The Microbial Metabolite Butyrate Induces Expression of Th1-Associated Factors in CD4+ T Cells

Short-chain fatty acids (SCFAs), which are generated by the bacterial fermentation of dietary fibers, promote expansion of regulatory T cells (Tregs). Potential therapeutic value of SCFAs has been recently highlighted in the experimental models of T cell-mediated autoimmunity and allergic inflammation. These studies suggest that physiological intestinal concentrations of SCFAs within the millimolar range are crucial for dampening inflammation-mediated processes. Here, we describe opposing effects of SCFAs on T cell-mediated immune responses. In accordance with published data, lower butyrate concentrations facilitated differentiation of Tregs in vitro and in vivo under steady-state conditions. In contrast, higher concentrations of butyrate induced expression of the transcription factor T-bet in all investigated T cell subsets resulting in IFN-γ-producing Tregs or conventional T cells. This effect was mediated by the inhibition of histone deacetylase activity and was independent of SCFA-receptors FFA2 and FFA3 as well as of Na+-coupled SCFA transporter Slc5a8. Importantly, while butyrate was not able to induce the generation of Tregs in the absence of TGF-β1, the expression of T-bet and IFN-γ was triggered upon stimulation of CD4+ T cells with this SCFA alone. Moreover, the treatment of germ-free mice with butyrate enhanced the expression of T-bet and IFN-γ during acute colitis. Our data reveal that, depending on its concentration and immunological milieu, butyrate may exert either beneficial or detrimental effects on the mucosal immune system.

that regulation of colonic Tregs by SCFAs via engagement of free fatty acid receptor FFA2 (GPR43) is one of the mechanisms by which commensal bacteria help to maintain intestinal immune homeostasis (6,7). Aside from direct interaction of SCFAs with FFA2 on Tregs, butyrate has been shown to initiate differentiation of Tregs either via involvement of high-affinity SCFA transporter Slc5a8 in dendritic cells (DCs) or through activation of cell surface receptor GPR109a on colonic macrophages and DCs (8,9). In addition, two recent studies demonstrated that the ability of SCFAs to promote induction of Foxp3 expression strongly correlated with their histone deacetylase (HDAC) inhibitory activity (3,4). Collectively, these novel findings suggest that SCFAs exhibit pleiotropic effects on immune system, including the induction of Treg differentiation. Therefore, SCFAs such as butyrate, propionate, or acetate have recently been considered as potential therapeutic tool to modulate inflammatory responses (10,11). Interestingly, increased consumption of dietary fibers led not only to SCFA-mediated expansion of Tregs but also suppressed the onset of carcinogenesis in the colon and dampened allergic responses in the lung of mice (8,12). A recent study describing the impact of SCFAs on autoimmune reactions in the central nervous system (CNS) suggests that bacterial metabolites have a therapeutic potential for disorders such as multiple sclerosis (MS) (13). In spite of growing evidence of the immunomodulatory capacity for SCFAs, several questions concerning dosage and potential toxic effects remain unanswered. Furthermore, butyrate did not ameliorate inflammation in dextran sodium sulfate (DSS)-induced colitis model, thus raising the question if beneficial effects of SCFAs might be overridden by potentially deleterious, SCFA-mediated activities (14,15).
In this study, we analyzed the dose-dependent impact of the microbial metabolite butyrate on Tregs and conventional CD4 + T cells. Low butyrate concentrations (0.1-0.5 mM) facilitated differentiation of Foxp3 + Tregs in the presence of TGF-β1, while, at a concentration of 1 mM, butyrate induced expression of T-bet and IFN-γ in Tregs and conventional T cells. Furthermore, during the DSS-induced colitis, the treatment of germ-free (GF) mice with butyrate increased the colonic expression of the proinflammatory molecules T-bet and IFN-γ.
MaTerials anD MeThODs animals 6-to 10-week-old wild-type (WT) C57BL/6 mice were obtained from Charles River Laboratories (Sulzfeld, Germany) and were maintained under specific pathogen-free (SPF) or GF conditions. GF mice were kept in adequate isolators (Metall + Plastic, Radolfzell-Stahringen, Germany). The bedding, water, and food were routinely autoclaved, and the sterility of GF mice was checked biweekly. All experimental procedures with GF animals were carried out in a laminar flow hood under sterile conditions. Ffar2 −/− Ffar3 −/− , Slc5a8 −/− , and Tbx21 −/− mice on C57BL/6 background were bred at the animal facility of the Biomedical Research Center, University of Marburg, Germany. Ffar2 −/− Ffar3 −/− mice were generated by Prof. Stefan Offermanns (Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany) (16).
Slc5a8 −/− mice were received from Dr. Thomas Boettger (Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany). All animal experiments were conducted according to the German animal protection law.

