The type III histone deacetylase Sirt1 is essential for maintenance of T cell tolerance in mice

Although many self-reactive T cells are eliminated by negative selection in the thymus, some of these cells escape into the periphery, where they must be controlled by additional mechanisms. However, the molecular mechanisms underlying peripheral T cell tolerance and its maintenance remain largely undefined. In this study, we report that sirtuin 1 (Sirt1), a type III histone deacetylase, negatively regulates T cell activation and plays a major role in clonal T cell anergy in mice. In vivo, we found that loss of Sirt1 function resulted in abnormally increased T cell activation and a breakdown of CD4⁺ T cell tolerance. Conversely, upregulation of Sirt1 expression led to T cell anergy, in which the activity of the transcription factor AP-1 was substantially diminished. Furthermore, Sirt1 interacted with and deacetylated c-Jun, yielding an inactive AP-1 factor. In addition, Sirt1-deficient mice were unable to maintain T cell tolerance and developed severe experimental allergic encephalomyelitis as well as spontaneous autoimmunity. These findings provide insight into the molecular mechanisms of T cell activation and anergy, and we suggest that activators of Sirt1 may be useful as therapeutic agents for the treatment and/or prevention of autoimmune diseases.

Many self-reactive T cells are eliminated by negative selection during development in the thymus (central tolerance), but leaking of autoreactive T cells into the periphery can occur. One of the additional mechanisms to inactivate self-reactive T cells in the periphery is clonal anergy (peripheral tolerance), which is induced by partial or suboptimal stimulation (1–3). A breakdown of peripheral tolerance is considered an important mechanism in autoimmunity. Activation of T cells requires the cooperative interactions of several transcription factors, including AP-1, NF-κB, and NFAT. Among these transcription factors, AP-1 is selectively inhibited in peripheral T cell tolerance (4). However, the molecular mechanisms by which AP-1 transcriptional activity is inhibited in tolerized autoreactive T cells remain largely unknown.

Sirtuin 1 (Sirt1) is the human ortholog of the yeast Sir2 protein, which is the prototypic class III histone deacetylase (HDAC) (5). This protein contains one HDAC domain that has the deacetylation activity, one nuclear localization sequence, and a coiled-coil–like domain. Sirt1 is highly expressed in the heart, brain, and skeletal muscle and is expressed at very low levels in the kidney and lung (6). In vitro studies indicated that Sirt1 deacetylates a variety of proteins including histones H1, H3, and H4 and may mediate heterochromatin formation (7). Several other proteins besides histones can serve as substrates for Sirt1 (8). Indeed, Sirt1 regulates the tumor suppressor proteins p53 and FOXO3 to suppress apoptosis and promote cell survival. Also, it plays a role in several biological processes including stress resistance, metabolism, differentiation, and aging (5). Mice carrying 2 null alleles of the Sirt1 gene are significantly smaller than wild-type animals at birth and exhibit notable developmental defects of the retina and heart, and both sexes are sterile (9, 10).

Sirt1 is expressed in all tissues but is abundant in the thymus, particularly in CD4⁺CD8⁺ thymocytes, suggesting an involvement of Sirt1 in T cell development. CD4⁺CD8⁺ thymocytes from Sirt1^–/– mice exhibit increased sensitivity to γ irradiation–induced apoptosis (10). Moreover, several studies suggest that Sirt1 may negatively regulate T cell activation. Indeed, treatment of T cells with resveratrol, a Sirt1 activator, suppresses proliferation and cytokine production in vitro (11). Resveratrol suppresses immune functions by inducing lymphocyte apoptosis (12, 13). Downregulation of APC functions is another possible mechanism for the immune-suppressive functions of resveratrol (14). While the mechanisms of resveratrol action remain debatable, its interference with immune function is well established and provides a potential avenue for treatment of autoimmune diseases as well as allograft rejections.

In the present study, we demonstrate that Sirt1 functions as an anergic factor in peripheral CD4⁺ T cell tolerance. Sirt1^–/– mice have elevated immune responses and fail to maintain peripheral tolerance to autoantigens, as exemplified by the presence of anti-nuclear antibodies, systemic lymphocyte infiltration, and increased susceptibility to experimental autoimmune encephalomyelitis (EAE). Sirt1 suppression of AP-1 transcriptional activity likely represents a central mechanism for control of T cell activation and induction of anergy. Indeed, we found that Sirt1 inhibits AP-1 transcriptional activity by deacetylating the AP-1 family transcription factor c-Jun. This previously unrecognized observation provides a molecular mechanism for modulation of T cell activation and manifestation of anergy.

Sirt1 inhibits T cell activation. Sirt1 was highly expressed in lymphoid tissues including the thymus, bone marrow, lymph nodes, and spleen (Supplemental Figure 1; supplemental material available online with this article; doi: 10.1172/JCI38902DS1). However, disruption of Sirt1 expression in mice appeared not to affect T cell development, because the cell surface expression of CD4 or CD8 in Sirt1^–/– thymocytes was comparable to that in heterozygous mice (Supplemental Figure 2A). Similarly, the percentage of CD4⁺ and CD8⁺ mature T cells did not change in peripheral lymphoid tissues such as the spleen. The ratios of B220⁺ B cells to CD3⁺ T cells in the spleens and lymph nodes were also comparable in Sirt1^+/– and Sirt1^–/– mice (Supplemental Figure 2A).

