Sirtuin-1 targeting promotes Foxp3+ T-regulatory cell function and prolongs allograft survival - PubMed (original) (raw)

Sirtuin-1 targeting promotes Foxp3+ T-regulatory cell function and prolongs allograft survival

Ulf H Beier et al. Mol Cell Biol. 2011 Mar.

Abstract

Sirtuin 1 (Sirt1), a class III histone/protein deacetylase, is central to cellular metabolism, stress responses, and aging, but its contributions to various host immune functions have been little investigated. To study the role of Sirt1 in T cell functions, we undertook targeted deletions by mating mice with a floxed Sirt1 gene to mice expressing CD4-cre or Foxp3-cre recombinase, respectively. We found that Sirt1 deletion left conventional T-effector cell activation, proliferation, and cytokine production largely unaltered. However, Sirt1 targeting promoted the expression of Foxp3, a key transcription factor in T-regulatory (Treg) cells, and increased Treg suppressive functions in vitro and in vivo. Consistent with these data, mice with targeted deletions of Sirt1 in either CD4(+) T cells or Foxp3(+) Treg cells exhibited prolonged survival of major histocompatibility complex (MHC)-mismatched cardiac allografts. Allografts in Sirt1-targeted recipients showed long-term preservation of myocardial histology and infiltration by Foxp3(+) Treg cells. Comparable results were seen in wild-type allograft recipients treated with Sirt1 inhibitors, such as EX-527 and splitomicin. Hence, Sirt1 may inhibit Treg functions, and its targeting may have therapeutic value in autoimmunity and transplantation.

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Figures

FIG. 1.

FIG. 1.

Differential effects of cell activation on sirtuin gene expression by conventional T cells versus Tregs. Expression of sirtuin genes 1 to 7 (qPCR, mean ± SD, 3/group) by resting and activated T-effector cells and Tregs is shown. Activation was induced for 24 h using CD3ɛ MAb (1 μg/ml) and irradiated APC, and data from stimulated versus unstimulated cells (n = 3/group) were assessed (*, P < 0.05; **, P < 0.01; ***, P < 0.001 [versus results for unstimulated cells]).

FIG. 2.

FIG. 2.

Targeted deletion of Sirt1 in T cells and Tregs. (A) Expression of Sirt1 by WT Tregs (CD4+ CD25+, isolated by magnetic beads, 85% Foxp3+ Treg purity) versus corresponding cells from fl-Sirt1/CD4cre or fl-Sirt1/Foxp3cre mice (qPCR, mean ± SD, 3/group; **, P < 0.01 versus results for WT). (B and C) Flow cytometric analysis of T cell populations in thymi, peripheral lymph nodes (LN), and spleens of WT mice or mice with deletion of Sirt1 using CD4cre (B) or Foxp3cre (C); the percentage of each population is indicated, and data are representative of 3 mice/group.

FIG. 3.

FIG. 3.

Sirt1 deletion does not alter T-effector cell activation. (A) In vitro stimulation of CFSE-labeled CD4+ CD25− T-effector cells showed no difference in proliferation between fl-Sirt1/CD4cre and WT cells upon activation by CD3ɛ ± CD28 MAb and APC (1 × 106/ml). (B) Parent-to-F1 assay: CFSE-labeled T-effector cells from WT or fl-Sirt1/CD4cre mice (both H-2b) were injected into H-2b/d mice. After 3 days, injected cells (H-2d negative) were analyzed for proliferation, cellular activation, and cytokine production. For all parameters shown, data from fl-Sirt1/CD4cre and WT T-effector cells were comparable; error bars represent results of 3 independent assays (P > 0.05 in all cases).

FIG. 4.

FIG. 4.

Sirt1 and Treg function. (A) Comparison of the ability of Sirt1−/− versus WT Tregs to suppress proliferation of CFSE-labeled WT effector T cells in vitro, showing the enhanced suppressive function of Sirt1−/− Tregs (*, P < 0.05; **, P < 0.01 [versus results for WT Tregs]). (B) In vitro suppression assay showing effect of the sirtuin inhibitor splitomicin on WT Treg function; residual proliferation of CFSE-labeled T cells is shown in each panel. (C) In vitro suppression assay showing effect of the Sirt1-specific inhibitor EX-527 on Treg function; residual proliferation of CFSE-labeled T cells is shown in each panel. (D) Homeostatic proliferation showed enhanced suppressive function of Sirt1 deletion Tregs in vivo (**, P < 0.01; ***, P < 0.005). Abbreviations: SC, fl-Sirt1/CD4cre; SF, fl-Sirt1/Foxp3cre.

