Histone Deacetylase Inhibitors Modulate Interleukin 6-dependent CD4+ T Cell Polarization in Vitro and in Vivo *

Background: HDAC inhibitors exert anti-inflammatory properties. Results: ITF2357 shifts the balance from Th17 cells toward regulatory T cells via suppression of the IL-6R expression on naïve T cells. Conclusion: The HDAC inhibitor ITF2357 modulates T cell polarization in experimental colitis. Significance: Learning the mode of action of this compound class will serve to optimize future therapeutic strategies. Histone deacetylase (HDAC) inhibitors have been associated primarily with an anti-proliferative effect in vitro and in vivo. Recent data provide evidence for an anti-inflammatory potency of HDAC inhibitors in models of experimental colitis. Because the balance of T cell subpopulations is critical for the balance of the mucosal immune system, this study explores the regulatory potency of HDAC inhibitors on T cell polarization as a mechanistic explanation for the observed anti-inflammatory effects. Although HDAC inhibition suppressed the polarization toward the pro-inflammatory T helper 17 (Th17) cells, it enhanced forkhead box P3 (FoxP3)+ regulatory T cell polarization in vitro and in vivo at the site of inflammation in the lamina propria. This was paralleled by a down-regulation of the interleukin 6 receptor (IL-6R) on naïve CD4+ T cells on the mRNA as well as on the protein level and changes in the chromatin acetylation at the IL6R gene and its promoter. Downstream of the IL-6R, HDAC inhibition was followed by a decrease in STAT3 phosphorylation as well as retinoic acid receptor-related orphan receptor γT (RORγT) expression, thus identifying the IL-6/STAT3/IL-17 pathway as an important target of HDAC inhibitors. These results directly translated to experimental colitis, where IL-6R expression was suppressed in naïve T cells, paralleled by a significant reduction of Th17 cells in the lamina propria of ITF2357-treated animals, resulting in the amelioration of disease. This study indicates that, in experimental colitis, inhibition of HDAC exerts an anti-inflammatory potency by directing T helper cell polarization via targeting the IL-6 pathway.

Epigenetic modifications represent an additional regulatory level for gene expression and, thus, comprise potential therapeutic targets for autoinflammatory diseases or cancer. This work focuses on the modification of histone acetylation that is under a physiological state regulated by the balance of histone acetyltransferases and histone deacetylases (HDACs). 2 HDAC inhibitors have been primarily described for their antiproliferative potency, as shown in animal models for colon, mammary, prostate, and bladder cancer (1,2). Subsequent clinical trials resulted in Food and Drug Administration approval of the HDAC inhibitor suberoylanilide hydroxamid acid (SAHA) for the treatment of cutaneous T cell lymphoma (3). Remarkably, these antiproliferative effects have been partially attributed to the inhibition of the nuclear factor-B pathway (4,5). At the same time, a strong anti-inflammatory capacity of this compound class was identified. Initial in vitro studies revealed a dose-dependent suppression of proinflammatory cytokine production by SAHA at concentrations 10-to 100-fold lower than required for apoptosis induction (6,7). These results were complemented in vivo by investigating inflammatory models such as concanavalin A-induced hepatitis or LPS-induced shock in mice (7). Following these initial data, the anti-inflammatory potency could be confirmed in models of nephritis (8), arthritis (9), intestinal inflammation (6,10), graft versus host disease (11), and asthma (12). In addition, the HDAC inhibitor ITF2357 has shown clinical efficacy in clinical trials for juvenile arthritis (13).
The maintenance of the mucosal homeostasis requires the balance of CD4 ϩ T cell subsets within the compartment of the lamina propria. In particular, proinflammatory T helper 17 (Th17) cells and anti-inflammatory regulatory T (Treg) cells have been identified as critical factors that define the course and severity of intestinal inflammation (14,15). This concept is supported by studies indicating a beneficial effect by targeting the Th17-inducing cytokine IL-23 in experimental colitis (16). This strategy has been proven successful in clinical trials for inflammatory bowel disease (17,18). In addition, transfer of regulatory T cells resulted in the amelioration in various models of experimental colitis (19). Thus, for inflammatory bowel disease as well as for other chronic inflammatory conditions, a therapeutic approach targeting both T cell subsets would be intriguing.
