Histone/protein Deacetylases Research Articles

Differential Effects of HDAC6 Inhibition Versus Knockout During Hepatic Ischemia-Reperfusion Injury Highlight Importance of HDAC6 C-terminal Zinc-finger Ubiquitin-binding Domain.

Ischemia-reperfusion injury (IRI) causes significant morbidity in liver transplantation among other medical conditions. IRI following liver transplantation contributes to poor outcomes and early graft loss. Histone/protein deacetylases (HDACs) regulate diverse cellular processes, play a role in mediating tissue responses to IRI, and may represent a novel therapeutic target in preventing IRI in liver transplantation. Using a previously described standardized model of murine liver warm IRI, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels were assessed at 24 and 48 h after reperfusion to determine the effect of different HDAC inhibitors. Broad HDAC inhibition with trichostatin-A (TSA) was protective against hepatocellular damage ( P < 0.01 for AST and P < 0.05 for ALT). Although HDAC class I inhibition with MS-275 provided statistically insignificant benefit, tubastatin-A (TubA), an HDAC6 inhibitor with additional activity against HDAC10, provided significant protection against liver IRI ( P < 0.01 for AST and P < 0.001 for ALT). Surprisingly genetic deletion of HDAC6 or -10 did not replicate the protective effects of HDAC6 inhibition with TubA, whereas treatment with an HDAC6 BUZ-domain inhibitor, LakZnFD, eliminated the protective effect of TubA treatment in liver ischemia ( P < 0.01 for AST and P < 0.01 for ALT). Our findings suggest TubA, a class IIb HDAC inhibitor, can mitigate hepatic IRI in a manner distinct from previously described class I HDAC inhibition and requires the HDAC6 BUZ-domain activity. Our data corroborate previous findings that HDAC targets for therapeutic intervention of IRI may be tissue-specific, and identify HDAC6 inhibition as a possible target in the treatment of liver IRI.

Nuclear Coregulatory Complexes in Tregs as Targets to Promote Anticancer Immune Responses.

T-regulatory (Treg) cells display considerable heterogeneity in their responses to various cancers. The functional differences among this cell type are heavily influenced by multiprotein nuclear complexes that control their gene expression. Many such complexes act mechanistically by altering epigenetic profiles of genes important to Treg function, including the forkhead P3 (Foxp3) transcription factor. Complexes that form with certain members of the histone/protein deacetylase (HDAC) class of enzymes, like HDACs 1, 2, and 3, along with histone methyltransferase complexes, are important in the induction and stabilization of Foxp3 and Treg identity. The functional behavior of both circulating and intratumoral Tregs greatly impacts the antitumor immune response and can be predictive of patient outcome. Thus, targeting these regulatory complexes within Tregs may have therapeutic potential, especially in personalized immunotherapies.

Control of Foxp3+ Treg Production, Stability and Function by the Nuclear Co‐regulator, Sin3A

