SIRT3-mediated deacetylation of NLRC4 promotes inflammasome activation.
SIRT3-mediated deacetylation of NLRC4 promotes inflammasome activation.
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作者：
Guan C;Huang X;Yue J;Xiang H;Shaheen S;Jiang Z;Tao Y;Tu J;Liu Z;Yao Y;Yang W;Hou Z;Liu J;Yang XD;Zou Q;Su B;Liu Z;Ni J;Cheng J;Wu X
鼠伤寒沙门氏菌（S.鼠伤寒沙门氏菌）感染的巨噬细胞诱导NLRC 4炎性体介导的促炎细胞因子IL-1β的产生。NLRC 4的翻译后修饰对其激活至关重要。sirtuin 3（SIRT 3）是研究最深入的线粒体烟酰胺腺嘌呤二核苷酸（NAD+）依赖性脱乙酰酶。我们想知道SIRT 3介导的去乙酰化是否可以参与NLRC 4炎性小体激活。方法：我们初步检测了鞭毛蛋白或S.通过免疫印迹和ELISA测定，在野生型和SIRT 3缺陷的原代腹膜巨噬细胞中检测鼠伤寒杆菌感染。这些结果在通过CRISPR-Cas9技术产生的SIRT 3缺陷型永生化骨髓源性巨噬细胞（iBMDM）中得到证实。此外，进行体内实验以证实SIRT 3在S.鼠伤寒沙门氏菌诱导的细胞因子产生。然后通过免疫荧光分析和ASC寡聚化分析来分析NLRC 4组装。进行免疫印迹、ELISA和流式细胞术以阐明SIRT 3在NLRP 3和AIM 2炎性小体活化中的作用。为了进一步研究SIRT 3在NLRC 4激活、免疫共沉淀（Co-IP）中的机制，我们进行了免疫印迹、细胞分级分离和体外脱乙酰化测定。最后，为了明确NLRC 4的乙酰化位点，我们进行了液相色谱-质谱（LC-MS）和免疫印迹分析。结果：SIRT 3缺陷导致体外和体内NLRC 4炎性小体活化和焦亡显著受损。此外，SIRT 3通过诱导更多的ASC斑点形成和ASC寡聚化来促进NLRC 4炎性体组装。然而，SIRT 3对于NLRP 3和AIM 2炎性体激活是不可或缺的。此外，SIRT 3与NLRC 4相互作用并使其脱乙酰化以促进其活化。最后，我们证明了NLRC 4在Lys 71或Lys 272处的脱乙酰化可以促进其活化。结论：我们的研究表明，SIRT 3介导的NLRC 4的脱乙酰化是NLRC 4激活的关键，NLRC 4的乙酰化开关可能有助于S.鼠伤寒感染
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.
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