After stimulation with anti-CD3 or anti-CD3 plus anti-CD28 antibodies, Sirt1^–/– T cells showed dramatically increased proliferation and produced more IL-2 compared with Sirt1^+/– T cells (Figure 1, A–C), suggesting that Sirt1 suppresses T cell activation. Sirt1 appeared to inhibit IL-2 transcription without affecting Il2 mRNA stability in T cells because the Il2 mRNA level, but not its half-life, was increased in Sirt1^–/– T cells during activation (Figure 1D). The enhanced activation of Sirt1^–/– T cells was not due to pre-existing activated T cells because the percentages of T cells bearing the activation markers CD69, CD44, and CD25 were similar in heterozygous and mutant mice (Supplemental Figure 2B). Also, the enhanced activation was not due to a higher level of cell surface TCR, as heterozygous and mutant mice displayed similar TCR expression (Supplemental Figure 2C). Thus, we determined that Sirt1 intrinsically inhibits T cell activation. Indeed, deletion of Sirt1 gene expression in vitro by tamoxifen treatment of CD4⁺ T cells from Sirt1^loxp/loxpESR-Cre^TG mice (Supplemental Methods) resulted in a dramatically increased CD4⁺ T cell proliferation (Supplemental Figure 3A), and ecotropic expression of Sirt1 inhibited both Sirt1^+/– and Sirt1^–/– T cell activation (Supplemental Figure 3B).

Figure 1

Sirt1 inhibits T cell activation in vitro Primary CD4⁺ T cells were isolated from splenocytes of Sirt1^+/– and Sirt1^–/– mice. Cells were stimulated with anti-CD3 or anti-CD3 plus anti-CD28, or with PMA plus ionomycin (P+I) as a control, for 3 days. (A) Proliferation was analyzed with a ³H-thymidine incorporation assay. (B) The production of IL-2 was analyzed by ELISA. In A and B, data are from 3 independent experiments (mean ± SD). (C) Flow cytometry of purified CD4⁺ T cells labeled with CFSE and left unstimulated or stimulated with anti-CD3 at the concentrations for 5 days. (D) Mouse primary T cells were isolated from Sirt1^–/– and Sirt1^+/– mice and cultivated with anti-CD3 plus anti-CD28 for 24 hours. Cells were then treated with 10 μg/ml of actinomycin D (Act D) for different amounts of time as indicated. The levels of mouse Il2 mRNA were analyzed by real-time RT-PCR. The production of IFN-γ (E) and IL-5 (F) by CD4⁺ T cells after 3-day stimulation was analyzed by ELISA. In E and F, data are from 3 independent experiments (mean ± SD).

It is likely that T cell hyperresponsiveness driven by Sirt1 deficiency is due to signals downstream of the TCR. This hypothesis was drawn from the observation that upon stimulation with PMA plus ionomycin, Sirt1^–/– T cells exhibited a significant increase in proliferation and IL-2 production (Figure 1, A and B). Since PMA directly activates protein kinase (15) and ionomycin forms a lipid-soluble calcium complex to convey Ca²⁺ across the hydrocarbon region of the cell membrane (16), Sirt1 likely targets signaling molecules or transcription factors downstream of the TCR. Furthermore, similar to Cbl-b^–/– T cells (17), the Sirt1^–/– T cells exhibited a full-scale activation when stimulated with anti-CD3 antibody alone, whereas Sirt1^+/– T cells showed only minimal activation under the same stimulation conditions (Figure 1, A–C). These results suggest that TCR signaling without costimulation is sufficient for activation of Sirt1^–/– T cells.

Next, we analyzed the effect of Sirt1 on the production of both Th1 and Th2 cytokines by CD4⁺ T cells. When stimulated with anti-CD3 or anti-CD3 plus anti-CD28 in vitro, Sirt1^–/– CD4⁺ T cells produced more Th1 cytokine IFN-γ and Th2 cytokine IL-5 than did Sirt1^+/– CD4⁺ T cells (Figure 1, E and F). The recall experiments using lymphocytes from mice immunized with OVA indicated substantial increases of both IFN-γ and IL-5 production by Sirt1^–/– T cells compared with Sirt1^+/– T cells (Figure 2, C and D). These results suggest that Sirt1 inhibits the productions of both Th1 and Th2 cytokines by CD4⁺ T cells.

Figure 2

Analysis of T cell–mediated immune responses. Sirt1^+/– and Sirt1^–/– mice were immunized with 200 μg OVA protein in CFA. (A–D) Seven days later, mice were euthanized and total splenocytes were cultured with different amounts of OVA. (A–D) Proliferation (A) and production of IL-2 (B), IFN-γ (C), and IL-5 (D) were determined. Experiments were repeated 3 times using 6 Sirt1^+/– and 6 Sirt1^–/– mice. Representative data are shown. (E and F) Sera were obtained 10 days after immunization with OVA/CFA (E) or 7 days after a second immunization with OVA/IFA (F). The concentrations of each isotype of immunoglobulin were determined by ELISA. The arbitrary units of IgG1 shown in the figure were determined after 1:1,000 dilution. Data are from 5 Sirt1^+/– and 5 Sirt1^–/– mice (mean ± SD).

Sirt1 suppresses T cell–dependent immunity in mice. To determine the effect of Sirt1 deficiency on T cell activation in vivo, 6- to 8-week-old Sirt1^–/– mice and their heterozygous littermates were immunized subcutaneously with chicken OVA protein emulsified in CFA, and their OVA-specific T cell responses were analyzed 7 days later. The results indicated that proliferation as well as IL-2 production by Sirt1^–/– T cells were dramatically increased compared with Sirt1^+/– T cells (Figure 2, A and B), suggesting that Sirt1 functions as a negative regulator of antigen-specific T cell activation in vivo.