PurposeFoxp3+ T‐regulatory (Treg) cells are well established as a crucial component of immune homeostasis and self‐tolerance. Histone/protein deacetylase (HDAC) activity within Foxp3+ Tregs plays an important role in regulation of gene expression. HDAC1 and ‐2 regulate Foxp3 expression and play critical roles in the control of Treg functions and properties. Less understood, are the contributions made by proteins which form large complexes with HDAC1 and ‐2, such as the paired amphipathic helix protein, Sin3a.MethodsWe sought to evaluate the contributions Sin3a makes to Tregs by undertaking its conditional deletion in Foxp3+ cells.ResultsSin3a‐/‐Foxp3Cre mice developed severe, systemic autoimmunity, leading to death within 2‐3 weeks of birth. These mice had greatly enlarged superficial lymph nodes, massive inflammatory lesions in lungs, skin, livers and kidneys and developed autoantibodies to islet cells, striated muscles, keratin, endomysium and gastric parietal cells. Percentages of Foxp3+ Tregs in the secondary lymphoid organs of Sin3a‐/‐Foxp3Cre mice were vastly lower than in controls (p values ranging from &gt;0.01 to &gt;0.0001). Further, the amount of Foxp3 mRNA expression (&gt;20‐fold) and Foxp3 protein per Treg (&gt;3‐fold) were also significantly reduced within Sin3a deficient animals. The ratios of T effectors to Tregs, and follicular T effectors to follicular Tregs were drastically increased within the spleens and superficial lymph nodes (ranging from 87‐154% and 152‐160%, respectively). Sin3a deficient Tregs produced significant amounts of pro‐inflammatory cytokines, including Granzyme B, IFN‐γ and IL‐2 (ranging from 12‐45% of cells). Tregs lacking Sin3a had impaired suppressive function against CD4+ and CD8+ responders (p&lt;0.01 and p&lt;0.05 respectively, compared to WT Tregs) in vitro. In addition, we found that Sin3a was essential for peripheral iTreg conversion since conventional CD4+CD25‐ cells from Sin3a‐/‐FoxP3Cre mice completely lacked the ability to convert to Foxp3+ iTregs ex vivo in the presence of IL‐2, TGFb, and CD3/CD28 beads. Consistent with impaired Treg function, CD4+ and CD8+ T cells from Sin3a‐/‐Foxp3Cre mice showed increased activation ex vivo (Ki67+ and CD69+ increased 120% and 230%, respectively) and enhanced production of Granzyme B and cytokines IFN‐γ and IL‐2 upon stimulation in vitro. To gain a more complete understanding of the regulatory role of Sin3a in Tregs, we are in the process of performing RNAseq. To address the vast reduction of Foxp3+ Tregs within Sin3a‐/‐FoxP3Cre mice we are investigating methylation of the CNS2 intronic region of Foxp3 by bisulphite conversion and determining the fate of Tregs in vivo by lineage tracing.ConclusionsThese data show that the nuclear co‐regulator, Sin3a, functions in modulating Treg gene expression and helps maintain the unique properties of these key immune cells. Our results indicate a potential for therapeutic modulation of Treg functions by pharmacologic targeting of components within the Sin3a complex.

Plasma cell dependence on histone/protein deacetylase 11 reveals a therapeutic target in multiple myeloma.

The clinical utility of histone/protein deacetylase (HDAC) inhibitors in combinatorial regimens with proteasome inhibitors for patients with relapsed and refractory multiple myeloma (MM) is often limited by excessive toxicity due to HDAC inhibitor promiscuity with multiple HDACs. Therefore, more selective inhibition minimizing off-target toxicity may increase the clinical effectiveness of HDAC inhibitors. We demonstrated that plasma cell development and survival are dependent upon HDAC11, suggesting this enzyme is a promising therapeutic target in MM. Mice lacking HDAC11 exhibited markedly decreased plasma cell numbers. Accordingly, in vitro plasma cell differentiation was arrested in B cells lacking functional HDAC11. Mechanistically, we showed that HDAC11 is involved in the deacetylation of IRF4 at lysine103. Further, targeting HDAC11 led to IRF4 hyperacetylation, resulting in impaired IRF4 nuclear localization and target promoter binding. Importantly, transient HDAC11 knockdown or treatment with elevenostat, an HDAC11-selective inhibitor, induced cell death in MM cell lines. Elevenostat produced similar anti-MM activity in vivo, improving survival among mice inoculated with 5TGM1 MM cells. Elevenostat demonstrated nanomolar ex vivo activity in 34 MM patient specimens and synergistic activity when combined with bortezomib. Collectively, our data indicated that HDAC11 regulates an essential pathway in plasma cell biology establishing its potential as an emerging theraputic vulnerability in MM.

