TBK1 kinase acts like a parking brake to prevent premature activation of NLRP3 inflammasome
Lead Research Organisation:
University of Oxford
Department Name: Kennedy Institute
Abstract
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 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.
Technical Summary
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.
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.
Publications
Bezbradica J
(2023)
Inflammasomes as regulators of mechano-immunity
in EMBO Reports
Bezbradica JS
(2022)
Activation of the Non-canonical Inflammasome in Mouse and Human Cells.
in Methods in molecular biology (Clifton, N.J.)
Danielli S
(2023)
The ion channel CALHM6 controls bacterial infection-induced cellular cross-talk at the immunological synapse.
in The EMBO journal
Demarco B
(2022)
How Pyroptosis Contributes to Inflammation and Fibroblast-Macrophage Cross-Talk in Rheumatoid Arthritis.
in Cells
Fischer F
(2021)
TBK1 and IKKe act like an OFF switch to limit NLRP3 inflammasome pathway activation
in Proceedings of the National Academy of Sciences
Fischer FA
(2021)
Posttranslational and Therapeutic Control of Gasdermin-Mediated Pyroptosis and Inflammation.
in Frontiers in immunology
McKee C
(2021)
PHOrming the inflammasome: phosphorylation is a critical switch in inflammasome signalling
in Biochemical Society Transactions
Reinke S
(2023)
Emulsion and liposome-based adjuvanted R21 vaccine formulations mediate protection against malaria through distinct immune mechanisms
in Cell Reports Medicine
Description | BBSRC iCASE studentship, PI, to support Katherine Heighes, DPhil student |
Amount | £200,000 (GBP) |
Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
Sector | Public |
Country | United Kingdom |
Start | 10/2023 |
End | 10/2027 |
Description | EMBO Fellowship for Postdoc Benjamin Demarco |
Amount | € 60,000 (EUR) |
Organisation | European Molecular Biology Organisation |
Sector | Charity/Non Profit |
Country | Germany |
Start | 09/2021 |
End | 08/2022 |
Description | Salary Support for Assoc. Prof. Jelena Bezbradica |
Amount | £132,684 (GBP) |
Funding ID | KENN212202 |
Organisation | The Kennedy Trust |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 05/2023 |
End | 04/2026 |
Description | The Kennedy Trust studentship, PI, to support Su Qi, DPhil student |
Amount | £220,000 (GBP) |
Organisation | The Kennedy Trust |
Sector | Charity/Non Profit |
Country | United Kingdom |
Start | 10/2023 |
End | 10/2027 |
Description | Travel award to attend the International Cytokine & Interferon Society meeting Cardiff 2021, Fabian Fischer, DPhil student |
Amount | $500 (USD) |
Organisation | International Cytokine and Interferon Society |
Sector | Charity/Non Profit |
Country | United States |
Start | 10/2021 |
End | 10/2021 |
Title | Comissioned Methods Chapter for Methods in Molecular biology |
Description | Comissioned Methods Chapter for Methods in Molecular biology on method for Activation of the Non-canonical Inflammasome in Mouse and Human Cells |
Type Of Material | Technology assay or reagent |
Year Produced | 2022 |
Provided To Others? | Yes |
Impact | Generated bradly available and standardised method to improve reproducibility in the field |
URL | https://link.springer.com/protocol/10.1007/978-1-0716-2144-8_5 |
Description | Dr Dave Boucher |
Organisation | University of York |
Department | York Trials Unit |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Collaboration on innate sensing and signalling, resulted in two publications, in 2021 and 2022 |
Collaborator Contribution | Intelectual Contribution |
Impact | Joint publicaiton DOI: 10.1007/978-1-0716-2144-8_5 |
Start Year | 2021 |
Description | Dr Rebecca Coll |
Organisation | Queen's University Belfast |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Collaboration on innate sensing and signalling, resulted in two publications, in 2021 and 2022 |
Collaborator Contribution | Intelectual Input |
Impact | Two publications DOI: 10.1007/978-1-0716-2144-8_5 and DOI: 10.1042/bst20200987 |
Start Year | 2021 |
Description | Elena Di Daniel - Oxford Target Discovery Institute |
Organisation | University of Oxford |
Department | Target Discovery Institute (TDI) |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Co-correspnding author on primary research paper |
Collaborator Contribution | Co-corresponding author contributed data for primary research paper |
Impact | Primary research paper PMID: 34518217 |
Start Year | 2021 |
Description | Prof Kevin Foskett |
Organisation | University of Pennsylvania |
Country | United States |
Sector | Academic/University |
PI Contribution | Co-corresponding author on the EMBO primary research paper |
Collaborator Contribution | Electrophysiology studies of a novel ion channel on macrophages |
Impact | Joined Publication PMID: 36861806 EMBO J. 2023 Mar 2;e111450. doi: 10.15252/embj.2022111450. |
Start Year | 2022 |
Description | BSI annual meeting Session Chair |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Postgraduate students |
Results and Impact | Session Chair at Annual BSI meeting, Session on Innate Immune Sensing and Signalling |
Year(s) Of Engagement Activity | 2022 |
URL | https://www.bsicongress.com/bsi/frontend/reg/tAgendaWebsite.csp?pageID=7307&daySelector=2&eventID=10 |
Description | EMBO meeting Session Chair |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Postgraduate students |
Results and Impact | Chaired a half day session at international EMBO conference |
Year(s) Of Engagement Activity | 2022 |
URL | https://meetings.embo.org/event/21-signaling |
Description | European Congress of Immunology Session Chair |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Public/other audiences |
Results and Impact | Chaired a half day session at European Congress of Immunology |
Year(s) Of Engagement Activity | 2021 |
URL | https://www.immunology.org/events/6th-european-congress-immunology-eci-2021 |
Description | Public Engagement at Natural Hustory Museum Art and Science Day by Reuben College, participation by Ada Liebenau, DPhil student |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Public/other audiences |
Results and Impact | Public Engagement at Natural Hustory Museum Art and Science Day by Reuben College, participation by Ada Liebenau, DPhil student |
Year(s) Of Engagement Activity | 2023 |
URL | https://reuben.ox.ac.uk/event/art-science-day |