For the in vitro generation of both Th17 and inducible Treg cells from naïve CD4 ϩ cells, TGF␤ has been identified as a mandatory cytokine (20,21). Adding IL-6 to low doses of TGF␤ results in the differentiation of Th17 cells in vitro (22)(23)(24), thus identifying IL-6 as the key cytokine defining the polarization of these two cell types. IL-6 is a pleiotropic cytokine and was first described to function as a B cell differentiation factor being produced by several cell types, such as macrophages, monocytes, and T cells (25). In acute inflammation such as sepsis, IL-6 is critical for the induction of acute phase reactions (26).
IL-6 deploys its effects through binding to the IL-6 receptor (IL-6R). The IL-6⅐IL-6R complex then signals through the ubiquitously expressed subunit gp130, leading to activation of the JAK/STAT pathway (classic signaling) (27). Remarkably, the expression of the membrane-bound IL-6R is restricted to naïve T cells and hepatocytes, thus emphasizing the significance of this cytokine for these cell types. However, many other cell types can be activated by IL-6. To enable signaling in cells not expressing the IL-6R, the soluble IL-6R forms a complex with IL-6 that binds to gp130 and, thereby, activates cells that would be naturally unresponsive to IL-6 (28).
The aim of this study was to define the effect of HDAC inhibition on CD4 ϩ T cell polarization and to provide an explanation for the anti-inflammatory effects observed in vivo. In fact, we were able to link HDAC inhibition to decreased IL-6R expression, resulting in suppression of the associated signaling pathway and, ultimately, the suppression of an inflammatory T cell response.
HDAC Inhibitors-ITF2357 was synthesized by the Chemical Department of Italfarmaco (Cinisello Balsamo, Italy) and processed as described previously (5). SAHA was provided by MSD (Haar, Germany). SAHA and ITF2357 were diluted in sterile distilled water and heated to 95°C for complete dissolution before being added to the cultures. For use in animal models, SAHA or ITF2357 were freshly dissolved in sterile PBS, heated to 95°C, and kept at room temperature.
Mice-Six-to eight-week-old female BALB/c mice were obtained from Harlan Winkelmann (Borchen, Germany). DO11.10 mice were obtained from the Bundesinstitut für Risikobewertung (Berlin, Germany). Animal protocols were approved by the regional animal study committee of Berlin (Germany).
Experimental Colitis-Colitis was induced by administering 4% dextran sulfate sodium (DSS; molecular mass 40 kDa; MP Biomedicals, Illkirch, France) dissolved in drinking water from day 1 to day 7, followed by 2 days of regular drinking water.
ITF2357 and SAHA treatment were initiated simultaneously at the start of colitis induction via oral gavage. Final doses were 10 mg/kg body weight (ITF2357) and 50 mg/kg body weight (SAHA), administered in 200 l of PBS once daily.
Assessment of Colitis Severity-Body weight and the presence of occult or gross blood per rectum were determined daily. Bleeding was scored as follows: 0, no blood using hemocare (Fresenius, Friedberg, Germany); 2 points, positive hemocare samples; and 4 points, gross bleeding. Mice were sacrificed by cervical dislocation. The entire colon was removed from the cecum to the anus, and colon length was measured as a marker of inflammation. Whole colon tissue (1 cm) was cut open longitudinally and cultured overnight in 1 ml of serum-free medium. Cytokine concentrations were assessed by ELISA and calculated as a ratio versus total protein concentration.
To generate murine bone marrow-derived macrophages, the cavities of femur and tibia bones of BALB/c mice were flushed with PBS. The single cell suspensions were cultured in high-glucose DMEM supplemented with 10% fetal calf serum, 100 units/ml penicillin, 100 g/ml streptomycin, and 20 ng/ml macrophage colony-stimulating factor (Peprotech, Hamburg, Germany). After 24 h of incubation, non-adherent macrophage progenitor cells were isolated and incubated for another 6 days. Thereafter, non-adhesive cells were discarded. Only populations with Ͼ98% of CD11b ϩ F4/80 ϩ cells were used for further analysis. Cells (10 6 /ml) were then cultured in medium devoid of macrophage colony-stimulating factor and incubated with the indicated concentration of ITF2357 for 1 h before further processing. LPS (1 g/ml) was added for 18 h as described below.