HDAC2 targeting stabilizes the CoREST complex in renal tubular cells and protects against renal ischemia/reperfusion injury

Histone/protein deacetylases (HDAC) 1 and 2 are typically viewed as structurally and functionally similar enzymes present within various co-regulatory complexes. We tested differential effects of these isoforms in renal ischemia reperfusion injury (IRI) using inducible knockout mice and found no significant change in ischemic tolerance with HDAC1 deletion, but mitigation of ischemic injury with HDAC2 deletion. Restriction of HDAC2 deletion to the kidney via transplantation or PAX8-controlled proximal renal tubule-specific Cre resulted in renal IRI protection. Pharmacologic inhibition of HDAC2 increased histone acetylation in the kidney but did not extend renal protection. Protein analysis demonstrated increased HDAC1-associated CoREST protein in HDAC2-/- versus WT cells, suggesting that in the absence of HDAC2, increased CoREST complex occupancy of HDAC1 can stabilize this complex. In vivo administration of a CoREST inhibitor exacerbated renal injury in WT mice and eliminated the benefit of HDAC2 deletion. Gene expression analysis of endothelin showed decreased endothelin levels in HDAC2 deletion. These data demonstrate that contrasting effects of HDAC1 and 2 on CoREST complex stability within renal tubules can affect outcomes of renal IRI and implicate endothelin as a potential downstream mediator.

Decreased SIRT1 expression in the peripheral blood of patients with Graves' disease.

SIRT1, a class III histone/protein deacetylase (HDAC), has been associated with autoimmune diseases. There is a paucity of data about the role of SIRT1 in Graves’ disease. The aim of this study was to investigate the role of SIRT1 in the pathogenesis of GD. Here, we showed that SIRT1 expression and activity were significantly decreased in GD patients compared with healthy controls. The NF-κB pathway was activated in the peripheral blood of GD patients. The reduced SIRT1 levels correlated strongly with clinical parameters. In euthyroid patients, SIRT1 expression was markedly upregulated and NF-κB downstream target gene expression was significantly reduced. SIRT1 inhibited the NF-κB pathway activity by deacetylating P65. These results demonstrate that reduced SIRT1 expression and activity contribute to the activation of the NF-κB pathway and may be involved in the pathogenesis of GD.

HDAC10 deletion promotes Foxp3+ T-regulatory cell function

Foxp3+ T-regulatory (Treg) cells are capable of suppressing immune responses. Lysine acetylation is a key mechanism of post-translational control of various transcription factors, and when acetylated, Foxp3 is stabilized and transcriptionally active. Therefore, understanding the roles of various histone/protein deacetylases (HDAC) are key to promoting Treg-based immunotherapy. Several of the 11 classical HDAC enzymes are necessary for optimal Treg function while others are dispensable. We investigated the effect of HDAC10 in murine Tregs. HDAC10 deletion had no adverse effect on the health of mice, which retained normal CD4+ and CD8+ T cell function. However, HDAC10−/− Treg exhibited increased suppressive function in vitro and in vivo. C57BL/6 Rag1−/− mice adoptively transferred with HDAC10−/− but not wild Treg, were protected from developing colitis. HDAC10−/− but not wild-type mice receiving fully MHC-mismatched cardiac transplants became tolerant and showed long-term allograft survival (>100 d). We conclude that targeting of HDAC10 may be of therapeutic value for inflammatory disorders including colitis and also for transplantation.

Sirtuin-1 in immunotherapy: A Janus-headed target.

Sirtuin-1 (Sirt1), a member of the NAD-dependent sirtuin family of histone/protein deacetylases (HDAC), is an important target for immunotherapy due to its role in deacetylating the transcription factors Foxp3 and thymic retinoid acid receptor related orphan receptor gamma (RORγt). Sirt1 inhibition can increase Foxp3 acetylation and promote the production and functions of Foxp3+ T-regulatory (Treg) cells, whereas the acetylation of RORγt decreases its transcriptional activity DNA binding and decreases the differentiation of proinflammatory Th17 cells. Pharmacologic inhibitors of Sirt1 increase allograft survival and decrease autoimmune colitis and experimental allergic encephalomyelitis. However, in contrast to its role in T cells, Sirt1 has anti-inflammatory effects in myeloid cells, and, context dependent, in Th17 cells. Here, inhibition of Sirt1 can have proinflammatory effects. In addition to effects arising from the central role of Sirt1 in cellular metabolism and NAD-dependent reactions, such proinflammatory effects further complicate the potential of Sirt1 for therapeutic immunosuppression. This review aims to reconcile the opposing literature on pro- and anti-inflammatory effects of Sirt1, provides an overview of the role of Sir1 in the immune system, and discusses the pros and cons associated with inhibiting Sirt1 for control of inflammation and immune responses.

