SIRT1 deacetylates RORγt and enhances Th17 cell generation

Abstract

The balance of effector and regulatory T cell function, dependent on multiple signals and epigenetic regulators, is critical to immune self-tolerance. Dysregulation of T helper 17 (Th17) effector cells is associated with multiple autoimmune diseases, including multiple sclerosis. Here, we report that Sirtuin 1 (SIRT1), a protein deacetylase previously reported to have an antiinflammatory function, in fact promotes autoimmunity by deacetylating RORγt, the signature transcription factor of Th17 cells. SIRT1 increases RORγt transcriptional activity, enhancing Th17 cell generation and function. Both T cell-specific Sirt1 deletion and treatment with pharmacologic SIRT1 inhibitors suppress Th17 differentiation and are protective in a mouse model of multiple sclerosis. Moreover, analysis of infiltrating cell populations during disease induction in mixed hematopoietic chimeras shows a marked bias against Sirt1-deficient Th17 cells. These findings reveal an unexpected proinflammatory role of SIRT1 and, importantly, support the possible therapeutic use of SIRT1 inhibitors against autoimmunity.

© 2015 Lim et al.

Figures

Figure 1.

SIRT1 promotes Th17 differentiation ex vivo and in vivo. (A) Freshly isolated naive T (NVT) cells from C57BL/6 (B6) mice were differentiated ex vivo into various effector T cells as indicated (Materials and methods). Thy, thymocytes. SIRT1 expression was determined by Western blot using β-actin as an internal control. (B and C) Naive CD4 T cells from WT B6 mice were differentiated into Th17 cells in the presence of various amounts of nicotinamide (B) or Ex-527 (C). (D) Relative gene expression between 1.25 mM nicotinamide treated and untreated cells was determined for the indicated genes by qPCR. (E) Th17 cell protein lysates from WT and Sirt1−/− mice were subjected to Western blot analysis to visualize SIRT1 knock-out efficiency. (F) Naive T cells from WT and Sirt1−/− mice were differentiated into Th17 cells with the indicated amounts of TGF-β1. (G) Naive CD4 T cells from WT and Sirt1−/− mice were differentiated into Th17 cells with 2.5 ng/ml of TGFβ1 in the presence of 12.5 µM of Ex-527. (H) 12 wk after engraftment, total splenocytes from CD45.1 WT/CD45.2 Sirt1−/− (1:1) mixed hematopoietic chimeras were stained with antibodies against IL-17A, IL-17F, IL-22, IFN-γ, IL-2, and Foxp3, together with CD4, CD45.1, and CD45.2. Percentage of cytokine-expressing cells was measured by flow cytometry 3–6 d after differentiation. Error bars represent (±SEM) for data from three (A, B [right], C [right], D, and G), four (B [left], C [left]), or five (H, total 14 mice/condition) independent experiments. Also, representative data from one (E), two (F), and three (A and G) independent experiments is shown. Significance values are based on a two-tailed Student’s t test. *, P < 0.05; **, P < 0.01.

Figure 2.

SIRT1 interacts with RORγt. (A) Flag-tagged WT or H363Y mutant SIRT1 was immunoprecipitated from transfected 293T cells and probed as indicated. (B) RORγt was immunoprecipitated from thymocytes and Th17 cells, and probed with antibody against SIRT1. (C) Immunoprecipitation using lysates of 293T cells co-transfected with constructs encoding SIRT1 and various deletion mutants of RORγt. Relative binding was calculated by normalizing the ratio of immunoprecipitated RORγt/SIRT1 to the ratio of input RORγt/SIRT1. (D) Acetylation of Flag-tagged RORγt immunoprecipitated from 293T cells transfected with various acetyltransferases and Flag-RORγt. (E and F) Acetylation of Flag-RORγt co-transfected with p300 and WT or H363Y mutant SIRT1, in the absence (E) or in the presence (F) of nicotinamide and Ex-527. Equal amounts of Flag-RORγt were loaded (D–F). Representative data are shown from four (A), three (B and E), and two (D) independent experiments, and combined data are shown from three (C and F) independent experiments with error bars representing ±SEM.

Figure 3.