To determine the effects of Sirt1 on T cell–dependent humoral immune responses, OVA-specific antibodies were measured after the primary immunization with OVA plus CFA as well as after boosting with OVA plus incomplete Freund’s adjuvant (IFA). The results indicated that Sirt1^–/– mice had increased antigen-specific antibodies of both IgM and IgG isotypes in the primary and secondary responses, suggesting that Sirt1 deficiency sustained a more vigorous T cell–dependent humoral response (Figure 2, E and F).

The elevated immune response of Sirt1^–/– T cells was not the consequence of altered APC function in Sirt1^–/– mice, because the proliferation of Sirt1^–/– T cells showed comparable levels when they were stimulated with Sirt1^+/– or Sirt1^–/– APCs (Supplemental Figure 4A). In addition, expression of costimulatory molecules such as CD80 and CD86 on APCs was not affected by Sirt1 deficiency (Supplemental Figure 4B). Overall, these findings suggest that Sirt1 is a negative regulator of T cell activation.

Sirt1 is required for peripheral CD4⁺ T cell tolerance. TCR ligation in the absence of costimulation gives rise to a state of long-term functional unresponsiveness known as anergy (1–3). Similar to Cbl-b^–/– T cells (17), Sirt1^–/– T cells were fully activated when they were stimulated with anti-CD3 alone without any costimulations (Figure 1, A–C). This suggests that loss of Sirt1 function overrides costimulation, leading to breakdown of peripheral T cell tolerance. To test whether loss of Sirt1 leads to a breakdown of tolerance in vivo, we bred the Sirt1^+/– mice with OT-II TCR transgenic mice (18) and generated OT-II TCR Sirt1^+/– and OT-II TCR Sirt1^–/– mice. The animals (with 90% or higher TCR Vβ5 chain expression) were then given OVA_323–339 peptide in PBS intravenously, and their splenic T cells were tested for antigen-induced proliferation and IL-2 production. The results showed that while Sirt1^+/– OT-II T cells were unresponsive, the Sirt1^–/– OT-II cells had significant proliferative responses and increased IL-2 production (Figure 3, A and B, and Supplemental Figure 5, A and B). Similar results were obtained when purified CD4⁺ T cells from OVA_323–339 peptide–treated mice were cocultured with APC and OVA peptide (Supplemental Figure 6). Therefore, we concluded that Sirt1 deficiency causes a breakdown of CD4⁺ T-cell tolerance in vivo.

Figure 3

Sirt1 is required for CD4⁺ T cell tolerance. (A and B) In vivo tolerance was induced by treatment of Sirt1^+/–OTII (left) and Sirt1^–/–OTII (right) mice with OVA_323–339 peptides, or with PBS as a control, by tail vein injection. Total lymphocytes from lymph nodes were isolated from treated mice, and proliferation was analyzed. Data are from 5 Sirt1^+/– and 5 Sirt1^–/– mice. (C and D) CD4⁺ T cells were purified from Sirt1^+/–OTII (left) and Sirt1^–/–OTII (right) mice and adoptively transferred into T cell–null mice. CD4⁺ T cell tolerance in the recipient mice was analyzed as described in A and B. (E) The proliferation of Sirt1^–/– and Sirt1^+/– CD4⁺ T cells that underwent in vitro anergy induction was determined. (F and G) Freshly purified CD4⁺ T cells (naive), CD4⁺ T cells after 24 hours of stimulation with anti-CD3 plus anti-CD28 (active), and CD4⁺ T cells tolerized in vitro by ionomycin treatment (anergic) were used. (F) The mRNA levels of Sirt1 in naive, activated, and anergic T cells were analyzed with real-time RT-PCR, using β-actin as a reference standard. Data are from 3 independent experiments. (G) Whole cell lysates of naive, activated, and anergic T cells were serially diluted to 1:2 and 1:4. The protein expression of Sirt1 was analyzed by Western blot, and the same membrane was reprobed with anti–β-actin antibody. Data are from 3 independent experiments (mean ± SD).

Using an in vitro T cell anergy induction assay (19) we show that ionomycin treatment failed to induce anergy of Sirt1^–/– T cells. In contrast, proliferation and IL-2 production of Sirt1^+/– T cells were inhibited by ionomycin treatment, and this was reversible by adding exogenous IL-2 (Figure 3E and Supplemental Figure 5C). Ecotropic expression of Sirt1, as described in the Supplemental Methods, inhibited the activation and restored the anergic induction of Sirt1^–/– CD4⁺ T cells (Supplemental Figures 3B and 7A). In addition, in vitro deletion of Sirt1 from CD4⁺ T cells using an inducible Cre expression system resulted in the breakdown of T cell tolerance (Supplemental Figure 7B). Therefore, Sirt1 appears to regulate tolerance independent of its function in T cell development. The resistance of Sirt1^–/– CD4⁺ T cells to tolerance was probably not the result of decreased cell death because annexin V staining revealed comparable percentages of apoptotic cells among Sirt1^+/– and Sirt1^–/– CD4⁺ T cells (Supplemental Figure 8). Together, these results indicate that Sirt1 is required for CD4⁺ T cell tolerance in vitro.

To further assess the role Sirt1 deficiency plays in the breakdown of T cell tolerance, CD4⁺ T cells were isolated from Sirt1^–/– and Sirt1^+/– OT-II mice and adoptively transferred into T cell–null mice, and CD4⁺ T cell tolerance was analyzed. As shown in Figure 3, C and D, injection of OVA_323–339 peptide into the host mice inhibited Sirt1^+/– but not Sirt1^–/– OT-II T cell activation. To determine whether this breakdown of Sirt1^–/– CD4⁺ T cell tolerance was due to increased homeostatic proliferation in the lymphopenic host, we adoptively transferred CFSE-labeled CD4⁺ T cells into T cell–null mice and analyzed cell division. The findings indicate that homeostatic proliferation of Sirt1^+/– and Sirt1^–/– CD4⁺ T cells in the hosts was indistinguishable 7 days after transfer (Supplemental Figure 9).

