N Naser(1), A Aronova(1), F Tosato(1), S Steffens(2), A Imhof(3), Y Asare(1), M Dichgans(1)

1. Institute for Stroke and Dementia Research (ISD), LMU Munich, Munich, Germany; 2. Institute for Cardiovascular Prevention (IPEK), LMU Munich, Munich, Germany; 3. Protein Analysis Unit, Faculty of Medicine, Biomedical Center, LMU, Martinsried, Germany.

Background: Atherosclerosis is driven by chronic inflammation. Targeting IL-1βreduced cardiovascular events, however, led to higher infection rates. GWAS identified HDAC9 as a key cardiovascular risk factor, and we demonstrated its link to inflammasome activation. However, the precise mechanism and therapeutic targeting remain undefined.

Methods and results: To determine the specific mechanism by which HDAC9 activates the inflammasome, we stimulated BMDMs with LPS and nigericin and found a reduction of cleaved caspase-1, cleaved gasdermin D and IL-1β levels in both Hdac9-deficient and TMP195 (class lla inhibitor)-treated BMDMs, indicating reduced inflammasome activation. We then screened for HDAC9 interactions with inflammasome components by immunoprecipitation and found that HDAC9 specifically binds to NLRP3, but not NLRC4 and AIM2. These findings were confirmed by mass spectrometry in both overexpression model and endogenously in human macrophages. Motivated by HDAC9’s molecular scaffolding function, we immunoprecipitated NLRP3 from HDAC9-deficient and control BMDMs, identifying NEK9 as an HDAC9-dependent interactor that partially shares share the binding site on NLRP3 with NEK7, a key regulator of the NLRP3 inflammasome. We then examined NLRP3 oligomerisation in the presence or absence of HDAC9 and found that HDAC9 promoted the oligomerization of NLRP3. Then, we screened for HDAC inhibitors and identified CHDI, a class lla HDAC inhibitor, with higher potency than TMP195 in cell-free and cell-based assays.

Conclusion: We hereby demonstrate that HDAC9 drives vascular inflammation by activating the NLRP3 inflammasome. Additionally, we identified NEK9 as an HDAC9-dependent interactor of NLRP3, and CHDI as a potent class lla inhibitor with high therapeutic potential.