HDAC10 Targeting Regulates Foxp3 Promoter, Enhances T-regulatory (Treg) Function and Suppresses Autoimmune Colitis

Abstract Targeting histone/protein deacetylases (HDACs), especially by isoform-selective targeting, has major promise as a means to enhance the function of Tregs and thereby suppress immune-related disorders, such as colitis. We present data on the functions of HDAC10 in Treg cells. We found that HDAC10 binds to the Foxp3 promoter in the Tregs, is able to physically bind and deacetylate Foxp3 (at K31), and regulates the transcriptional activity of the Foxp3 promoter. Next, we evaluated the function of HDAC10 using HDAC10 KO mice. Overall, such mice were healthy and HDAC10 deletion: (i) enhanced Treg suppressive function both in vitro (Treg suppression assays) and in vivo (homeostatic proliferation assays), compared to wild-type (WT) Tregs; (ii) modestly enhanced the stability of monomeric Foxp3 (reducing gels) but the oligomeric forms, which are more relevant functionally, were increased by more than two-fold (native gels); (iii) increased H3K4Me3 (activating) marks on Foxp3 promoter and the key regulatory region of Foxp3 promoter-CNS2; and (iv) enhanced binding of ATF/CREB and FoxO1 at the Foxp3 promoter and CNS2 sites. We then evaluated the effects of HDAC10 deletion in Tregs in colitis models using adoptively transferred Rag1 KO mice. We found that HDAC10 KO Tregs were superior to WT Tregs in: (i) a ‘prevention of induction’ model of colitis, whereby WT T effector cells were injected with a limiting ratio of Tregs that allowed for weight loss as an indicator of colitis; and (ii) rescuing from colitis. Studies to develop and test HDAC10-specific inhibitors are currently underway. Overall, we show that HDAC10 has a key role in Foxp3+ Treg cells, and is an important new target for therapeutic intervention in autoimmune diseases, including colitis.

Histone/protein deacetylase inhibitor therapy for enhancement of Foxp3+ T-regulatory cell function posttransplantation.

T-regulatory (Treg) cells are like other cells present throughout the body in being subject to biochemical modifications in response to extracellular signals. An important component of these responses involves changes in posttranslational modifications (PTMs) of histones and many nonhistone proteins, including phosphorylation/dephosphorylation, ubiquitination/deubiquitination, and acetylation/deacetylation. Foxp3, the key transcription factor of Tregs, is constantly being rapidly turned over, and a number of these PTMs determine its level of expression and activity. Of interest in the transplant setting, modulation of the acetylation or deacetylation of key lysine residues in Foxp3 can promote the stability and function, leading to increased Treg production and increased Treg suppressive activity. This mini-review focuses on recent data concerning the roles that histone/protein deacetylases (HDACs) play in control of Treg function, and how small molecule HDAC inhibitors can be used to promote Treg-dependent allograft survival in experimental models. These data are discussed in the light of increasing interest in the identification and clinical evaluation of isoform-selective HDAC inhibitors, and their potential application as tools to modulate Foxp3+ Treg cell numbers and function in transplant recipients.