SIRT1 deacetylates RORγt. (A) Flag-RORγt immunoprecipitated from 293T cells expressing p300 and RORγt (black bars), or p300, RORγt, and SIRT1 (red bars) was analyzed by mass spectrometry, identifying the indicated acetylated lysines and changes in their respective MS1 precursor ion abundances. (B) Structural model of acetylated RORγt with DNA. The N-terminal DNA-binding domain of RORγt with DNA is shown. The blue globules indicate side chains of the K69, K81, and K99 residues that are strongly acetylated by p300 and deacetylated by SIRT1. (C and D) Acetylation of endogenous RORγt immunoprecipitated from thymocytes (C) and Th17 cells (D) was determined by Western blot. (E) MS/MS spectrum of acetylated peptide containing Kac-99 from RORγt. (F) Targeted MRM-HR showing the protein level of RORγt in Th17 cells (top panel, nonacetylated peptide ELFSTDVESPEGLSK, m/z at 819.402+), as well as potential RORγt acetylation level changes at residue Kac-99 (bottom, acetylated peptide DSLHAEVQKacQLQQQQQQEQVAK, m/z at 659.09++++). (G and H) Acetylated tryptic peptides were monitored by MRM-HR, in technical duplicates between WT and Sirt1−/− pooled mice (10 mice per each genotype), to quantify differences in RORγt acetylation levels in Th17 cells (G) and in thymocytes (H). Peptides assayed for each acetylated lysine were as follows: Kac-69, LQKacCLALGMoxSR, m/z at 667.852+ (thymocytes only); Kac-81, DAVKacFGR, m/z at 417.732+; Kac-87/88, MSKacKacQR, m/z at 431.232+; and Kac-99, DSLHAEVQKacQLQQQQQQEQVAK, m/z at 878.45+++ and 659.09++++. Peak areas were normalized to account for RORγt protein level changes. Significance was assessed using a two-tailed Student’s t test (P < 0.05). Combined data from two (A) independent experiments or representative data from two (D; 3 mice/genotype) and three (C; 10 mice/genotype) independent experiments are shown. *, P < 0.05.

Figure 4.

SIRT1 modulates IL-17A and IL-2 transcriptional activity via deacetylation of RORγt. (A) Jurkat cells were transiently transfected with RORγt, a TK-driven Renilla luciferase vector, and either IL-17A (blue symbols) or IL-2 (red symbols) promoter-driven firefly luciferase reporter constructs. (B and C) Jurkat cells were transiently transfected with TK Renilla luciferase and either an IL-17A firefly reporter construct (B) or an IL-2 firefly reporter construct (C), as well as WT, single, double, or triple RORγt mutants. Firefly luciferase values were normalized to Renilla luciferase values. Error bars represent ±absolute value of differences between duplicate samples. (D) CD4 T cells from B6 mice were infected with retrovirus carrying either Thy1.1 alone or Thy1.1 together with WT or mutant RORγt as indicated, and further differentiated under Th0 (top) or Th17 (bottom) conditions. IL-17A secretion was measured. (E) Naive CD4 T cells from WT and Sirt1−/− mice were differentiated into Th17 cells in the presence of either isotype control antibody or IL-2 neutralizing antibody. The relative percentage of Th17 cells was examined. (F) Secreted IL-2 from WT and Sirt1−/− cells cultured under Th17 differentiation conditions was measured by ELISA. Error bars represent ±SEM from triplicate samples. Representative data from two (A and F), or three (B–D) independent experiments or combined data from four (E) independent experiments are shown.

Figure 5.

Deletion of SIRT1 in T cells or chemical inhibition of SIRT1 protects mice from EAE. (A) EAE clinical scores of WT and Sirt1−/− mice after MOG35-55 peptide immunization. (B) EAE clinical scores of WT mice immunized with MOG35-55 peptide followed by treatment with DMSO or Ex-527 at 0, 1, and 2 d after immunization. (C) Representative histological sections of spinal cords of DMSO (left) and Ex-527–treated mice (right) from (B). 28 d after immunization, panels were stained for demyelination (LFB/PAS) and lymphocytic infiltration (H&E). Bars, 200 µm. (D) Quantification of demyelination (left) and number of inflammatory foci (right) for DMSO and Ex-527–treated mice. (E) Quantification of spinal cord infiltrated T cell subsets from DMSO and Ex-527 treated mice. (F–H) WT: Sirt1−/− mixed hematopoietic chimeras were immunized with MOG35-55 peptide. Relative CD4 T cell ratio of Sirt1−/− to WT was calculated (F), spinal cords were analyzed for Foxp3+ and IL-17A+ CD4 T cells (G), or Foxp3+ CD4 T cells producing the indicated cytokines (H) were examined, when the mice reached an EAE clinical score of 3. Error bars represent mean ±SEM. Combined data from two (A; 15 mice/each experiment) and three (B; 5–10 mice/each experiment and F, total 10 mice) independent experiments are shown. Representative or combined data from four (C and D), five (E and H), and seven (G, left) mice are shown. *, P < 0.05, **, P < 0.01.