Increased Sirt1 expression in anergic CD4⁺ T cells. The fact that CD4⁺ T cells of Sirt1^–/– mice were unable to be tolerized suggests that Sirt1 could function as an anergic factor in T cells. Recent studies suggest that anergy induction requires upregulation of the expression of inhibitory proteins such as the E3 ligase Cbl-b (20). We therefore determined whether expression of Sirt1 changes in anergic T cells. Real-time PCR analysis revealed that expression of Sirt1 mRNA was increased 4- to 5-fold in anergic versus naive T cells but increased only slightly in activated T cells (Figure 3F). Western blotting analysis also showed a similar increase at the protein level (Figure 3G). Thus, Sirt1 is upregulated in anergic T cells. In support of this is the finding that TCR stimulation alone, which presumably induces tolerance in vivo, is sufficient for Sirt1 upregulation (data not shown).

Previous studies have identified several genes that are upregulated in anergic CD4⁺ T cells when there is NFAT but not AP-1 transcriptional activation (20–27). We therefore determined whether Sirt1, as a HDAC, induces and/or maintains CD4⁺ T cell tolerance by altering the expression of these anergic genes. Real-time PCR analysis indicated that the expression levels of Cbl-b, DGK-a, EGR2, and EGR3 were comparable between Sirt1^+/– and Sirt1^–/– CD4⁺ T cells even when treated with ionomycin. Interestingly, the transcription of Grail and IKAROS-1 in Sirt1^–/– T cells was reduced, suggesting a functional linkage of Sirt1 with Grail and IKAROS-1 in anergic T cells (Supplemental Figure 10).

Sirt1 inhibits AP-1 transcriptional activity in T cells. The transcription factor AP-1, usually made of c-Jun homodimers or c-Jun/Fos heterodimers, has been identified as a molecular target in T cell clonal anergy (4). However, the underlying molecular mechanism remains largely unknown. Sirt1 is a nuclear protein and has been found to suppress the transcriptional activity of several transcription factors, such as p53. We thus asked whether Sirt1 induces anergy by suppressing AP-1 transcriptional activity in T cells. Indeed, using a luciferase AP-1 reporter system we demonstrated that overexpression of Sirt1 inhibited AP-1 transcriptional activity in a dose-dependent manner (Figure 4A). This suggests that Sirt1 can function as a suppressor of AP-1 transcription factor. To determine whether Sirt1 inhibits AP-1 transcriptional activity in primary T cells, we bred Sirt1^+/– mice with AP-1 luciferase transgenic (AP-1luc^TG) mice (28) and used this specific reporter to evaluate the effect of Sirt1^–/– on AP-1 transcriptional activity. As shown in Figure 4B, after stimulation with anti-CD3 or anti-CD3 plus anti-CD28 for 24 hours, AP-1 luciferase activities were increased in Sirt1^–/–AP-1luc^TG T cells compared with Sirt1^+/–AP-1^TG cells. Also, gel shift experiments demonstrated a significant increase of AP-1 promoter DNA-binding activity in Sirt1^–/– T cells after TCR/CD28 stimulation (Figure 4C). This increased AP-1 transcriptional activation does not appear to result from the elevated activation of the upstream MAPK pathway because both JNK and Erk activation were indistinguishable between Sirt1^+/– and Sirt1^–/– T cells (Supplemental Figure 11). These results indicate that Sirt1 functions as a suppressor of AP-1 in T cells.

Figure 4

Sirt1 inhibits AP-1 transcriptional activity. (A) AP-1 luciferase, control luciferase, and c-Jun expression plasmids were cotransfected into HEK-293 cells with different amounts of Sirt1 expression plasmid DNA. The ratios of AP-1 luciferase activities to control luciferase activity (fold change) are shown. (B) Primary T cells were isolated from Sirt1^+/–AP-1luc and Sirt1^–/–AP-1luc mice and left unstimulated or stimulated with anti-CD3 or anti-CD3 plus anti-CD28 for 24 hours. The luciferase activity of stimulated cells was analyzed. (C) Increased DNA-binding activity in Sirt1^–/– T cells. Nuclear extracts from primary T cells stimulated with anti-CD3 or anti-CD3 plus anti-CD28 were used for gel-shift analysis. Data are from 3 independent experiments (mean ± SD).

Sirt1 inhibits c-Jun acetylation to sustain T cell anergy. Since Sirt1 functions as a deacetylase and c-Jun requires acetylation for its activity, it is possible that Sirt1 operates T cell anergy by suppressing c-Jun acetylation. To test this premise, c-Jun acetylation was compared in activated versus anergic T cells. Indeed, c-Jun was highly acetylated in activated CD4⁺ T cells, while its acetylation was diminished in anergic T cells (in which anergy was induced either in vitro [Figure 5A] or in mice [Figure 5B]) to levels comparable to those of naive T cells. These results provide a direct link between the regulation of c-Jun acetylation and T cell anergy.