Deletion of a Class IIb Histone/protein Deacetylase (HDAC), HDAC10, Enhances FoxP3+ T-regulatory (Treg) Cell Suppressive Function, Gene Expression and Metabolism, and Promotes Allograft Survival

Abstract Treg cells control immune responses that cause unwarranted inflammation and promote allograft rejection, leading to interest in their therapeutic applications. Targeting HDACs to modulate the function of Tregs has practical advantages over injecting ex-vivo-expanded Tregs that may not survive long post-injection. Several of the 11 classical HDAC enzymes are necessary for optimal Treg function while others are dispensable. We evaluated the function of HDAC10 in murine Tregs. We showed that HDAC10 bound to Foxp3 in co-immunoprecipitation assays, and that germ-line knockout of this hitherto enigmatic HDAC had no effect on the health of targeted mice. We found that HDAC10 deletion: (i) enhanced Treg suppressive function both in vitro (Treg suppression assays) and in vivo (homeostatic proliferation assays), compared to wild-type (WT) Tregs; (ii) increased Treg expression of genes including Foxp3, Granzyme-B, IL-10 and others (microarray, qPCR); (iii) increased acetylation of NFkB/p65 at lysine 310 and thereby prolonged the nuclear retention of p65 and increased transcription from p65-dependent promoters; and (iv) increased oxidative phosphorylation in Tregs compared to WT Tregs (assays of mitochondrial function on the Seahorse platform). Lastly, heterotopic cardiac allograft studies (BALB/c-&amp;gt;C57BL/6) showed that compared to WT recipients, HDAC10−/− allograft recipients had markedly prolonged allograft survival (p&amp;lt;0.01). While additional studies of the in vivo effects of HDAC10 deletion in transplant and autoimmune models, including colitis, are underway our data show promise for the therapeutic targeting of HDAC10 as a novel approach for transplant rejection and other immunologically mediated disorders.

HDAC5 controls the functions of Foxp3(+) T-regulatory and CD8(+) T cells.

Histone/protein deacetylases (HDACs) are frequently upregulated in human malignancies and have therefore become therapeutic targets in cancer therapy. However, inhibiting certain HDAC isoforms can have protolerogenic effects on the immune system, which could make it easier for tumor cells to evade the host immune system. Therefore, a better understanding of how each HDAC isoform affects immune biology is needed to develop targeted cancer therapy. Here, we studied the immune phenotype of HDAC5(-/-) mice on a C57BL/6 background. While HDAC5(-/-) mice replicate at expected Mendelian ratios and do not develop overt autoimmune disease, their T-regulatory (Treg) cells show reduced suppressive function in vitro and in vivo. Likewise, CD4(+) T-cells lacking HDAC5 convert poorly to Tregs under appropriately polarizing conditions. To test if this attenuated Treg formation and suppressive function translated into improved anticancer immunity, we inoculated HDAC5(-/-) mice and littermate controls with a lung adenocarcinoma cell line. Cumulatively, lack of HDAC5 did not lead to better anticancer immunity. We found that CD8(+) T cells missing HDAC5 had a reduced ability to produce the cytokine, IFN-γ, in vitro and in vivo, which may offset the benefit of weakened Treg function and formation. Taken together, targeting HDAC5 weakens suppressive function and de-novo induction of Tregs, but also reduces the ability of CD8(+) T cells to produce IFN-γ.

Class-Specific Histone/Protein Deacetylase Inhibition Protects Against Renal Ischemia Reperfusion Injury and Fibrosis Formation

Renal ischemia-reperfusion injury (IRI) is a common cause of renal dysfunction and renal failure. Histone/protein deacetylases (HDACs) regulate gene accessibility and higher order protein structures and may alter cellular responses to a variety of stresses. We investigated whether use of pan- and class-specific HDAC inhibitors (HDACi) could improve IRI tolerance in the kidney. Using a model of unilateral renal IRI, we investigated early renal function after IRI, and calculated fibrosis after IRI using an automated scoring system. We found that pan-HDAC inhibition using trichostatin (TSA) yielded significant renal functional benefit at 24-96 hours (p < 0.001). Treated mice developed significantly less fibrosis at 30 days (p < 0.0004). Class I HDAC inhibition with MS-275 yielded similar effects. Protection from fibrosis formation was also noted in a cold ischemia transplant model (p < 0.008) with a trend toward improved cold ischemic survival in TSA-treated mice. These effects were not accompanied by induction of typical ischemic tolerance pathways or by priming of heat shock protein expression. In fact, heat shock protein 70 deletion or overexpression did not alter renal ischemia tolerance. Micro-RNA 21, known to be enhanced in vitro in renal tubular cells that survive stress, was enhanced by treatment with HDACi, pointing to possible mechanism.

