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.

Keywords: NLRC4; S. typhimurium infection; SIRT3; deacetylation; inflammasome.

Figure 1

SIRT3 deficiency impairs NLRC4 inflammasome activation. (A) LPS-primed WT and SIRT3^-/- peritoneal macrophages were treated with 1 µg/mL flagellin for 6 h. Cell lysates and culture supernatants (Sup) were collected and immunoblotted with the indicated antibodies. (B) Peritoneal macrophages from littermate control (WT) and SIRT3^-/- mice were infected with S. typhimurium at an MOI of 20 for 2 h. Cell lysates and culture supernatants were collected and immunoblotted with the indicated antibodies. (C) ELISA of IL-1β in cell-free supernatants from WT and SIRT3^-/- peritoneal macrophages that were either pretreated with LPS (500 ng/mL) for 4 h followed by transfected with Flagellin (1 µg/mL) or infected with S. typhimurium at an MOI of 20 for 2 h. (D) LDH in cell free supernatants from WT and SIRT3^-/- peritoneal macrophages that were treated as stated in C. (E-F) Immunoblot analysis of full-length(53kD) and cleaved (30kD) GSDMD protein in lysate of macrophages treated as stated in C. (G) ELISA of IL-6 in cell free supernatants from WT and SIRT3^-/- peritoneal macrophages that were treated as stated in C. (H) ELISA analysis of IL-1β levels in sera of littermate control (WT n=9) and SIRT3^-/- mice (n=10) 6 h after S. typhimurium infection. (I) ELISA analysis of IL-1β from supernatants of overnight-cultured equal number of macrophages isolated from flushed peritoneal cells of WT and SIRT3^-/- mice which were infected by S. typhimurium for 6 h. Data are presented as means ± SEM; *, P < 0.05; ***, P <0.001 (Student's t test) all conditions were determined in technology triplicate. In each panel, data are representative of at least three independent experiments.

Figure 2

SIRT3 promotes NLRC4 activation in iBMDMs. (A) Immortalized bone marrow derived macrophages (iBMDMs) were transfected with control (Ctrl) or HA-tagged SIRT3. These cells were transfected with flagellin or infected with S. typhimurium at an MOI of 20 for 2 h, then the cell lysates and culture supernatants (Sup) were collected and immunoblotted with the indicated antibodies. (B) ELISA analysis of IL-1β from the cell-free supernatant of cells treated as stated in A. (C-D) Immunoblot analysis of full-length (53kD) and cleaved (30kD) GSDMD protein in lysate of iBMDMs that were treated as stated in A. (E) LDH release in cell free supernatants from iBMDMs that were treated as stated in A. (F-G) SIRT3-deficient iBMDMs were generated by CRISPR-Cas9-mediated deletion. These cells were primed with LPS followed by transfected with 1 μg/mL flagellin for 6 h (F) or infected with S. typhimurium at an MOI of 20 for 2 h (G). Cell lysate and culture supernatants were collected and immunoblotted with the indicated antibodies. (H-I) SIRT3-deficient iBMDMs were treated as stated in F and G. Cells were lysed and detected with antibody to GSDMD by immunoblot. (J) LDH in cell free supernatants from these SIRT3-deficient iBMDMs that were treated as stated in F-G. Data information: In B, E, J data are presented as means ± SEM; ***, P < 0.001 (Student's test). All the results are of at least three independent experiments. Each condition was performed in technology triplicate.

Figure 3

SIRT3 promotes ASC speck formation and assembly of NLRC4 inflammasome. (A) LPS-primed WT and SIRT3-/- peritoneal macrophages were either cytosolic transfected with 1 µg/mL flagellin or infected with S. typhimurium at an MOI of 20. ASC speck formation was assayed by ASC immunofluorescent staining, and cells were counterstained by DAPI (blue). Fluorescent images were analyzed by confocal microscopy. Percentages of macrophages containing ASC foci was quantified (right), with at least 200 cells counted in each experiment. Bars, 20 µm. Arrows in the figures indicate the ASC speck. (B) Peritoneal macrophages from littermate control (WT) and SIRT3-/- mice were cytosolic delivery of 1μg/mL flagellin or infected with S. typhimurium at an MOI of 20 for 2 h. Cells were dissolved with Triton X-100-containing buffer followed by cross-linkage of insoluble fractions with DSS to capture ASC oligomers. Immunoblots of those insoluble fractions (Insoluble + DSS) and soluble fractions were detected with an antibody to ASC. Data information: In A, data are shown as means ± SEM; ***, P < 0.001 (Student's test). All results are representative of three independent experiments.