Figure 5

Sirt1 deacetylates c-Jun in T cells. (A) c-Jun protein was immuno­precipitated from the lysates of T cells activated or anergized in vitro. The acetylation of c-Jun was detected by anti–acetyl-lysine antibody (top). The same membrane was reprobed with anti–c-Jun antibody (bottom). (B) In vivo T cell anergy was induced by treatment of OT-II TCR transgenic mice with OVA_323–339 peptide, using PBS as a control. CD4⁺ T cells from peptide-treated (anergic) and control (naive and activated with anti-CD3 plus anti-CD28) mice were isolated and restimulated with anti-CD3 plus anti-CD28 for 2 hours. c-Jun acetylation was analyzed as described in A. (C) Primary T cells were isolated from Sirt1^+/– and Sirt1^–/– mice. Purified T cells were stimulated with anti-CD3 or anti-CD3 plus anti-CD28 for 24 hours. c-Jun acetylation in the stimulated Sirt1^+/– and Sirt1^–/– T cells was determined as described in A. (D) T cells from Sirt1^+/– and Sirt1^–/– mice were infected with a retrovirus that carries an empty vector (MIG) or the Sirt1 gene. c-Jun acetylation in these infected T cells was analyzed. (E) HEK-293 cells were transfected with c-Jun, p300 with Sirt1 expression plasmids. c-Jun acetylation in the transfected HEK-293 cells was analyzed as described in A.

The fact that Sirt1 expression is upregulated in anergic T cells implies that Sirt1 may be responsible for impaired c-Jun acetylation. If this hypothesis were correct, increased c-Jun acetylation would be observed in Sirt1^–/– compared with Sirt1^+/– CD4⁺ T cells. Indeed, acetylation of c-Jun was detected in Sirt1^+/– T cells under stimulation with anti-CD3 plus anti-CD28 but not with anti-CD3 alone. In contrast, c-Jun acetylation was increased in Sirt1^–/– T cells (Figure 5C). These results indicate that Sirt1 inhibits T cell activation by suppressing c-Jun acetylation. In particular, anti-CD3 stimulation is sufficient to induce c-Jun acetylation in Sirt1^–/– T cells, indicating that TCR-mediated c-Jun acetylation is inhibited by Sirt1 (Figure 5C). To support this conclusion, we further demonstrated that expression of Sirt1 in T cells inhibited c-Jun acetylation (Figure 5D). TCR stimulation alone usually induces T cell tolerance, which is a state of anergy. Given the observation that Sirt1 expression is upregulated in anergic T cells and that the lack of Sirt1 function causes breakdown of T cell tolerance, these results suggest that Sirt1 maintains T cell tolerance by suppressing c-Jun acetylation. In support of this statement is the observation that overexpression of Sirt1 inhibited c-Jun acetylation in transiently transfected HEK-293 cells (Figure 5E).

Sirt1 interacts with c-Jun independently of JNK. To gain further insight into the interaction of Sirt1 with c-Jun in mouse primary T cells, we analyzed the binding of Sirt1 with c-Jun by coimmunoprecipitation. HEK-293 cells were transfected with Flag-tagged Sirt1 plasmid and/or c-Jun, and anti-flag immunoprecipitation was performed. The findings indicate that c-Jun protein was detected in anti-Flag immunoprecipitates when the cells were transfected with Sirt1 and c-Jun but not in cells transfected with c-Jun alone (Figure 6, A and B). This suggests that Sirt1 interacts with c-Jun. Furthermore, when mouse primary T cells were stimulated with anti-CD3 plus anti-CD28, Sirt1/c-Jun interaction was detectable (Figure 6C). However, Sirt1/c-Jun interaction could not be detected in naive unstimulated T cells. Consistent with this finding, colocalization of Sirt1 with c-Jun in the nuclei was observed in the activated but not naive T cells (Supplemental Figure 13). These results indicate that Sirt1 interacts with c-Jun both in transiently transfected HEK-293 cells and in mouse primary T cells. More interestingly, a constitutive and significantly increased interaction of Sirt1 with c-Jun was detected in anergic T cells (Figure 6D), indicating that Sirt1 constitutively suppresses AP-1 transcription to maintain CD4⁺ T cell tolerance. To support this, immunostaining experiments revealed a brighter Sirt1 expression in anergic T cells (Supplemental Methods), and Sirt1 was well colocalized with c-Jun in the nuclei (Supplemental Figure 14). We determined that the C terminus of Sirt1 was responsible for its interaction with c-Jun because deletion of the C terminus completely abolished Sirt1/c-Jun interaction as well as its ability to inhibit c-Jun acetylation, while deletion of the N terminus did not affect their interaction (Figure 6, A, E, and F), and the C terminus alone was sufficient for its interaction with c-Jun (data not shown).

Figure 6

The Sirt1 C terminus mediates its interaction with c-Jun. (A) Schematic representations of Sirt1 and Sirt1 mutants. The c-Jun interaction patterns of Sirt1 and its mutants as well as their deacetylase activity to inhibit c-Jun acetylation are summarized. (B) Xpress–c-Jun was cotransfected with or without Flag-Sirt1 in HEK-293 cells. Sirt1 protein in the lysates was immunoprecipitated with an anti-Flag antibody. The interaction of c-Jun with Sirt1 was determined with an anti-Xpress antibody (first panel). The expression levels of Sirt1 (second panel), c-Jun (third panel), and actin (fourth panel) in whole cell lysates were analyzed. (C) Primary T cells were activated by anti-CD3 plus anti-CD28 for 16 hours. The interaction of Sirt1 with c-Jun was analyzed as described in B. (D) Expanded T cells were treated with (anergic) or without (rested) ionomycin for 16 hours, followed with 30 minutes of stimulation with anti-CD3 plus anti-CD28. The interaction of Sirt1 and c-Jun was analyzed as described in B. (E) The interaction of c-Jun with Flag-tagged Sirt1 or each of its deletion mutants was analyzed as described in A. (F) c-Jun, p300, and Sirt1 or Sirt1 mutants were cotransfected into HEK-293 cells. c-Jun acetylation was analyzed by immunization of c-Jun and Western blotting with anti–acetyl-lysine antibodies (top panel). The expression levels of c-Jun and Sirt1 or Sirt1 mutants in the whole cell lysates were detected with anti–c-Jun (middle panel) and anti-Flag (bottom panel), respectively.