FOXP3+ regulatory T cell development and function require histone/protein deacetylase 3.

Treg dysfunction is associated with a variety of inflammatory diseases. Treg populations are defined by expression of the oligomeric transcription factor FOXP3 and inability to produce IL-2, a cytokine required for T cell maintenance and survival. FOXP3 activity is regulated post-translationally by histone/protein acetyltransferases and histone/protein deacetylases (HDACs). Here, we determined that HDAC3 mediates both the development and function of the two main Treg subsets, thymus-derived Tregs and induced Tregs (iTregs). We determined that HDAC3 and FOXP3 physically interact and that HDAC3 expression markedly reduces Il2 promoter activity. In murine models, conditional deletion of Hdac3 during thymic Treg development restored Treg production of IL-2 and blocked the suppressive function of Tregs. HDAC3-deficient mice died from autoimmunity by 4-6 weeks of age; however, injection of WT FOXP3+ Tregs prolonged survival. Adoptive transfer of Hdac3-deficient Tregs, unlike WT Tregs, did not control T cell proliferation in naive mice and did not prevent allograft rejection or colitis. HDAC3 also regulated the development of iTregs, as HDAC3-deficient conventional T cells were not converted into iTregs under polarizing conditions and produced large amounts of IL-2, IL-6, and IL-17. We conclude that HDAC3 is essential for the normal development and suppressive functions of thymic and peripheral FOXP3+ Tregs.

Insulin Resistance due to Nutrient Excess: Mechanisms of AMPK Downregulation

Excesses of glucose and the branched chain amino acids lead to insulin resistance in skeletal muscle by activating mTOR/p70S6 kinase (p70S6K). In a recent study we demonstrated that glucose and leucine may produce these effects by downregulating AMP‐activated protein kinase (AMPK) which, when activated, diminishes mTOR/p70S6 K signaling. How these excesses of glucose and leucine decrease AMPK activity is unclear, since they do not alter the cell's energy state (AMP/ATP or CrP). We explored whether the decrease in AMPK activity might be due to decreases in the activity of SIRT1, a histone protein deacetylase that can activate AMPK or by changes in the activity of an unidentified kinase or phosphatase that results in the phosphorylation of Ser485/491 on the catalytic subunit of AMPK leading to its inhibition. Incubation of extensor digitorum longus muscle, with high glucose (25 mM) or 5 mM glucose and leucine (100 μM) persistently diminished AMPK activity within 30 min an effect that lasted for 2 h. Likewise both high glucose and leucine decreased the activity of SIRT1as reflected by increases in the deacetylation of histone H3 and a decrease in the activity of the rate‐limiting enzyme for SIRT1 activation, nicotinamide phosphoribosyltransferase (NAMPT). In addition, SIRT1 abundance was decreased. These changes only become evident after 2 h, however. In contrast, excess glucose and leucine caused increased phosphorylation of AMPK at Ser485/491 after 1 h. In studies with intact rats, we found that prolonged (3–8 h) hyperglycemia, produced by a glucose infusion, downregulates AMPK Thr172 phosphorylation and causes insulin resistance as early as 3 h. However, SIRT1, AMPK (Ser485/491) and NAMPT activity only decreased between 5–8 h. The results indicate that both leucine and high glucose downregulate AMPK by at least two mechanisms. This could account for sustained decreases in AMPK activity; however, temporally they do not account for the initial decrease in AMPK activity caused by these fuels.

