and the world-leader imaging expert collaborator at Kennedy (Prof Michael Dustin). Critically, preliminary data upon which this application is predicated are currently in revision for publication.

The aim of this work is to understand what sites in the NLRP3 inflammasome are regulated by TBK1, the mechanism by which TBK1 controls NLRP3 inflammasome, and how this mechanism may be dysregulated in COVID-19 disease.

Objective 1: Identify TBK1-controlled sites in NLRP3 complex and map at which stage of priming or activation TBK1 blocks the pathway activation.
We found that TBK1 controls NLRP3 pathway, independently of the known inhibitory phosphorylation of NLRP3-serine 3. TBK1 had no such control over other inflammasomes, suggesting that its client protein is either NLRP3 or its upstream interacting partner, e.g NEK7. We will test whether TBK1 phosphorylates mouse and human NLRP3 directly (using in vitro kinase assay), identify the TBK1 regulated phospho-sites in NLRP3 complex (by proteomics), and test at which stage of pathway activation TBK1 blocks the NLRP3 activity (by imaging and functional assays).

Objective 2 Describe whether viruses such as Coronavirus SARS-CoV-2 that are known to inhibit TBK1 activation, cause NLRP3 pathway overactivation by releasing the TBK1-regulated NLRP3 brake.
NLRP3 inflammasome is activated in response to SARS-CoV-2 infection and higher levels of NLRP3 activity are associated with poor clinical outcome. Papain-like protease (PLpro) from SARS-CoV-2 was recently described to inhibit TBK1 activation. We will test whether PLpro from SARS-CoV-2, when ectopicaly expressed in macrophages (as a part of the inert vector), inhibits TBK1, and boosts NLRP3 pathway activity. As controls we will use cataliticaly inactive PLpro and PLpro from SARS-CoV-1. We showed that TLRs introduce TBK1-dependent brake on NLRP3, to prevent spontaneous NLRP3 activation during NLRP3 priming. We will also test the role of TBK1 in NLRP3 activation upon viral sensing by monitoring NLRP3 phosphorylation and activation in BMDMs upon pharmacological activation of cytosolic viral sensors in the presence or absence of TBK1 inhibition.