SIRT3-mediated deacetylation of NLRC4 promotes inflammasome activation

Abstract

Salmonella typhimurium ( S. typhimurium ) infection of macrophage induces NLRC4 inflammasome-mediated production of the pro-inflammatory cytokines IL-1β. Post-translational modifications on NLRC4 are critical for its activation. Sirtuin3 (SIRT3) is the most thoroughly studied mitochondrial nicotinamide adenine dinucleotide (NAD + ) -dependent deacetylase. We wondered whether SIRT3 mediated-deacetylation could take part in NLRC4 inflammasome activation. Methods: We initially tested IL-1β production and pyroptosis after cytosolic transfection of flagellin or S. typhimurium infection in wild type and SIRT3-deficient primary peritoneal macrophages via immunoblotting and ELISA assay. These results were confirmed in SIRT3-deficient immortalized bone marrow derived macrophages (iBMDMs) which were generated by CRISPR-Cas9 technology. In addition, in vivo experiments were conducted to confirm the role of SIRT3 in S. typhimurium -induced cytokines production. Then NLRC4 assembly was analyzed by immune-fluorescence assay and ASC oligomerization assay. Immunoblotting, ELISA and flow cytometry were performed to clarify the role of SIRT3 in NLRP3 and AIM2 inflammasomes activation. To further investigate the mechanism of SIRT3 in NLRC4 activation, co-immunoprecipitation (Co-IP), we did immunoblot, cellular fractionation and in-vitro deacetylation assay. Finally, to clarify the acetylation sites of NLRC4, we performed liquid chromatography-mass spectrometry (LC-MS) and immunoblotting analysis. Results: SIRT3 deficiency led to significantly impaired NLRC4 inflammasome activation and pyroptosis both in vitro and in vivo . Furthermore, SIRT3 promotes NLRC4 inflammasome assembly by inducing more ASC speck formation and ASC oligomerization. However, SIRT3 is dispensable for NLRP3 and AIM2 inflammasome activation. Moreover, SIRT3 interacts with and deacetylates NLRC4 to promote its activation. Finally, we proved that deacetylation of NLRC4 at Lys71 or Lys272 could promote its activation. Conclusions: Our study reveals that SIRT3 mediated-deacetylation of NLRC4 is pivotal for NLRC4 activation and the acetylation switch of NLRC4 may aid the clearance of S. typhimurium infection.

Mitochondrial sirtuin 3 and various cell death modalities

“…On the other hand, in primary peritoneal macrophages, Sirt3 deficiency had no significant effect on NLRP3 inflammasome activation. Interestingly, in this study, Sirt3 was shown to directly deacetylate another inflammasome component, NLR domain-containing protein 4 (NLRC4), to promote pyroptosis (Guan et al, 2021).…”

Posttranslational Regulation of Inflammasomes, Its Potential as Biomarkers and in the Identification of Novel Drugs Targets

“…SIRT3 deficiency leads to significantly impaired NLRC4 inflammasome activation, ASC oligomerization, and pyroptosis both in vitro and in vivo . SIRT3 interacts with and deacetylates NLRC4 at Lys71 or Lys272 to promote its activation ( Guan et al, 2021 ). However, SIRT3 is dispensable for NLRP3 and AIM2 inflammasome.…”

Nod-like Receptors: Critical Intracellular Sensors for Host Protection and Cell Death in Microbial and Parasitic Infections

“…Its expression is upregulated by TNF and the stress-mediated p53 activation [ 143 , 144 , 145 ]. NLRC4 contains a CARD domain, and therefore, can directly recruit pro-caspase 1 via CARD–CARD interaction to activate caspase 1, associates or co-localizes with ASC [ 143 , 146 ] for proteolytic cleavage of proinflammatory cytokines (pro–IL-1β and pro–IL-18) and GSDMD [ 147 , 148 ]. The pathogen recognition of NLRC4 is usually via a sensor NAIP (NLR family, apoptosis inhibitory proteins) [ 149 ].…”

“…In addition, leucine-rich repeat kinase 2 LRRK2 is an intrinsic regulator of NLRC4 and LRRK2 formed a complex with NLRC4 for its optimal phosphorylation at Ser533, which promotes inflammasome activation during S. typhimurium infection in macrophages [ 157 ]. Interestingly, just as a report showed, acetylation-induced NLRP3 activation can be reversed by SIRT2 [ 158 ]; also SIRT3 was found to promote NLRC4 inflammasome activation by deacetylation [ 148 ]. The involvement of NLRC4 in pathogen recognition was largely thought to be restricted to bacterial infections but recent studies revealed that dendritic cell NLRC4 regulates T cell response during influenza A virus infection.…”