Histone Deacetylase 6 and Heat Shock Protein 90 Control the Functions of Foxp3+ T-Regulatory Cells

Foxp3(+) T-regulatory cells (Tregs) are key to immune homeostasis such that their diminished numbers or function can cause autoimmunity and allograft rejection. Foxp3(+) Tregs express multiple histone/protein deacetylases (HDACs) that regulate chromatin remodeling, gene expression, and protein function. Pan-HDAC inhibitors developed for oncologic applications enhance Treg production and Treg suppression function but have limited nononcologic utility given their broad actions and various side effects. We show, using HDAC6-deficient mice and wild-type (WT) mice treated with HDAC6-specific inhibitors, that HDAC6 inhibition promotes Treg suppressive activity in models of inflammation and autoimmunity, including multiple forms of experimental colitis and fully major histocompatibility complex (MHC)-incompatible cardiac allograft rejection. Many of the beneficial effects of HDAC6 targeting are also achieved by inhibition of the HDAC6-regulated protein heat shock protein 90 (HSP90). Hence, selective targeting of a single HDAC isoform, HDAC6, or its downstream target, HSP90, can promote Treg-dependent suppression of autoimmunity and transplant rejection.

Epigenetic Regulation of Regulatory T-Cells: Impact on Autoimmunity and Graft Rejection

AbstractAbstract SCI-23Mutations of Foxp3, a transcription factor characteristic of T regulatory (Treg) cells, often cause lethal autoimmunity, leading to much research into how Foxp3+ Tregs control inflammatory and immune responses. This presentation will emphasize therapeutic aspects of our ongoing studies showing that the functions of Foxp3 are regulated by histone/protein deacetylases (HDAC), histone acetyltransferases (HAT), and DNA methyltransferases (DNMT), and for the sake of time will focus on the effects of HDAC inhibitors (HDACi). We have found that Foxp3 acetylation promotes DNA binding and can induce or suppress expression of multiple Foxp3-dependent genes in Tregs. Acetylation is catalyzed by specific HATs whose neutralization can diminish Treg function, with relevance to cancer and HIV, whereas use of selected HDAC inhibitors (HDACi) can increase acetylation and Treg suppression, with relevance to control of autoimmunity and transplant rejection. Many HDAC inhibitors (HDACi) were evaluated for their effects on Treg function. As class I-selective HDACi agents (e.g. benzamides) had little or no effect on Treg function, but pan-HDACi (e.g. hydroxymates) enhanced Treg function, we focused on class II HDACs. There are 2 class II subfamilies; class IIa members are thought to largely function in a tissue-specific manner through recruitment of other proteins since they display only weak catalytic activity, whereas class IIb family members display bona fide catalytic activity. We therefore analyzed class IIb members, of which HDAC6 is the best established and for which selective HDACi are available. HDAC6 exists in the cytoplasm and regulates acetylation of alpha-tubulin and other proteins, including HSP90. Blocking HDAC6 through the use of an HDAC6-specific inhibitor promotes HSP90 acetylation and release of HSP90 client proteins. Use of HDAC6 or HSP90 inhibitors increased Foxp3 expression and enhanced Treg function in vitro and in vivo, and could prevent, or treat pre-existing, autoimmunity in a Treg-dependent manner. We have also investigated the various class IIa family members, of which HDAC9 is of particular interest since its expression is increased 30-fold in Treg vs. regular T cells. HDAC9 decreases Foxp3 expression and function, and its neutralization promotes Treg survival by regulating expression of HSP70 and related HSPs. Our ongoing studies show that HDAC6 neutralization leads to acetylation of HSP90, release of HSF-1 and induction of HSP70, and also suggest that HDAC9 may regulate the acetylation and stabilization of HSF-1. Once produced, HSP70 can chaperone and promote Foxp3 nuclear translocation and function, such that the HDAC6 and HDAC9 pathways are closely intertwined with regard to control of Treg biology. In summary, acetylation, methylation and other epigenetic mechanisms in Tregs are being probed using genetic and pharmacologic approaches. Various currently approved drugs influence Foxp3-dependent Treg functions by affecting epigenetic mechanisms, and while additional HDAC-specific regulators are needed, a rationale is now in place for use of HDAC inhibitors as powerful tools to promote the development and functions of Foxp3+ Tregs in vitro and in vivo. Disclosures:No relevant conflicts of interest to declare.