FIG. 5.

FIG. 5.

Sirt1 and Foxp3 expression. (A) qPCR showing upregulation of Foxp3, CTLA-4, and herpesvirus entry mediator (HVEM) mRNA in Sirt1−/− versus WT Tregs (n = 4/group; *, P < 0.05; **, P < 0.01; ***, P < 0.005). (B) Western blotting showing increased Foxp3 protein in Sirt1−/− versus WT Tregs (actin loading control). (C) Immunoprecipitation of Foxp3,= followed by Western blotting for acetylated lysine showed increased Foxp3 acetylation in Sirt1−/− versus WT Tregs. (C) Western blot comparing Sirt1 and p65, both total and acetylated at lysine 310, showing that Sirt1−/− Tregs exhibit more acetylated p65 and total p65. Abbreviations: SC, fl-Sirt1/CD4cre; SF, fl-Sirt1/Foxp3cre.

FIG. 6.

FIG. 6.

Microarray analysis of expression of genes relevant to Treg function. Data displayed are ≥2× differentially expressed Sirt1−/− Tregs compared to WT Tregs (P < 0.05). Data are displayed after z-score transformation; see the text for details. Abbreviations: SC, fl-Sirt1/CD4cre; SF, fl-Sirt1/Foxp3cre; Ifng, IFN-γ; Ccr/ccl, chemokine (C-C motif) receptor/ligand; Xcl, chemokine (C motif) ligand; Icam, intercellular adhesion molecule; Il6ST, IL-6 signal transducer; Fasl, Fas ligand; Hsp, heat shock protein; Tnfrsf, tumor necrosis factor receptor superfamily; Ikbkb, inhibitor of κB kinase beta; Prf, perforin; Traf, TNFR-associated factor; Itga4, integrin alpha 4; Dnmt, DNA methyltransferase; Tgfbr, transforming growth factor β receptor; Pdk, pyruvate dehydrogenase kinase; Sdh, succinate dehydrogenase; Vps33b, vacuolar protein sorting 33B; Idh, isocitrate dehydrogenase; Sucl, succinate-coenzyme A ligase; Cs, citrate synthase; Fh, fumarate hydratase; CyP51, cytochrome P450 family 51; Idi1, isopentenyl-diphosphate delta isomerase; Hmgcs1, 3-hydroxy-3-methylglutaryl-coenzyme A synthase 1; Sqle, squalene epoxidase; Dhcr7, 7-dehydrocholesterol reductase; Lss, lanosterol synthase; Hdac, histone deacetylase.

FIG. 7.

FIG. 7.

Sirt1 and allograft survival. (A) Kaplan-Meier survival curves showing prolonged survival of MHC-mismatched cardiac allografts following Sirt1 deletion in CD4+ T cells (fl-Sirt1/CD4cre) or Sirt1 inhibition versus WT recipients (4 to 5 grafts/group). (B) Allograft histology at 9 days posttransplant shows a similar degree of lymphocyte infiltration but better myocyte preservation, with intact nuclei and cross striations of fl-Sirt1/CD4cre recipients. For low-power (upper) or high-power (lower) magnifications, scale bar equals 200 μm or 50 μm, respectively. (C) Corresponding immunoperoxidase detection at 9 days posttransplant of CD4+, CD8+, and Foxp3+ T cells in cardiac allografts in WT mice or mice with deletion of Sirt1 using CD4cre; considerably more infiltrating Foxp3+ cells are noted in interstitial areas of fl-Sirt1/CD4cre versus WT recipients; scale bar = 50 μm. (D) qPCR analysis of whole-tissue RNA obtained from allografts and native BALB/c hearts (gray box) as a control, showing increased intragraft Foxp3 and decreased IFN-γ and Sirt1 in Sirt1−/− recipients. (E) Flow cytometric analysis of splenic CD4+ T cells shows decreased activation in Sirt1−/− versus WT allograft recipients. (B to E are representative of 2 independent experiments). (F) B6/RAG1−/− cardiac allograft recipients were adoptively transferred with T-effector cells and WT versus fl-Sirt1/CD4cre Tregs; a significant benefit of Sirt1 deletion in Tregs was shown. (G) Likewise, fl-Sirt1/Foxp3cre recipients exhibited prolonged allograft survival compared to WT controls. Statistical analysis: *, P < 0.05; **, P < 0.01; ***, P < 0.005; Mantel-Cox test versus results for untreated WT (A, F, and G) or Student t test versus results for BALB/c heart control (D).

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