Since activation signals of T cells provided by TCR/CD28 promote c-Jun phosphorylation by JNK (29, 30), we then tested whether this phosphorylation event was responsible for c-Jun interaction with Sirt1. Treatment of cells with a JNK-specific inhibitor, SP600125, which dramatically inhibited c-Jun phosphorylation, did not affect Sirt1/c-Jun interaction (Supplemental Figure 12A). In addition, deletion of the JNK-docking site or the δ domain of c-Jun, or mutation of the predominant phosphorylation serine and threonine within c-Jun, did not affect its interaction with Sirt1 (Supplemental Figure 12B). Immunostaining experiments revealed that c-Jun is mainly distributed in the cytoplasm of naive T cells and that TCR/CD28 stimulation relocated c-Jun into the nucleus (Supplemental Figure 13). In contrast, Sirt1 remained in both cytoplasm and nucleus in naive unstimulated T cells. Therefore, TCR/CD28 stimulation might enhance c-Jun/Sirt1 interaction by promoting c-Jun nuclear translocation. Interestingly, consistent with our finding of increased Sirt1 expression in anergic T cells, a brighter Sirt1 staining that was well colocalized with c-Jun was observed in anergic T cells (Supplemental Figure 14). Based on these findings, we conclude that Sirt1 is a deacetylase of c-Jun, and Sirt1-mediated deacetylation of c-Jun inhibits AP-1 transcriptional activation.

Sirt1 deficiency results in autoimmunity in mice. Breakdown of self-tolerance in CD4⁺ T cells plays a major role in the development of EAE in mice as well as MS in humans (31). To further investigate the role of Sirt1 in T cell tolerance, Sirt1^–/– and Sirt1^+/– mice were immunized with myelin oligodendrocyte glycoprotein 35–55 (MOG_35–55) peptide to induce EAE and the animals were monitored for signs of paralysis. The findings indicated that Sirt1^–/– mice were more susceptible to EAE, as 87.5% (14 of 16) manifested clinical signs of disease after immunization (Table 1). In contrast, 55% (11 of 20) of the Sirt1^+/– mice showed signs of EAE. In addition, the onset of EAE in the Sirt1^–/– mice was 2 days earlier relative to the onset in Sirt1^+/– mice, and the average clinical score was significantly higher than in control mice (P < 0.005) (Figure 7A and Table 1). Histological analysis of the spinal cord sections revealed more lymphocyte infiltration in Sirt1^–/– mice (Figure 7B). Most of the infiltrating cells were T cells, as confirmed by immunostaining (Figure 7C). Thus, we concluded that Sirt1 deficiency promotes susceptibility to EAE.

Figure 7

EAE development in Sirt1^+/– and Sirt1^–/– mice. EAE was induced by immunization of Sirt1^+/– and Sirt1^–/– mice with MOG peptide. (A) Disease scores were obtained daily for 55 days after immunization, and the average score is shown. (B and C) Spinal cord sections of sick mice were analyzed by H&E staining (B), or infiltrated cells were detected by Alexa Fluor 488–conjugated anti-CD4 antibody (C). Representative images are shown. (D) Sirt1^+/– and Sirt1^–/– mice were immunized with MOG peptide emulsified with CFA. CD4⁺ T cells were isolated from the immunized mice 7 days after immunization. CD4⁺ cells (5 × 10⁶) were adoptively transferred into T cell–null mice, followed by pertussis toxin injection 1 and 2 days after transfer. EAE was analyzed as described in A. (E) Naive CD4⁺ T cells were isolated from Sirt1^+/– and Sirt1^–/– mice and adoptively transferred into T cell–null mice at 5 × 10⁶ cells per mouse. EAE induction and analysis in the recipient mice was performed as described in A.

Table 1

Clinical observation of immunized Sirt1^–/– and Sirt1^+/– mice

Next, we determined whether Sirt1-deficient CD4⁺ T cells are able to transfer EAE to normal animals. Sirt1^+/– and Sirt1^–/– mice were immunized with MOG peptide in CFA. CD4⁺ T cells were isolated and adoptively transferred into T cell–deficient mice. As shown in Figure 7D, mice recipients of Sirt1^–/– CD4⁺ T cells showed signs of EAE as early as 5 days after transfer. In contrast, disease development in mice that received Sirt1^+/– T cells was delayed by 3–4 days. In addition, a significantly increased average clinical score was observed in recipients of Sirt1^–/– versus Sirt1^+/– T cells. Furthermore, experiments using naive Sirt1^+/– and Sirt1^–/– T cells for the adoptive transfer demonstrated that the clinical severity of EAE was also increased in mice that received Sirt1^–/– T cells (Figure 7E). Therefore, increased T cell activation and breakdown of T cell tolerance appear to be critical for the development of autoimmunity in Sirt1^–/– mice.