Histone/protein deacetylase inhibitors increase suppressive functions of human FOXP3+ Tregs

Histone/protein deacetylases (HDACs) decrease histone and protein acetylation, typically leading to suppression of gene transcription and modulation of various protein functions. We found significant differences in expression of HDAC before and after stimulation of human T regulatory (Treg) and T effector cells, suggesting the potential for future selective targeting of Tregs with HDAC inhibitors (HDACi). Use of various HDACi small molecules enhanced, by up to 4.5-fold (average 2-fold), the suppressive functions of both freshly isolated and expanded human Tregs, consistent with our previous murine data. HDACi use increased Treg expression of CTLA-4, a key negative regulator of immune response, and we found a direct and significant correlation between CTLA-4 expression and Treg suppression. Hence, HDACi compounds are promising pharmacologic tools to increase Treg suppressive functions, and this action may potentially be of use in patients with autoimmunity or post-transplantation.

Inhibition of HDAC9 Increases T Regulatory Cell Function and Prevents Colitis in Mice

Foxp3+ T regulatory cells (Tregs) help prevent autoimmunity, and increases in their numbers of functions could decrease the development of inflammatory bowel disease. Like other cells, Foxp3+ Tregs express histone/protein deacetylases (HDACs), which regulate chromatin remodeling and gene expression. We investigated whether disruption of a specific class IIa HDAC, HDAC9, activity in Tregs affects the pathogenesis of colitis in mice. We tested the effects of various HDAC inhibitors (HDACi) in models of colitis using wild-type mice. We also transferred Tregs and non-Treg cells from HDAC9-/- or wild-type mice to immunodeficient mice. HDAC9 contributions to the functions of Tregs were determined during development and progression of colitis. Pan-HDACi, but not class I-specific HDACi, increased the functions of Foxp3+ Tregs, prevented colitis, and reduced established colitis in mice, indicating the role of class II HDACs in controlling Treg function. The abilities of pan-HDACi to prevent/reduce colitis were associated with increased numbers of Foxp3+ Tregs and their suppressive functions. Colitis was associated with increased local expression of HDAC9; HDAC9-/- mice resistant to development of colitis. HDAC9-/- Tregs expressed increased levels of the heat shock protein (HSP) 70, compared with controls. Immunoprecipitation experiments indicated an interaction between HSP70 and Foxp3. Inhibition of HSP70 reduced the suppressive functions of HDAC9-/- Tregs; Tregs that overexpressed HSP70 had increased suppressive functions. Strategies to decrease HDAC9 expression or function in Tregs or to increase expression of HSP70 might be used to treat colitis and other autoimmune disorders.

Use of Epigenetic Modification to Induce FOXP3 Expression in Naïve T Cells

We investigated whether epigenetic modification agents can convert naïve T cells to regulatory T cells (T regs) which are responsible for limiting immune responses and maintaining self-tolerance. We treated splenic CD4 +/CD25 − naïve T cells from BALB/c mice with the DNA-methyltransferase inhibitor 5-aza-2′-deoxycytidine (5AzaD) or the histone protein deacetylase (HDAC) inhibitor Trichostatin A (TSA), and measured the effects on the expression of FOXP3, which encodes a transcription factor ( FOXP3) that regulates T reg development. FOXP3 expression in naïve T cells was increased by 5AzaD or TSA treatment, administered 72 hours after T-cell receptor (TCR) stimulation with anti-CD3 plus anti-CD28. The T regs induced by 5AzaD or TSA expressed greater amounts of the FOXP3 protein than the control and the natural T regs. The analysis of T reg-associated markers also showed T reg phenotypes (CD25 +/CTLA4 +/GITR +/CD127 −). Finally, the induced T reg population also displayed T-cell suppression. These data suggested that epigenetic modification agents can induce FOXP3 expression, promoting the conversion of naïve T cells to T regs.