Flow Cytometry Analysis-For intracellular staining, cells were incubated for 4 h with 50 ng/ml phorbol myristate acetate and 500 ng/ml ionomycin, plus 2 mg/ml brefeldin A during the last 2 h. The cells were kept in a tissue culture incubator at 37°C. Cells were fixed with 4% paraformaldehyde for 5 min at 37°C and subsequently stained for cytokines in 0.5% saponin for 25 min at room temperature. Intranuclear FoxP3 as well as ROR␥T staining were performed using the FoxP3 staining kit from eBioscience (San Diego, CA) according to the manual. STAT3 staining was performed using 100% methanol as the permeabilization reagent. Surface staining was performed for 10 min on ice. Anti-CD4, anti-CD62L, anti-IFN␥, and anti-IL-4 were purchased from BD Biosciences. Anti-IL-17A, anti-FoxP3, and anti-phosphorylated STAT3 (anti-pSTAT3) were from eBioscience. Fixable live/dead dye (aqua) was purchased from Invitrogen and used as indicated.
Western Blot Analysis-For pSTAT3, 10 6 cells were lysed in SDS loading buffer (Cell Signaling Technology, Beverly, MA), heated to 95°C for 5 min, and loaded onto a 7% polyacrylamide gel. The proteins were transferred to a PVDF membrane and detected with ECL reagent (GE Healthcare). The following antibodies were applied: anti-STAT3 and anti-pSTAT3 (Cell Signaling Technology) and anti-␤-actin and horseradish peroxidase-labeled rabbit anti-goat and goat anti-rabbit antibodies (Dako, Hamburg, Germany). Densitometric analysis was done using Fuji MultiGauge software (Fujifilm, Düsseldorf, Germany).
ChIP-Cells were lysed in SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris (pH 8.1)) and fixed with 0.5% formaldehyde, followed by sonication. ChIP was performed using protein A MicroBeads (Miltenyi Biotec) and anti-acetyl-histone 3 antibody (Lys9, Cell Signaling Technology) according to the protocol of the manufacturer.
Statistical Analysis-The data are expressed as mean Ϯ S.E. Statistical significance of differences between treatment and control groups were determined by factorial analysis of vari-

HDAC Inhibition Impacts CD4 ϩ T Cell Polarization-We
recently demonstrated an anti-inflammatory effect of HDAC inhibitor treatment in experimental colitis (6). To reveal the mechanism behind this anti-inflammatory effect on a cellular level at the site of inflammation, LPMCs were stimulated with concanavalin A in the presence or absence of increasing concentrations of the HDAC inhibitors ITF2357 or SAHA. HDAC inhibition dose-dependently decreased IFN␥ synthesis down to 20% relative to control samples (Fig. 1A). This effect could be reproduced in purified CD4 ϩ T cells, with the rate of inhibition ranging from 40 -70% (Fig. 1B). Besides IFN␥, IL-6 synthesis was almost completely suppressed (up to 90%), whereas the release of the Th2 cytokine IL-4 remained unaffected (Fig. 1B).
This suppression of IFN␥ and IL-6 production mediated by HDAC inhibition on a cellular level could be translated to ex vivo colon cultures from colitic mice that were treated with HDAC inhibitors (Fig. 1C). Having shown that HDAC inhibition altered the quality of the T cell response, and considering the impact of this cell type in intestinal inflammation, we next asked whether HDAC inhibitors exhibit the potency to regulate T cell polarization. To address this question, naïve CD4 ϩ T cells were polarized in the presence or absence of the HDAC inhibitor ITF2357 into four different T helper cell subsets, namely Th1, Th2, Th17, and induced Treg cells. No influence on the "classic" T cell polarization to either Th1 or Th2 cells was observed (data not shown). In contrast, the presence of 200 nM ITF2357 resulted in increased Treg cell generation and a profound suppression of Th17 cell polarization (Fig. 2).