We then asked whether aging Sirt1^–/– mice (11 months old or older) develop spontaneous autoimmune responses (Supplemental Methods). Sera from Sirt1^–/– mice had higher amounts of anti-nuclear antibodies than did sera from Sirt1^+/– (Supplemental Figure 15A). This was supported by the stronger fluorescence in the nuclei of NIH3T3 cells when the staining was made with the sera from Sirt1^–/– versus Sirt1^+/– mice (Supplemental Figure 15B), which is similar to findings from a recent study of Sirt1^–/– mice on a mixed genetic background (32). Next, we tested whether self-reactive antibodies deposit in the kidney glomerules. Kidney tissue sections were prepared, frozen with OCT, fixed, saturated with normal goat IgM and IgG, and stained with fluorescence-labeled goat anti-mouse IgM or IgG. As shown in Supplemental Figure 17C, deposition of both IgM and IgG was evident in the kidney glomerules of Sirt1^–/– mice, while only background staining was observed in kidney sections of Sirt1^+/– mice. Lymphocyte infiltration was observed in the liver, lung, and kidney of all 5 Sirt1^–/– mice examined. In contrast, no obvious lymphocyte infiltration was observed in tissues from any Sirt1^+/– mice (Supplemental Figure 15D). These results indicate that Sirt1 deficiency results in the development of an autoimmune syndrome in mice.

The CD4⁺CD25⁺FoxP3⁺ Tregs that suppress autoreactive T cells are critical for autoimmune suppression (33, 34), and in some instances TGF-β is essential for Treg function (35). Interestingly, Sirt1 has been shown to destabilize Smad7, a suppressive transcription factor in the TGF-β pathway (36). Smad7 expression strongly affects in vitro Treg differentiation induced by TGF-β (37, 38). Therefore, loss of Sirt1 function might impair the development and/or function of Tregs and consequently contribute to the autoimmune phenotype in Sirt1^–/– mice. To test this hypothesis, we compared the percentages of Tregs between Sirt1^+/– and Sirt1^–/– mice. The percentages of CD4⁺FoxP3⁺ populations, as well as their suppressive functions, were indistinguishable between the 2 strains (Supplemental Figure 16, A and B), indicating that Sirt1 deficiency does not affect Treg development and function.

An alternative for Sirt1^–/– involvement in autoimmunity may be increased Th17 differentiation. To test this premise, we compared the percentages of IL-17⁺ populations between heterozygous and Sirt1^–/– CD4⁺ T cells upon polarization with TGF-β and IL-6 (39). CD4⁺ T cells were purified from Sirt1^+/– and Sirt1^–/– mice and cultured with anti-CD3, anti-CD28, TGF-β, and IL-6 for 5 days. The production of IL-17 and IFN-γ was detected by intracellular staining with anti–IL-17–PE and anti–IFN-γ–FITC, respectively. As shown in Supplemental Figure 18C, the percentages of IL-17⁺ cells were indistinguishable between Sirt1^+/– and Sirt1^–/– T cells, indicating that loss of Sirt1 function does not affect Th17 polarization in vitro. Collectively, our findings suggest that breakdown of CD4⁺ T cell tolerance due to the lack of Sirt1 functions is related to uncontrolled activation of autoreactive T cells.

The findings presented in this report suggest that anergic signals induce upregulation of Sirt1 expression, which suppresses AP-1 transcriptional activity, leading to inhibition of T cell activation and maintenance of peripheral T cell tolerance. A lack of Sirt1 causes a breakdown of peripheral tolerance, and Sirt1^–/– mice are more susceptible to autoimmune diseases.

Autoreactive T cells are generally eliminated by negative selection during thymic development (central tolerance). Self-reactive thymic escapees remain harmless due to the lack of costimulation when they detect antigen, a phenomenon known as peripheral tolerance (1–3). Although the molecular mechanisms underlying peripheral tolerance remain largely unknown, progress has been made that sheds light on how TCR stimulation without CD28 signaling induces unresponsiveness of autoimmune T cells (1, 2). This imbalanced stimulation of autoreactive T cells activates the transcription factor NFAT, possibly together with other unknown transcription factors, for the transcription of genes to induce and maintain peripheral T cell tolerance (19, 40). Recent studies reported that anergy induction is a process that upregulates expression of a cascade of inhibitory proteins including the E3 ubiquitin ligases Cbl-b, Itch, and Grail (20, 41). These upregulated E3 ubiquitin ligases selectively target T cell activators for ubiquitination-mediated degradation and/or functional suppression. Our study here defines what we believe is a new anergic gene, Sirt1, for peripheral T cell tolerance, because TCR-mediated signaling alone was sufficient for its transcription and Sirt1 suppressed T cell responses to TCR/CD28 stimuli. One interesting observation was that Sirt1 transcription was significantly higher in T cells with TCR stimulation alone than with both TCR and CD28 together, suggesting that CD28 stimulation may suppress Sirt1 expression to allow T cells to be activated. This CD28-mediated Sirt1 downregulation seems to depend on TCR signaling, because Sirt1 was not transcribed when naive T cells were stimulated with anti-CD28 in the absence of TCR stimulation (data not shown). Thus, it is likely that binding of the MHC/peptide complex to TCRs without costimulation induces Sirt1 expression and the consequent T cell anergy, while ligation of the TCR and CD28 costimulatory molecule leads to downregulation of Sirt1, allowing for T cell activation. How the CD28-mediated signal blocks Sirt1 transcription remains to be defined.