Figure 4

SIRT3 is dispensable for NLRP3 and AIM2 inflammasome activation. (A) WT and SIRT3-/- peritoneal macrophages were primed with 500 ng/mL LPS for 4 h and then treated with ATP (5 mM) or nigericin (5 μM) for 1 h. Cell lysates and culture supernatants were collected and immunoblotted with the indicated antibodies. (B-C) ELISA of IL-1β (B) and IL-6 (C) in cell-free supernatants from WT and SIRT3-/- peritoneal macrophages that were treated as (A). (D-E) Peritoneal macrophages were primed with LPS for 4 h followed by activated by ATP (5 mM) or nigericin (5 μM) for 1 h and then stained with Mitotracker green and Mitotracker deep red (D) or MitoSOX (E) for 30 min and analyzed by flow cytometry. (F) WT and SIRT3-/- peritoneal macrophages were primed with 500 ng/mL LPS for 4 h and then transfected with poly (dA: dT) (2 μg/mL) for 6 h. ELISA analysis of IL-1β in cell-free supernatants from these cells. (G) Cells were treated as (F), cell lysates and culture supernatants were collected and immunoblotted with the indicated antibodies. (H) ELISA of IL-6 in cell-free supernatants from WT and SIRT3-/- peritoneal macrophages that were treated as (F). Data information: In B-C, F and H, data are presented as means ± SEM. Data are representative of three independent experiments.

Figure 5

SIRT3 interacts with and deacetylates NLRC4 to promote its activation. (A) HEK293T cells were transfected with HA-tagged SIRT3 along with or without Flag-tagged NLRC4. Cell lysates were immunoprecipitated with anti-Flag and analyzed by immunoblot. (B) Subcellular distribution of NLRC4 and SIRT3 before and after S. typhimurium infection in peritoneal macrophages. ATP5a as a mitochondria marker. HSP 90 serves as cytosol marker. (C and D) HEK293T cells ectopically expressing NLRC4 were co-transfected with or without CBP. Cell lysates were then immunoprecipitated (IP) using pan-acetyl-lysine antibody (C) or anti-Flag NLRC4 antibody (D) and were immunoblotted with the indicated antibodies. Arrows in D indicate the Ac-NLRC4 band, * in D indicates non-specific band. (E) Peritoneal macrophages were infected with S. typhimurium at an MOI of 20 for 2 h. Uninfected cells were included as controls. Cell lysates were then immunoprecipitated (IP) using pan-acetyl-lysine antibody and were immunoblotted with the indicated antibodies. Arrow indicates the NLRC4 band. (F) HEK293T cells were transfected with Flag-tagged NLRC4 along with or without HA-tagged SIRT3. Whole-cell lysates were then immunoprecipitated (IP) using pan-acetyl-lysine and were immunoblotted with the indicated antibody. (G) SIRT3 deacetylates NLRC4 in vitro. NLRC4 (0.2 mg/μL) was purified and incubated with SIRT3 (0.1 mg/mL) with or without NAD ⁺ (1 mM). The acetylation levels were determined by Western blotting. (H) HEK293T cells were transfected with WT or H248Y mutant SIRT3 along with NLRC4. Cell lysates were then immunoprecipitated (IP) using pan-acetyl-lysine and were immunoblotted with the indicated antibodies. Data information: All the results are representative of at least three independent experiments.

Figure 6

Identification of NLRC4 acetylation. (A) List of the identified acetylation Sites of NLRC4. HEK293T cells were transfected with Flag-tagged NLRC4, these cells were harvested and NLRC4 was immunoprecipitated and analyzed by mass spectrometer. (B) NLRC4 acetylation conserved sites. The sequences of NLRC4 in seven species were aligned. K71, K272, K469, K674, K763 of NLRC4 were highlighted in red.

Figure 7

Deacetylation at K71/K272 enhances NLRC4 activation. (A) Flag-tagged NLRC4 and its mutants were transiently expressed in HEK293T cells, and lysates were subjected to immunoprecipitated using pan-acetyl-lysine antibody followed by Western blotting. (B-C) The MS/MS spectrum of modified peptides that include K71 and K272. (D-E) HEK293T cells were transfected with Flag-tagged WT NLRC4 and its acetylation-mimetic (K-to-Q) mutants (D) or K-to-R mutants (E) along with Flag-tagged-pro-caspase-1. Whole-cell lysates were analyzed by Western blotting for the presence of cleaved caspase-1. All results are representative of three independent experiments.