HDAC Inhibition in Vivo Regulates T Helper Cell Polarization at the Site of Inflammation-To evaluate whether the effect of HDAC inhibition on T helper cell subsets might contribute to the anti-inflammatory potency during intestinal inflammation, colitis was induced by DSS, and mice received either the HDAC inhibitor ITF2357 or vehicle during the entire experimental time course (Fig. 3G). At the end of the experiment, the ratio of CD4 ϩ /IL-17 ϩ as well as CD4 ϩ /FoxP3 ϩ T cells within the LPMCs and the MLNs was analyzed (Fig. 3, A-F). In healthy mice, no considerable changes in the experimental groups were detected. In contrast, in mice suffering from colitis, inhibition of HDAC was followed by a significant increase of Treg cells in the lamina propria as well as in the draining MLNs (Fig. 3, A-C). In line with the in vitro data, this increase in Treg cells was paralleled by a significant decrease of Th17 cells at both sites to levels of healthy control mice (Fig. 3, D-F).
HDAC Inhibition Suppresses IL-6R Expression and Downstream Signaling-When polarizing naïve CD4 ϩ T cells with TGF␤, the presence of IL-6 determines the direction toward Th17 cells, whereas the absence of IL-6 results in inducible Treg cells. Thus, the ability of naïve CD4 ϩ T cells treated with ITF2357 to react to IL-6 was analyzed. In the presence of ITF2357, naïve CD4 ϩ T cells down-regulated IL-6R expression on the cell surface in a time-and dose-dependent manner, as shown in protein as well as mRNA levels (Fig. 4, A and B). Confirming the impact of the HDAC inhibitor, a hyperacetylation of histone 3 at the site of the IL-6R locus was detected, whereas the IL-6R promoter region was deacetylated after ITF2357 treatment. The IL-6 promoter region remained unaffected (Fig. 4C). In parallel to the in vitro findings, the IL-6R was down-regulated significantly in the naïve CD4 ϩ T cells from MLNs of ITF2357-treated mice (Fig. 4D). This effect was observed in healthy as well as in diseased mice but appeared statistically stronger in the DSS experiments.
To reveal whether or not the decrease in IL-6R expression affects downstream signaling, phosphorylation of STAT3 was evaluated (30). Fig. 5 indicates that the presence of the HDAC inhibitor ITF2357 resulted in a dose-dependent reduction of IL-6-induced STAT3 phosphorylation in naïve CD4 ϩ T cells. These findings are complemented by additional signaling data indicating that ROR␥T expression downstream of STAT3 is  F) is shown. G, weight was determined daily and expressed as the percentage of initial body weight. The bleeding score was determined daily. At the end of the experiment, colon length was measured. All three groups were fed daily via oral gavage, either ITF2357 or vehicle (PBS), whereas the H 2 O group was given regular drinking water instead of DSS. Horizontal lines represent the mean (n ϭ 8). *, p Ͻ 0.05; **, p Ͻ 0.01; ***, p Ͻ 0.001. also down-regulated in ITF2357-treated, Th17-polarized T cells (Fig. 6).
HDAC Inhibition Modulates IL-6 and IL-6R Expression of Macrophages-Having shown that HDAC inhibition leads to a suppressed response to IL-6 in naïve CD4 ϩ T cells and a subsequent redirection of T cell polarization, one has to consider the effect of HDAC inhibition on other IL-6-producing cell popu-lations. Macrophages are known to release IL-6 as well as soluble IL-6R upon stimulation and represent a critical cell type within the lamina propria and for directing T cell polarization (31,32). LPS stimulation of macrophages in the presence of ITF2357 resulted in a dose-dependent reduction of IL-6R and soluble IL-6R expression. Remarkably, the expression of gp130 remained unaffected (Fig. 7, A-C). In addition to receptor  FEBRUARY 28, 2014 • VOLUME 289 • NUMBER 9 JOURNAL OF BIOLOGICAL CHEMISTRY 6147 expression, IL-6 synthesis itself was strongly down-regulated by HDAC inhibition (Fig. 7D).