One signature of Sirt1^–/– T cells was that full-scale activation did not require costimulation, suggesting that a “short-cut” signal transduction pathway that links TCR to CD28 elements is put in place by the lack of Sirt1. This short-cut pathway likely plays a major role in the breakdown of T cell tolerance. Similar results have been observed in Cbl-b^–/– T cells showing a vigorous T cell activation independent of CD28 stimulation (17). However, in Sirt1^–/– T cells, unlike Cbl-b^–/– T cells, CD28 stimulation further enhances activation, suggesting that Sirt1 inhibits the signal transduction mediated by both TCR and CD28 in T cells. Signal transduction was inhibited at multiple stages when anergic T cells were stimulated by self-antigen and costimulators. For instance, altered expression of Fyn and Lck protein tyrosine kinases and altered patterns of early tyrosine phosphorylation have been correlated with deficient IL-2 production in anergic T cells (42–47). Initial interpretations suggested that the defect in IL-2 production of anergic T cells emanates from translational regulation because stimulation of anergic cells failed to induce Il2 mRNA (48). A milestone in the understanding of how IL-2 transcription is silenced in anergic T cells transpired from the finding that anergic T cells display selective inhibition of AP-1 transcriptional activity (4). This is consistent with our recent report showing that AP-1 is a molecular target for FoxP3 to maintain the unresponsiveness of Tregs (49). AP-1 transcriptional activation, which is crucial for IL-2 transcription, is triggered by both TCR and CD28 stimulation in T cells (50, 51). When self-reactive T cells see antigen in the absence of CD28 stimulation, Sirt1 expression is upregulated, which inhibits the TCR-mediated AP-1 transcriptional activity. However, costimulation through CD28 counters Sirt1 activity, leading to T cell activation. The study in this report elucidates the mechanism underlying AP-1 inhibition and indicates that Sirt1-mediated c-Jun acetylation leads to AP-1 inhibition in anergic T cells.

Acetylation and deacetylation of transcription factors represent critical processes that dynamically regulate gene transcription (52). In the case of c-Jun, a recent study found that acetylation at lysines 268, 271, and 273 is required for c-Jun transcriptional activation, and mutations of these 3 lysines to arginine completely abolished c-Jun transcriptional activity (53). The finding that c-Jun acetylation is diminished in anergic T cells indicates that AP-1 transcriptional activity is suppressed by c-Jun deacetylation. Sirt1 is solely responsible for the suppression of c-Jun acetylation during induction of T cell anergy because the lack of Sirt1 resulted in hyperacetylation of c-Jun and the breakdown of T cell tolerance. Also, it should be noted that TCR stimulation alone could not induce c-Jun acetylation in naive CD4⁺ T cells, possibly because TCR-mediated signaling upregulates Sirt1 transcription. Indeed, when Sirt1^–/– T cells were stimulated with anti-CD3 antibody alone, c-Jun acetylation was highly detected. Given that TCR stimulation in the absence of costimulation induces T cell tolerance (2), the finding of Sirt1-mediated AP-1 deacetylation defines what we believe is a novel molecular mechanism underlying T cell tolerance.

Recent studies have found that Sirt1 interacts with AP-1 in fibroblasts and epithelial cells (54, 55). We demonstrate here that Sirt1 interacts with c-Jun to form a protein complex that catalyzes c-Jun deacetylation in anergic T cells. Protein-protein interactions are specifically regulated by extracellular stimuli. Indeed, Sirt1/c-Jun interaction requires TCR/CD28 stimulation because their interaction is not detectable in naive T cells. It is surprising that Sirt1 interacts with c-Jun independent of JNK activation, because treatment with a JNK-specific inhibitor or mutation of the phosphorylation sites within c-Jun did not affect the interaction, suggesting that TCR/CD28 signaling regulates Sirt1/c-Jun interaction by other mechanisms, which is interesting to further characterize. Therefore, TCR/CD28 signaling regulates T cell activation and tolerance not only by altering Sirt1 transcription but also by controlling its access to substrate proteins such as c-Jun. Our laboratory is currently investigating the precise mechanisms underlying the regulation of Sirt1/c-Jun interaction by activation and/or anergic signals in T cells.

Overall, the findings that Sirt1 inhibits T cell activation and is required for T cell tolerance imply that activators of Sirt1 might be useful as therapeutic reagents for the treatment/prevention of autoimmune diseases such as MS, rheumatoid arthritis, and type 1 diabetes. Indeed, a Chinese herbal medicine, Huzhang (polygonum cuspidatum), which is one of the richest known sources of Sirt1 activator, resveratrol, has been widely used for the treatment of autoimmune diseases, particularly rheumatoid arthritis, in China. Resveratrol has been found to attenuate EAE development by suppressing T cell activation in mice (56). Also, Sirt1 activators have been successfully used for treatment of type 2 diabetes (57). Finally, the findings that Sirt1 inhibits T cell activation and is required for T cell tolerance suggest that a Sirt1 activator may help in the treatment of both autoimmune diseases and type 2 diabetes.

View Supplemental data

We thank Eric Verdin (Gladstone Institute of Virology and Immunology, University of California, San Francisco, California, USA) for Sirt1 expression plasmid and Fernando Macian (Albert Einstein College of Medicine, Bronx, New York, USA.) for providing the protocol of in vitro T cell anergy induction. This study was supported by NIH grants RO1AI079056 and DK083050 (“Type I Diabetes Pathfinder Award”) (to D. Fang) and 2RO1 NS37406 and 1RO1 NS057194 (to H. Zaghouani). S. Shannon was supported by a Fellowship from University of Missouri College of Biological Science.

Address correspondence to: Deyu Fang, Department of Otolaryngology — Head and Neck Surgery and Department of Molecular Microbiology and Immunology, University of Missouri School of Medicine, M616 Medical Sciences Bldg., Columbia, Missouri 65212, USA. Phone: (573) 882-4593; Fax: (573) 882-4287; E-mail: fangd@health.missouri.edu.

Conflict of interest: The authors have declared that no conflict of interest exists.

Reference information: J. Clin. Invest.119:3048–3058 (2009). doi:10.1172/JCI38902.

Jinping Zhang’s present address is: Department of Microbiology, Columbia University College of Physicians and Surgeons, New York, New York, USA.

Sang-Myeong Lee, Beixue Gao, Weimin Chen, and Deyu Fang’s present address is: Department of Pathology, Northwestern University Feinberg School of Medicine, Chicago, Illinois, USA.

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