Histone deacetylases 6 and 9 and sirtuin-1 control Foxp3+ regulatory T cell function through shared and isoform-specific mechanisms - PubMed (original) (raw)

Histone deacetylases 6 and 9 and sirtuin-1 control Foxp3+ regulatory T cell function through shared and isoform-specific mechanisms

Ulf H Beier et al. Sci Signal. 2012.

Abstract

Therapeutic inhibition of the histone deacetylases HDAC6, HDAC9, or sirtuin-1 (Sirt1) augments the suppressive functions of regulatory T cells (T(regs)) that contain the transcription factor Foxp3 (Forkhead box P3) and is useful in organ transplant patients or patients with autoimmune diseases. However, it is unclear whether distinct mechanisms are involved for each HDAC or whether combined inhibition of HDACs would be more effective. We compared the suppressive functions of T(regs) from wild-type C57BL/6 mice with those from mice with either complete or cell-specific deletion of various HDACs, as well as with those of T(regs) treated with isoform-selective HDAC inhibitors. The improvement of T(reg) suppressive function mediated by inhibition of HDAC6, but not Sirt1, required an intact heat shock response. Although HDAC6, HDAC9, and Sirt1 all deacetylated Foxp3, each protein had different effects on transcription factors that control expression of the gene encoding Foxp3. For example, loss of HDAC9, but not other HDACs, was associated with stabilization of the acetylated form of signal transducer and activator of transcription 5 (STAT5) and promoted its transcriptional activity. Thus, targeting different HDACs increased T(reg) function through multiple and additive mechanisms, which suggests the therapeutic potential for using combinations of HDAC inhibitors in the management of autoimmunity and organ transplantation.

PubMed Disclaimer

Conflict of interest statement

Competing interests: None.

Figures

Fig. 1

Fig. 1

The heat shock response is required to improve Treg function in response to inhibition of HDAC6, but not Sirt1. (A) Increased Hsp90 K294 acetylation (aHsp90) in HDAC6−/− Tregs (H6) and HDAC9−/− Tregs (H9), and to a lesser degree in fl-Sirt1/CD4cre Tregs (SC). Densitometry data pooled from two experiments. Immunofluorescence of stimulated Tregs showed nuclear translocation of HSF1, mostly in HDAC6−/− cells, whereas fl-Sirt1/CD4cre Tregs exhibited the least amount of intranuclear HSF1. (B) Hsp70−/− Tregs had diminished suppressive function. Data are from three experiments. (C) Hsp70−/− Tregs were less responsive to ACY-738 (HDAC6i) than WT Tregs, especially under conditions of heat shock. Data are from three experiments. (D) Sirt1-deficient Tregs exhibited a trend to increased acetylation of Hsp90 compared to wild-type (WT) Tregs. Densitometry data pooled from two experiments. SF, fl-Sirt1/Foxp3cre. (E) Transcriptional repression of genes involved in the heat shock response in fl-Sirt1/CD4cre Tregs as analyzed by microarrays, with data shown after z-score transformation. (F) Apoptosis-free survival of Tregs after heat shock. (G) The suppressive function of Tregs was improved by inhibition of Sirt1 (with EX-527) in the absence of Hsp70 and under conditions of heat shock. Data are from four experiments. Effector T cells (Teff) and APCs used in the suppression assays were from (B) WT or (C and G) Hsp70−/− mice. WT refers to the appropriate control mouse strain, that is C57BL6 mice for (A), (D), (E), and (F) or B6/129 mice for (B), (C), and (G). *p<0.05, **p<0.01, and ***p<0.001. Scale bar = 10 μm. Additional statistics for (C) and (G) are available in tables S1 and S2.

Fig. 2

Fig. 2

Functions of HDAC6 in Foxp3+ Tregs that are independent of the heat shock response. (A) Immunofluorescence showing the translocation of HDAC6 into the nuclei of WT Tregs after stimulation with antibodies against CD3ε and CD28. (B) Western blotting analysis of Treg lysates indicated that Foxp3 is more abundant in HDAC6−/− cells than in WT cells. Densitometry data pooled from of three experiments. (C) Co-immunoprecipitation of HDAC6 and Foxp3 in WT Treg lysates. (D) Immunoprecipitation studies of Treg lysates indicated that Foxp3 is more acetylated in HDAC6−/− Tregs than in WT Tregs. Densitometry data pooled from two experiments. (E) Proximity ligation assays showed more acetylated Foxp3 in HDAC6−/− Tregs than in WT Tregs. (F) Quantification of proximity ligation assays with BlobFinder and statistical analysis with the Kruskal-Wallis test. *P<0.05, ***P<0.001. Data shown as boxplot with the median, 25th, and 75th quartiles; whiskers indicate the 10th and 90th percentiles. Scalebar: 10 μm.