DISCUSSION
Inhibition of HDAC results in the amelioration of colitis in a variety of models. However, the underlying mechanism remains largely to be defined. In these models, the severity, but even more the chronicity, of disease depends on the presence of CD4 ϩ T cells and the balance of distinct subpopulations at the site of inflammation (33). This study serves to identify the regulatory capacity of HDAC inhibition on T cell polarization and, consecutively, on this critical balance of T cell subpopulations. A regulatory role of T cell function was indicated by the suppression of IL-6 and IFN␥ synthesis in stimulated CD4 ϩ T cells in the presence of an HDAC inhibitor. More important, HDAC inhibition enhanced Treg cell generation and suppressed polarization toward Th17 cells in vitro as well as in vivo at the site of inflammation. Because Th1 and Th2 polarization was unaffected, the IL-6 pathway came into focus as potential target because IL-6 represents the key cytokine distinguishing polarization toward Th17 cells from inducible Treg cells. The subsequent studies revealed a unique anti-inflammatory mode of action via regulation of the IL-6R and downstream signaling.
Genetic as well as environmental factors contribute to the dysregulation of the mucosal immune system in Crohn disease and ulcerative colitis (34). Although the balance of T helper cell subpopulations has been identified to play a critical role in inflammatory bowel disease, recent studies provide evidence that Th17 cells, in particular, add to the inflammatory process in the lamina propria (35,36). Several studies identified IL-6 to be essential for the development of Th17 cells (22)(23)(24). Noguchi et al. (37) were able to demonstrate that blocking the IL-6 pathway through a specific IL-6R antibody resulted not only in the amelioration of experimental colitis but, furthermore, was par-alleled by a significant decrease in Th17 cells in the lamina propria.
Treatment with pan-HDAC inhibitors ameliorates experimental colitis in mice and inhibits the production of proinflammatory cytokines at the site of inflammation (6). Via stimulation of LPMCs in the presence of HDAC inhibitors, the local effect of these compounds on effector cells could be proven. A similar dose-dependent suppression of IFN␥ and IL-6 synthesis was observed when isolated CD4 ϩ cells were stimulated in the presence of HDAC inhibitors. Both experiments suggest that the local T helper cell population within the lamina propria represents a critical target for HDAC inhibition. In the majority of experimental colitis models, Th1 as well as Th17 cells have been shown to represent a key cell population in the disease course (38). The absence of IL-17-producing T cells in the transfer colitis model almost completely abolished the characteristic inflammation (39). In contrast, the presence of Treg cells has been demonstrated to exert an ameliorating effect in experimental colitis (40,41).
There are several options regarding how HDAC inhibition might modulate the T cell response toward an anti-inflammatory direction. Studying the effects of HDAC inhibition on T helper cell polarization, we were able to demonstrate, in vitro, that the release of Th1 cytokines was suppressed significantly by treatment with HDAC inhibitors (Fig. 1 and Refs. 6,7,42). Although the polarization toward either Th1 or Th2 cells was not affected by the presence of an HDAC inhibitor, the generation of Treg cells was enhanced significantly, and the polarization of Th17 cells was suppressed dose-dependently. In parallel to the in vitro findings, the analysis of T cell subsets in the lamina propria and MLN from mice with experimental colitis treated with ITF2357 showed a reduction in the frequency of Th17 cells within the CD4 ϩ T cell fraction, and, vice versa, the Treg cell population was increased. Hence, the anti-inflammatory shift in the Treg/Th17 cell balance demonstrated in vitro could be confirmed directly in the animal model at the site of inflammation, delivering an explicit mode of action for these compounds regarding the anti-inflammatory effect exerted in a variety of inflammation models (6 -12).
Complementary to our findings, Wayne Hancock and coworkers (10,43) showed an HDAC inhibitor-dependent increase in Treg cells that mediated the anti-inflammatory effect. Because experimental colitis was associated with an increased local expression of HDAC9, HDAC9 knockout mice were subsequently investigated in models of experimental colitis where they proved to be protected (10,43). For the IL-17producing T cells, Koenen et al. (44) described a reduced in vitro differentiation of Treg to Th17 cells in the presence of pan-HDAC inhibitors . Furthermore, Bosisio et al. (45) suggest that the production of Th1-and Th17-polarizing cytokines by dendritic cells is suppressed by HDAC inhibition. Together with our data regarding the lack of Th1 polarizing cytokines and the decrease in Th17 cells, an explanation for the anti-inflammatory potency in various models of inflammation is provided.
Considering the effect of HDAC inhibition on IL-6 production and the critical role of IL-6 in the polarization of naïve CD4 ϩ T cells toward Th17 cells, the suppression of IL-6 signaling could result in a consecutive decrease of Th17 cells. Thus, our following studies targeted the IL-6/IL-6R pathway. IL-6 exerts its function by binding to the IL-6R, starting a cascade, with STAT3 being one of the key signal transduction molecules downstream (30). HDAC inhibition not only resulted in a down-regulation of the IL-6R but, consecutively, lead to a dosedependent reduction of IL-6-induced STAT3 phosphorylation in naïve CD4 ϩ T cells. Modifications of this pathway lead directly to ROR␥T, the key transcription factor for IL17 production, and, therefore, to the polarization of Th17 cells (46). Consequently, ROR␥T is also down-regulated in ITF2357treated cells. To prove a direct impact of the HDAC inhibitor on the IL-6R expression on the chromatin level, we assessed changes in the histone acetylation pattern on the respective loci. Although our ChIP data revealed a hyperacetylation for the IL-6R gene locus, which is mostly associated with an increased transcription of the respective region, the IL-6R promoter locus was, in fact, deacetylated, indicating reduced gene transcription and, thus, confirming the proposed mechanism.
Furthermore, our data demonstrate that, also in macrophages, the expression of the IL-6R is diminished by ITF2357, even in its soluble form. Given that the secretion of IL-6 itself is reduced in macrophages, one of the main sources of this cytokine in the lamina propria, we were able to describe a massive down-modulation of the IL-6 signaling pathway in our models.
In view of these data and the literature discussed, we propose the IL-6/STAT3/IL-17 pathway as critical in the anti-inflammatory effect achieved by HDAC inhibition. Other studies have described IL-6 as the determining factor in the polarization of Th17 from naïve CD4 ϩ T cells, thus being crucial for obtaining the balance between Treg and Th17 cells (47). Because naïve FIGURE 6. ROR␥T expression is diminished in ITF2357-treated, Th17-polarized T cells. Naïve CD4 ϩ T cells were isolated from DO.11.10 mice, and antigen-specific T cell stimulation was performed under Th17-polarizing conditions in the presence of ITF2357 (200 mM). On day 5, intracellular staining was performed for ROR␥T. Exemplary cytometry data are shown as a dot blot (A) and histogram (B). The columns (C) represent percentages of ROR␥T-positive cells (two experiments, n ϭ 12 each). Data are mean Ϯ S.E. ***, p Ͻ 0.001. CD4 ϩ T cells are one of the few cell types in possession of the membrane-bound IL-6R, they are offering a possible target for the indicated regulation of T cell polarization (48). Complementary to the results presented here, it has been described that treatment of multiple myeloma cells with HDAC inhibitors resulted in down-modulation of IL-6R signaling (49,50). Here, the authors emphasized the functional consequences achieved by down-modulation of the IL-6R because the response to IL-6 was equally reduced, as determined by STAT3 phosphorylation (50). Clinical studies are underway to evaluate HDAC inhibitors as a treatment option for relapsed multiple myeloma (51), adding to the variety of other substances targeting the IL-6R in pharmacotherapy for different diseases, such as rheumatoid arthritis or Crohn disease, with remarkable clinical effects (47,52,53). Hence, HDAC inhibitors might provide a novel therapeutic approach for the treatment of autoimmune diseases and other diseases by targeting the IL-6 receptor pathway and, thereby, influencing the critical balance of T cell subpopulations at the site of inflammation.