experimental colitis and scFa administration
Following oral treatment with 100 mM sodium butyrate for 3 weeks, the acute colonic inflammation in GF mice was induced by adding 1.5% (w/v) DSS (MP Biomedicals, Eschwege, Germany) to the drinking water for 5 days (for the experiments with WT mice, 2% DSS was used). Animals were orally treated with 100 mM butyrate throughout the duration of the experiment based on the published protocol (6). In some experiments, mice were orally treated with 100 mM sodium butyrate for 3 weeks without colitis induction. In all SCFA experiments, the drinking water of control mice was pH-and sodium-adjusted. Colitis was scored using a previously published inflammation scoring system (18). Lamina propria mononuclear cells were purified from mice at day 8 after induction of colitis and were analyzed by FACS analysis and RT-PCR.

immunoblot analysis
For the preparation of cell lysates, 5 × 10 6 cells were harvested from Treg-inducing cell cultures and pelleted at 390 × g for 10 min at 4°C. Cell pellets were lysed with RIPA cell lysis buffer for 20 min on ice. Following the SDS-PAGE, protein samples were transferred to a PVDF membrane, and protein detection was performed in a chemiluminescence image station (MicroChemi, Biostep GmbH, Burkhardtsdorf, Germany). For the detection of histone acetylation in Tregs, anti-acetyl-Histone H3 and H4 Abs (Merck Millipore, Darmstadt, Germany) were used. As a loading control for total cell protein extracts, a monoclonal anti-mouse β-actin Ab (Sigma-Aldrich, Munich, Germany) was used.
hDac inhibitor activity assay CD4 + T cells were cultured under Treg-inducing conditions for 72 h. Subsequently, the cells were harvested in the lysis buffer and subjected to HDAC inhibition by adding 5 mM of SCFAs for 10 min at room temperature. Following initial inhibition of HDACs, the peptide substrate Ac-Arg-Gly-Lys-AMC (Bachem, Bubendorf, Switzerland) was added to the reaction tubes for next 30 min and finally the stop solution stopped the reaction mediated by HDAC enzymes. HDAC inhibition activity was determined by measuring the fluorescence intensity of free AMC at the spectrofluorometer FLUOstar Omega (BMG Labtech, Ortenberg, Germany).

chromatin immunoprecipitation (chiP)
Chromatin immunoprecipitation analysis was performed on CD4 + T cells which were cultured under optimal Tregpolarizing conditions for 3 days. A total of 1 × 10 6 Tregs were fixed with 1% formaldehyde for 10 min at room temperature. Subsequently, genomic DNA was extracted from precipitates using specific antibody for pan-acetylated H3 (Merk Millipore, Darmstadt, Germany). Control experiments were carried out with respective isotype antibodies as previously described (19). Samples were probed for the promoter regions of Ifnγ, Il17a, Tbx21, and Foxp3 by quantitative RT-PCR using the following primers: Ifnγ, forward CATACCCTTTCCTTGCTTTTC and reverse TTGTGGGATTCTCTGAAAGCA, Il17a, forward TGG TTCTGTGCTGACCTCAT and reverse GCTCTCCCTGGACT CATGTT, Tbx21 forward AGGTGGCAGGTTGACTCT and reverse CTGCTCCTGGGCTTTCTC, and Foxp3, forward TTC CCATTCAGCTTCA and reverse TGTTTGTGAGTGGAGG.

qrT-Pcr
In a first step, the total RNA is isolated from cell lysates. Subsequently, the RNA was transcribed into cDNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific) according to the manufacturer's instructions. Quantitative real-time PCR for Tbx21, Ifnγ, Gata3, and Rorγt was performed using a StepOnePlus device (Applied Biosystems, Darmstadt, Germany) with the following primers: Tbx21 forward CAACAACCCCTTTGCCAAAG, Tbx21 reverse TCCCCCAAGCAGTTGACAGT, Foxp3 forward TTCCTTCCCAGAGTTCTTCCA, Foxp3 reverse CATTGAGT GTCCTCTGCCTCT, Ifnγ forward GCAACAGCAAGGCGAAA AAG, Ifnγ reverse TTCCTGAGGCTGGATTCGG, Gata3 for ward AAGGCAGGGAGTGTGTGAAC, Gata3 reverse AGGATGTC CCTGCTCTCCTT, Rorγt forward TCCTGCCACCTTGAGTA TAGTCC, and Rorγt reverse GGACTATACTCAAGGTGGCA GGA. Quantitative analysis was performed by normalizing the target gene expression to expression of housekeeping gene Hypoxanthine-guanine phosphoribosyl transferase (Hprt1). The following primers were used for detection of Hprt1: forward, CTGGTGAAAAGGACCTCTCG, reverse, TGAAGTACTCATT ATAGTCAAGGGCA.

scFas analysis by Means of Ultra-high Performance liquid chromatography-Mass spectrometry (UhPlc-Ms)
Luminal content of colon (~20 mg) was weighted in sterile ceramic bead tubes (NucleoSpin ® Bead Tubes, Macherey-Nagel, Dueren, Germany) and extracted with 1 mL chilled methanol . The flow rate was set to 0.3 mL/min, injection volume to 5 µL, and column temperature to 30°C. After a pre-runtime of 2 min, a gradient profile was applied by starting at 1% B for 1 min, increasing to 25% B until 10 min, following an increase to 95% B within 7 min. The 95% B was hold for 2 min, returning to initial 1% B within 0.

statistical analysis
To compare the mean between two groups, statistical analysis was performed using an unpaired Student's t-test with Prism 5 software (GraphPad, La Jolla, CA, USA). P-Values of P < 0.05 were considered statistically significant. For the comparisons of the multiple groups, the one-way analysis of variance was used. The following P-values were used: ***P < 0.001, **P = 0.001-0.01, *P = 0.01-0.05. Where appropriate, the mean ± SEM is represented in graphs.

resUlTs
Butyrate Facilitates TgF-β1-Dependent generation of Foxp3 + Tregs Treatment of CD4 + T cells with butyrate has been shown to promote the differentiation of mucosal Tregs (3,4). Accordingly, we observed that WT mice treated orally with 100 mM sodium butyrate for 3 weeks were capable of locally expanding Tregs in colonic lamina propria but not in mesenteric lymph nodes (mLN) or spleen (Figures 1A,B). A similar increase in the frequency of colonic Tregs was observed when mice received 200 mM of sodium butyrate in the drinking water. In contrast, no enhanced Treg responses were observed after oral treatment of WT animals with 50 mM sodium butyrate ( Figure S1 in Supplementary Material). Furthermore, neither frequencies nor cell numbers of colonic Th1 and Th17 cells were affected by the butyrate treatment, suggesting that under steady-state conditions only colonic Tregs and not effector T cells are regulated by SCFAs ( Figure 1C and data not shown). Finally, oral administration of butyrate resulted in increased colonic mRNA levels of Il10 and Foxp3 but not of pro-inflammatory genes such as Tbx21 (Figure 1D).
In in vitro experiments, we confirmed that low concentrations of butyrate (0.1-0.25 mM) enhance expression of Foxp3 and differentiation of inducible (i) Tregs in the presence of TGF-β1 ( Figure 1E). Interestingly, suboptimal Treg-inducing TGF-β1 concentrations were essential for butyrate-mediated impact on Treg differentiation. The treatment of CD4 + T cells with butyrate in the absence of TGF-β1 did not lead to conversion into Foxp3 + Tregs, whereas optimal Treg-inducing TGF-β1 concentrations masked the effects of butyrate (Figure 1F). To exclude potential contamination by low numbers of already pre-existing Tregs in the CD4 + T cell culture, we sorted and cultured highly purified naïve GFP -CD62L + CD4 + T cells from DEREG mice in the presence of butyrate and TGF-β1. After 5 days of culture, we observed an increase in GFP + CD4 + T lymphocytes in butyrate-treated cells as compared to the control cell cultures indicating a direct effect of butyrate on de novo generation of Tregs from naïve T cells ( Figure S2 in Supplementary Material). Taken together, butyrate cooperates with TGF-β1 to enhance the expression of Foxp3 and differentiation into Tregs. This effect of butyrate was proposed to be based on its potent HDAC-inhibitory activity and ability to epigenetically regulate Foxp3 gene expression. In addition, butyrate was also able to stabilize Foxp3 protein by promoting its acetylation (3,20).

Butyrate induces expression of T-bet and iFn-γ during Treg Differentiation
Surprisingly, when performing a titration experiment with butyrate-treated T cells cultured under Treg-inducing conditions, we found that the frequencies of Foxp3 peaked at 0.25 mM butyrate concentration, while higher butyrate doses suppressed the differentiation of Foxp3 + Tregs (Figures 2A,B). Recently, it has been shown that SCFAs not only promote expansion of Tregs but impact also on Th1 and Th17 cells (21). Therefore, we examined whether SCFAs might trigger the production of pro-inflammatory cytokines under Treg-polarizing conditions. Notably, butyrate and to a lesser extent propionate were able to selectively induce expression of IFN-γ but not of IL-17A or IL-4 in a dose-dependent manner (Figures 2C,D; Figure S3A,B in Supplementary Material). Interestingly, this effect was independent of FFA2 and FFA3 receptors and of Slc5a8 transporter as the production of IFN-γ was not impaired in   (Figure 2F). To test whether the increase in IFN-γ production correlates with the HDAC inhibitory effects of SCFAs, we performed an HDAC activity assay on iTregs in the presence of butyrate, propionate, and acetate. While butyrate and propionate exerted a potent HDAC inhibitory effect, acetate almost completely lacked this inhibitory activity (Figure 2G).

Butyrate induces h3 acetylation at Tbx21
and Ifnγ locus during Treg Differentiation As previously described, CD4 + T cells cultured under Tregpolarizing conditions and treated with 1 mM butyrate showed significantly increased acetylation of histones H3 and H4 as compared to untreated Tregs ( Figure 3A). Under unpolarized conditions, histones are hypoacetylated at the Ifnγ locus in CD4 + T cells. During Th1 differentiation, proximal promoter sites of the Ifnγ gene become hyperacetylated (22,23). To analyze if the inhibition of HDAC activity by butyrate induces histone acetylation directly at the Ifnγ and Tbx21 locus during Treg differentiation, we performed ChIP assay using an anti-acetyl-H3 antibody. Our results revealed that butyrate was capable of promoting H3 acetylation at the Ifng and Tbx21 but not at Il17a locus in T cells cultured under Treg-inducing conditions ( Figure 3B). Of note, at a concentration of 0.25 mM, butyrate was already able to increase the pan-H3 acetylation during Treg differentiation ( Figure S5A in Supplementary Material). In the absence of butyrate, at the Foxp3 promoter region, H3 acetylation was observed only in TGF-β1-generated iTregs but not in naïve CD4 + T cells. This TGF-β1-mediated effect was enhanced after treatment of iTregs with 0.25 or 1 mM butyrate. Interestingly, at a concentration of 0.25 mM, butyrate was capable of upregulating H3 acetylation at T-bet and IFN-γ have been shown to directly oppose the induction of Foxp3 expression and negatively regulate differentiation of Tregs (24,25). FACS analysis confirmed induction of T-bet protein and partial reduction of Foxp3 expression in butyrate-treated cells during Treg differentiation (Figure 3C). By comparing the ability of WT and Tbx21 −/− CD4 + T cells cultured  under Treg-inducing conditions to induce IFN-γ after butyrate treatment, we found that T-bet was required for production of endogenous IFN-γ ( Figure 3D). Furthermore, we observed almost no reduction of Foxp3 frequencies in the absence of T-bet in T cells cultured under Treg-inducing conditions and treated with 1 mM butyrate. These data point to the role of this transcription factor in suppressing Foxp3 expression during the butyrate treatment (Figure 3E). To further prove the requirement of HDAC inhibition for IFN-γ induction, CD4 + T cells cultured under Treg-inducing conditions were treated with increasing concentrations of pan-HDAC inhibitor TSA. Indeed, the treatment with TSA caused the induction of IFN-γ production and reduction of Foxp3 expression during Treg differentiation (Figures 3F-I). In conclusion, our findings indicate that SCFAs might exhibit multiple effects during Treg differentiation including the induction of pro-inflammatory molecules.

Butyrate induces the expression of iFn-γ in Unpolarized and Polarized cD4 + T cells
To extend our analysis from the Treg-polarizing conditions to other effector T cells, we first examined possible effects of SCFA-induced IFN-γ expression under unpolarized conditions (Th0 cells). Butyrate was capable of selectively upregulating IFN-γ without inducing the expression of IL-17A ( Figure 4A). The treatment of Th0 cells with butyrate increased the expression of IFN-γ in a dose-dependent manner peaking at the concentration of 1 mM. Propionate was less effective than butyrate, while acetate was not able to induce the expression of IFN-γ in unpolarized T cells (Figures 4A,B). Furthermore, we were wondering whether pan-HDAC inhibitor TSA is capable of inducing IFN-γ expression in Th0 cells. Similar to butyrate, a strong induction of IFN-γ but not of Foxp3 or IL-17A was observed after treatment of cells with TSA (Figures 4C,D). Of note, this effect of HDAC inhibitors butyrate and TSA was mediated without contribution of additional factors, while the butyrate-triggered expansion of Tregs was TGF-β1 dependent ( Figure 1F).
Moreover, in the presence of butyrate, the IFN-γ expression was increased even in Th1 cells that already display high levels of this cytokine ( Figure S6 in Supplementary Material). Remarkably, we also observed a strong, butyrate-triggered induction of IFN-γ and T-bet and reduced IL-4 and IL-17A expression even under Th2and Th17-promoting conditions, respectively (Figures 5A-D; Figure S7A in Supplementary Material). This effect seems to be mediated via T-bet as Tbx21 −/− Th17 and Th2 cells did not switch toward IFN-γ production (Figures 5E,F). In accordance with these results, the inhibitory effect of butyrate on the expression of Gata3 and Rorγt, genes encoding for transcription factors controlling Th2 and Th17 development, was detected in WT but not in T-bet-deficient cells ( Figure S7B in Supplementary Material).

Butyrate increases T-bet and iFn-γ expression levels in acute colitis
A recent study has suggested that butyrate treatment is ineffective in ameliorating dextran sodium sulfate (DSS)-induced colitis in conventional mice pretreated with antibiotics (14). Although specific in vivo effects of SCFAs on Tregs have been proposed, the induction of Th1-associated genes by butyrate in an inflammatory milieu is also conceivable. To address this possibility, GF mice were orally treated with 100 mM butyrate [based on the published protocol (6)], during the course of DSSinduced colitis. As expected, in naïve GF mice, no SCFAs were found, owing to the absence of microbiota, whereas conventional (SPF) mice exhibited high luminal levels of acetate, propionate, and butyrate in the colon ( Figure 6A). Our data demonstrate that butyrate treatment does not attenuate acute intestinal inflammation in GF animals. Although the severity of colitis was similar in both groups, GF mice treated with butyrate and DSS had even slightly increased weigh loss as compared to only DSS-administered mice (Figures 6B,C). While the treatment of animals with butyrate did not impact on the Foxp3 levels during the course of inflammation, the expression of pro-inflammatory molecules Tbx21 and Ifnγ was significantly increased after butyrate administration as compared to only DSS-treated group ( Figure 6D). In addition, the FACS analysis confirmed a higher frequency of T-bet + cells within colonic CD4 + T lymphocytes after butyrate treatment of GF mice (Figures 6E,F). A similar trend was observed in WT mice treated with butyrate and DSS ( Figure   S8A,B in Supplementary Material). In summary, our present data challenge the current paradigm that butyrate-mediated effects on the host immune system are only beneficial. We conclude that, in the context of inflammatory bowel disease (IBD), the elevated levels of butyrate in lamina propria might be more harmful than previously thought.

DiscUssiOn
In the intestine, dietary fibers are fermented by commensal bacteria into SCFAs (26). Several beneficial effects of SCFAs such as suppression of intestinal inflammation, protection from colorectal cancer, and dampening allergic and autoimmune responses in the lung and CNS have been reported (6,8,13). Therefore, physiological concentrations of butyrate, propionate, and acetate (between 10 and 100 mM in the gut lumen) are thought to be essential for maintaining an immunological balance at mucosal surfaces. Recently, it has been shown that SCFAs promote expansion of colonic Tregs in conventionally reared and GF mice. Mechanistically, these effects of SCFAs were mediated through SCFA-receptor FFA2. In contrary to other Treg subpopulations, colonic Tregs were shown to express high levels of FFA2 and the observed SCFA-mediated effects were abrogated in mice lacking this receptor (6). Ability of butyrate to impact on differentiation of Tregs is likely mediated through its HDAC inhibitory activity because similar influence on Tregs was previously observed for pan-HDACi TSA (27,28).
In this study, we investigated the immunomodulatory effects of butyrate on Tregs and other CD4 + T cell subsets. Here, we demonstrate that butyrate is capable of triggering expression of T-bet and IFN-γ in all examined T cell populations. We further showed that GF mice treated with butyrate exhibited increased levels of pro-inflammatory molecules IFN-γ and T-bet during acute colonic inflammation. Thus, under homeostatic conditions, the coevolution of microbial community in the gut and mucosal immune system is reflected in the expansion of anti-inflammatory Tregs by microbial metabolites. Such anti-inflammatory activities of SCFAs and other metabolites might be overridden during the acute or chronic inflammation whereby SCFA-mediated pro-inflammatory effects might predominate.
In contrast to the previous work that has demonstrated that butyrate mainly modulates the histone acetylation at the Foxp3 locus but not at the Tbx21, Rorγt, and Gata3 genes (4), our novel data reveal that the promoters of some pro-inflammatory genes such as Ifnγ and Tbx21 are hyperacetylated by butyrate under Treg-inducing conditions. These discrepant findings might be due to different doses of butyrate used in the experimental protocols. Previously, butyrate and pan-HDAC inhibitor TSA were shown to establish the long-range histone acetylation across the Ifnγ gene in CD4 + T cells under neutral conditions or were even able to restore the histone acetylation of the Ifnγ and Tbx21 locus under Th2 conditions (23,29). These findings might explain the underlying mechanisms involved in vivo observations made by us and others showing that SCFAs are able to exacerbate DSS-induced colitis or to cause T cell-mediated tissue inflammation in the renal system (15,30).
Interestingly, physiological concentrations of SCFAs apart from the gut lumen and portal vein have not been assessed yet. Given that a large amount of luminal butyrate is consumed by colonic epithelial cells as energy source, physiological concentrations of butyrate in lamina propria are considered not to reach substantial levels. Alterations in intestinal permeability in patients with IBD due to disturbed epithelial barrier could lower the luminal levels of SCFAs and result in the influx of microbial metabolites in the lamina propria. In order to better understand various effects of SCFAs on the mucosal immune system, the measurement of the local concentrations of these metabolites in the intestinal-associated tissues such as mLN and Peyer's patches at steady-state and under inflammatory conditions should be addressed in future studies. Collectively, we show that Foxp3 + Tregs require the presence of TGF-β1 for butyrate-mediated expansion. In contrast, the butyrate treatment of CD4 + T cells in the absence of additional stimuli is sufficient for rapid and robust induction of Th1-associated factors T-bet and IFN-γ.

eThics sTaTeMenT
All animal experiments were conducted according to the German animal protection law. The study was approved by Regierungspräsidium Gießen, Germany, Nr. 70/2014. acKnOWleDgMenTs This study was supported by the UGMLC research collaborative grant (Ulrich Steinhoff and Stefan Offermanns) and by a research grant from the Fritz Thyssen Foundation (Alexander Visekruna). We thank Anne Hellhund and Elena Jenike for technical assistance. We are grateful to Dr. Stefanie Hagner (Philipps University of Marburg, Germany) for providing us with Tbx21 −/− mice.

FUnDing
This study was supported by the UGMLC research collaborative grant (Ulrich Steinhoff and Stefan Offermanns) and by a research grant from the Fritz Thyssen Foundation (Alexander Visekruna).