Fig. 3

Fig. 3

Deletion of HDAC9 stabilizes the acetylation, phosphorylation, and transcriptional activity of STAT5. (A) Analysis of Treg lysates indicated that STAT5 was relatively more phosphorylated in HDAC9−/− cells than in WT cells. Densitometry data pooled from three experiments. (B) Conceptual model of the acetylation of STAT5, which protects it from losing its transcriptionally active, phosphorylated dimeric form. (C) Proximity ligation assay showed prominent acetylation of STAT5 in HDAC9−/− Tregs compared to that in WT Tregs. (D) Quantification of the data in (C) was performed as described for Fig. 2F. (E) Microarray analysis showed STAT5-dependent signaling in HDAC9−/− Tregs compared to WT Treg (n=3/group) based on previously reported STAT5 targets (19). (F) Further STAT5 signaling as well as other transcriptional alterations promoted a suppressive phenotype in HDAC9−/− Tregs. Data are shown after z-score transformation. *IL-10 not significant. Scale bar: 10 μm. Trp, transformation-related protein; Mcl, myeloid leukemia cell differentiating protein; Tnfsf, tumor necrosis factor superfamily; Id, inhibitor of DNA; Lta, lymphotoxin-α; Nos, NO synthase; Mdm2, an E3 ubiquitin ligase; Scl2a1, GLUT1; Trim, tripartite motif; Klk, Kallikrein; ptgs, post-transcriptional gene silencing; bmp2k, bone morphogenetic protein inducible kinase-2; Cd2ap, CD2-associated protein; Ikbke, Inhibitor of nuclear factor κB kinase ε; Iigp, IFN-inducible GTPase; Xiap, X-linked inhibitor of apoptosis; Fas, CD95; Bcl, B-cell lymphoma; mettl, methyltransferase-like; St8sia, ST8 α-N-acetyl-neuraminide α-2,8-sialyltransferase-4.

Fig. 4

Fig. 4

Combined targeting of HDACs can further augment the suppressive function of Tregs. (A) Treg suppression assays with WT effector T cells and APCs, as well as with WT or HDAC-deficient Tregs with or without one or two specific HDAC inhibitors. We used EX-527 (5 μM) and ACY-738 (1 μM) to inhibit Sirt1 and HDAC6, respectively. (B) WT Tregs treated with Sirt1i, HDCA6i, or both showed increased suppressive function compared to Tregs treated with single HDAC6i or Sirt1i, or vehicle control treatment. (C) HDAC6−/− HDAC9−/− double knockout Tregs showed stronger suppressive function compared with WT and single knockout Tregs. Data are representative of three independent experiments.

Fig. 5

Fig. 5

Combined pharmacologic targeting of HDAC6 and Sirt1 is additive in improving the suppressive function of Tregs and preserving Foxp3 in vivo. (A) Homeostatic proliferation assays showed increased suppressive function of Tregs in mice after individual treatment with either Sirt1i (EX-527, 1 mg/kg/d i.p.) or HDAC6i (Tubastatin, 40 mg/kg/d i.p.) as well as further improvement with combined treatment. (B) Gating strategy to identify cell populations of interest in spleens harvested from B6/Rag1−/− mice; combined treatment sample shown. Red labeled boxes identify populations of cells subsequently analyzed in the indicated panels. (C) Ki67 is reduced in host non-CD4+ cells treated with Sirt1i or HDAC6i. (D) Foxp3+ was better preserved in Tregs from mice treated with Sirt1i and HDAC6i, especially in combination, than in untreated mice. (E) Tregs from mice treated with Sirt1i, HDAC6i, and combined treatment groups had a more activated phenotype (Ki67) than did untreated cells. (F) Neither Sirt1i nor HDAC6i improves the induction of Tregs from effector T cells. Data are from three mice per group (N=15). *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 6

Fig. 6

Combined genetic targeting of HDAC6 and HDAC9 does not substantially improve the suppressive function of Tregs in vivo. (A) Homeostatic proliferation assays showed improved suppression of effector T cells by Tregs with either individual deficiency in either HDAC6 or HDAC9, but no added benefit from combined deletion. (B) Preservation of Foxp3 was not changed by combined knockout of HDAC6 and HDAC9. (C) Ki67 abundance was increased in HDAC6−/− HDAC9−/− double knockout Treg compared to that in single knockout and WT cells. Data are from three to four mice per group (N=18). The gating strategy used was similar to that in Fig. 5B. *P < 0.05, **P < 0.01, and ***P <0.001.

Similar articles

Cited by

References

    1. Naesens M, Kuypers DR, Sarwal M. Calcineurin inhibitor nephrotoxicity. Clin J Am Soc Nephrol. 2009;4:481–508. - PubMed
    1. Fishman JA. Infection in solid-organ transplant recipients. N Engl J Med. 2007;357:2601–2614. - PubMed
    1. Feuerer M, Hill JA, Mathis D, Benoist C. Foxp3+ regulatory T cells: differentiation, specification, subphenotypes. Nat Immunol. 2009;10:689–695. - PubMed
    1. McMurchy AN, Bushell A, Levings MK, Wood KJ. Moving to tolerance: Clinical application of T regulatory cells. Semin Immunol. 2011;23:304–313. - PMC - PubMed
    1. Hori S, Nomura T, Sakaguchi S. Control of regulatory T cell development by the transcription factor Foxp3. Science. 2003;299:1057–1061. - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources