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International Immunology Advance Access originally published online on June 7, 2009
International Immunology 2009 21(7):881-889; doi:10.1093/intimm/dxp054
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© The Japanese Society for Immunology. 2009. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Gfi1 negatively regulates Th17 differentiation by inhibiting ROR{gamma}t activity

Kenji Ichiyama1, Masayuki Hashimoto1, Takashi Sekiya1, Ryusuke Nakagawa1, Yu Wakabayashi1,3, Yuki Sugiyama1,3, Kyoko Komai1, Ingrid Saba2, Tarik Möröy2 and Akihiko Yoshimura1,3

1 Department of Microbiology and Immunology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan
2 Institut de recherches cliniques de Montréal, Departement de Microbiologie et Immunologie, Université de Montréal, 110 Avenue des Pins West Montréal, Quebec H2W 1R7, Canada
3 Japan Science and Technology Agency, CREST, Chiyoda-ku, Tokyo 102-0075, Japan

Correspondence to: A. Yoshimura; E-mail: yakihiko{at}a6.keio.jp


   Abstract
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 Abstract
Introduction
 Methods
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 Funding
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Th cells have long been divided into two subsets, Th1 and Th2; however, recently, Th17 and inducible regulatory T (iTreg) cells were identified as new Th cell subsets. Although Th1- and Th2-polarizing cytokines have been shown to suppress Th17 and iTreg development, transcriptional regulation of Th17 and iTreg differentiation by cytokines remains to be clarified. In this study, we found that expression of the growth factor independent 1 (Gfi1) gene, which has been implicated in Th2 development, was repressed in Th17 and iTreg cells compared with Th1 and Th2 lineages. Gfi1 expression was enhanced by the IFN-{gamma}/STAT1 and IL-4/STAT6 pathways, whereas it was repressed by the transforming growth factor-β1 stimulation at the promoter level. Over-expression of Gfi1 strongly reduced IL-17A transcription in the EL4 T cell line, as well as in primary T cells. This was due to the blockade of recruitment of retinoid-related orphan receptor {gamma}t to the IL-17A promoter. In contrast, IL-17A expression was significantly enhanced in Gfi1-deficient T cells under Th17-promoting differentiation conditions as compared with wild-type T cells. In contrast, the impacts of Gfi1 in iTregs were not as strong as in Th17 cells. Taken together, these data strongly suggest that Gfi1 is a negative regulator of Th17 differentiation, which represents a novel mechanism for the regulation of Th17 development by cytokines.

Keywords: IL, Smad, STAT, transcription factor


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 Funding
 References
 
CD4+ Th cells play a central role in the immune response. Upon activation by antigens, Th cells undergo distinct developmental pathways, resulting in specialized properties and effector functions. CD4+ T cells are traditionally thought to differentiate into Th1 and Th2 cell subsets (1). Th1 cells require the transcription factor T-bet, secrete IFN-{gamma} and regulate cellular immunity. In contrast, Th2 cells express GATA3 and c-Maf, produce IL-4, IL-5 and IL-13 and mediate humoral responses (2–4).

Recently, a new subset of the polarized T cell subset Th17 cells, characterized by the production of IL-17A, was identified and found to play an important role in autoimmune diseases, elimination of extracellular bacteria and cancer (5–7). The differentiation of Th17 cells from naive T cells requires transforming growth factor-β1 (TGF-β1) and IL-6 both in vitro and in vivo (8–10). In addition, both IL-21 and IL-23 are thought to be critical for the maturation and/or maintenance of Th17 cells (11–13). Recently, the nuclear orphan receptor [retinoid-related orphan receptor {gamma}t (ROR{gamma}t)], which is induced by TGF-β1 and IL-6, has been shown to be indispensable for the differentiation of Th17 cells (14). Furthermore, we recently reported that ROR{gamma}t was directly involved in IL-17A transcription through binding to IL-17A promoter and that Foxp3 inhibits IL-17A expression through interaction with ROR{gamma}t (15).

TGF-β1 has also been shown to induce Foxp3, a master transcriptional factor of regulatory T (Treg) cells. Thymus-derived naturally occurring regulatory T (nTreg) cells predominantly express Foxp3 and are critically important for immune homeostasis (16). Likewise, in vitro TGF-β1-induced Treg (iTreg) cells can suppress effector T cell proliferation in vitro as well as in vivo, just as nTreg cells can (17, 18).

Differentiation of Th cells is regulated by various cytokines and transcription factors. For example, Th1 is suppressed by IL-4 and Th2 is suppressed by IFN-{gamma}. Moreover, inducible regulatory T (iTreg) is up-regulated by IL-2/STAT5 and down-regulated by IFN-{gamma}, IL-4 and IL-6/STAT3 (10, 19–21). We recently reported that IL-4/STAT6 inhibits TGF-β1-mediated Foxp3 induction through direct binding to the Foxp3 promoter (22). In the case of Th17 differentiation, it has been recently reported that Th17 is negatively regulated by the retinoic acid receptor RAR/RXR and positively regulated by the aryl hydrocarbon receptor AHR (23–25). In addition, IFN regulatory factor 4 is required for Th17 development, while Ets-1 negatively regulates Th17. Th17 development is also repressed by various cytokines, such as IL-2, IL-4, IL-27 and IFN-{gamma} (26–31). These cytokines activate STAT1, STAT5 and STAT6. However, the mechanisms underlying the blockade of Th17 differentiation by these factors are not entirely clear.

In this study, we found that the expression of growth factor independent 1 (Gfi1) was significantly up-regulated in Th1 and Th2 cells, whereas it was repressed in Th17 and iTreg cells. Gfi1 is a transcriptional repressor with a molecular weight of 47–55 kDa that recognizes the conserved AATC core sequence and binds to DNA through the C-terminal C2H2-type zinc finger domain. Gfi1 has been shown to play a role in granulocyte development, T cell differentiation and macrophage-dependent cytokine production (32). Recently, it was reported that Gfi1 could be induced in Th2 cells by IL-4 and STAT6 and that Gfi1 controls Th2 differentiation through the regulation of GATA3 protein stability (33, 34). However, the role of Gfi1 in regulating the differentiation of Th17 and iTreg cells remains unknown. We determined that over-expression of Gfi1 reduced IL-17A transcription in EL4 and primary T cells and inhibited ROR{gamma}t-mediated IL-17A promoter activity by inhibiting ROR{gamma}t activity. Moreover, Gfi1-deficient T cells produced an increased level of IL-17A under Th17 differentiation conditions, whereas the effect of Gfi1 on TGF-β1-mediated Foxp3 induction was partial. Taken together, our study provides a novel mechanism for regulation of Th17 development.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 Funding
 References
 
Mice
C57BL/6 mice were purchased from CLEA Japan, Inc. (Tokyo, Japan). The GFI1-deficient mice were described previously (35). The Smad3-deficient mice were generously provided by Dr Saika Shizuya (Wakayama University School of Medicine) and Dr Flanders (36). The T cell-specific Smad2-deficient mice will be described elsewhere. All mice were used between the ages of 6 and 12 weeks, and all experiments were approved by the Animal Ethics Committee of Keio University.

Plasmid constructs
PCR was done to generate Gfi1 promoter plasmid by using mouse genomic DNA as a template. In the case of Gfi1 promoter, an ~2 kb fragment corresponding to nucleotides from –1495 to +505 relative to the determined transcriptional staring site of Gfi1 gene was sub-cloned into pGV-basic 2 vector (TOYOINKI). The IL-17A promoter plasmids and the mouse ROR{gamma}t complementary DNA (cDNA)have been described previously (15). The Foxp3 promoter plasmids have been described previously (22). The constitutively active transforming growth factorβ receptor I (CA-TβRI) was provided by Dr Joan Massagué and has been described previously (37). Gfi1 cDNA and its mutant form have been described previously (38).

Luciferase assay
Both transfection into HEK 293T and EL4 cells and the luciferase assay have been described previously (15, 22). Briefly, HEK 293T cells were seeded on 6-well plates (2 x 105 cells per well), cultured for 24 h and transfected with various amounts of expression vector by PEI method (39). In contrast, EL4 cells (5 x 106 cells per well) were transfected by electroporation and then harvested 24 h later. All cells were harvested using 50 µl of lysis buffer. The luciferase assay was performed using a luciferase substrate kit (Promega), and luciferase activity was read with a Packard luminometer and normalized to an internal control, β-galactosidase (β-gal). Western blotting and immunoprecipitation were performed as reported previously (37).

Naive T cell preparation and differentiation
Single-cell suspensions from the mouse spleens and lymph nodes were negatively depleted by first staining with anti-B220-biotin, anti-CD11b-biotin, anti-CD11c-biotin, anti-Ter119-biotin, anti-DX5-biotin, anti-CD8{alpha}-biotin and anti-CD25-biotin (all from eBioscience) at a 1:125 dilution for 20 min on ice and then incubating with anti-biotin magnetic microbeads (Milteny Biotech) at a 1:20 dilution for 20 min on ice. The depleted fraction was stained with anti-CD62L-FITC, anti-CD25-PE, anti-CD4-PerCP and anti-CD44-APC. Cell sorting was performed using a FACSAria cell sorter (Becton Dickinson) to obtain a pure population of primary CD4+CD25–CD62LhighCD44– T cells (typically >98% purity). The CD4+CD25–CD62LhighCD44– T cells (1 x 106 cells per well) were cultured at 37°C (5% CO2) in RPMI-1640 (Invitrogen Life Technologies). The medium was supplemented with 10% FCS, 2 mM L-glutamine, 100 U ml–1 penicillin, 100 µg ml–1 streptomycin and 50 µM 2-mercaptoethanol. The cells were stimulated with the plate-bound anti-CD3 antibody (1 µg ml–1, clone 145-2C11) and the soluble anti-CD28 antibody (0.5 µg ml–1, eBioscience). For Th0 differentiation, the cells were treated with 1 µg ml–1 anti-IFN-{gamma} antibody and 1 µg ml–1 anti-IL-4 antibody. For Th1 differentiation, the cells were treated with 10 ng ml–1 mIL-12p70 (R&D Systems) and 1 µg ml–1 anti-IL-4 antibody. For Th2 differentiation, the cells were treated with 10 ng ml–1 mIL-4 (PeproTech) and 1 µg ml–1 anti-IFN-{gamma} antibody. For iTreg differentiation, the cells were treated with 2 ng ml–1 human TGF-β1 (R&D Systems), 1 µg ml–1 anti-IFN-{gamma} antibody and1 µg ml–1 anti-IL-4 antibody. For Th17 differentiation, the cells were treated with 1 ng ml–1 human TGF-β1, 20 ng ml–1 human IL-6 (R&D Systems), 1 µg ml–1 anti-IFN-{gamma} antibody and 1 µg ml–1 anti-IL-4 antibody. Cell surface marker and intracellular cytokine staining were performed as described previously (40).

Retroviral transduction
Sorted naive CD4+CD25–CD62LhighCD44– T cells were plated in the Th0 condition starting on day 0. On day 1, fresh retrovirus supernatant was added and the cells were spun at 2500 r.p.m. for 2 h at 35°C. After spin infection, the cells were cultured in iTreg or Th17 condition medium and harvested on day 4 or 5 for intracellular cytokine staining and real-time PCR analysis.

Reverse transcription–PCR and real-time PCR
The cells were lysed in RNAiso (Takara) for RNA extraction. Reverse transcription (RT)–PCR was performed according to standard procedures. The expression level of GAPDH was evaluated as an internal control. The primer sequences were as follows: GAPDH, 5'-ACCACAGTCCATGCCATCAC-3' and 5'-TCCACCACCCTGTTGCTGTA-3'; HPRT, 5'-TGAAGAGCTACTGTAATGATCAGTCAAC-3' and 5'-AGCAAGCTTGCAACCTTAACCA-3'; Gfi1, 5'-AGGAACGCAGCTTTGACTGT-3' and 5'-GATGAGCTTTGCACACTGGA-3'; ROR{gamma}t, 5'-ACCTCCACTGCCAGCTGTGTGCTGTC-3' and 5'-TCATTTCTGCACTTCTGCATGTAGACTGTCCC-3'; IL-17A 5'-CAGCAGCGATCATCCCTCAAAG-3' and 5'-CAGGACCAGGATCTCTTGCTG-3' and Foxp3, 5'-CCCAGGAAAGACAGCAACCTT-3' and 5'-TTCTCACAACCAGGCCACTTG-3'. Real-time PCR was performed on cDNA samples using the SYBR Green system (Applied Biosystems). The relative quantitation value is expressed as 2–delta Ct, where delta Ct is the difference between the mean Ct value of triplicates of the sample and of the endogenous HPRT control.

Chromatin immunoprecipitation assay
The chromatin immunoprecipitation (ChIP) assay was performed using ChIP-IT Express (Active Motif) according to the manufacturer's instructions. The cell extracts were immunoprecipitated with polyclonal rabbit anti-T7-tagged antibody (Medical & Biological Laboratories) or monoclonal mouse anti-FLAG-tagged antibody (Sigma) or rabbit anti-acetyl-Histone H4 antibody (Upstate) and protein G magnetic beads for 4 h at 4°C. The DNA–histone complexes were incubated for 15 min at 94°C for reverse cross-linking and then treated with protease K. Purified DNA fragments were subjected to real-time PCR, which addressed for the IL-17A promoter region +37 to –205. Real-time PCR was performed using the following primers: 5'-AACTTCTGCCCTTCCCATCT-3' and 5'-GTTTGCGCGTCCTGATCAGC-3'.

Statistical analysis
The Student's paired two-tailed t-test was used. Values of P < 0.05 were considered significant. All error bars shown in this article represent standard deviations.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 Funding
 References
 
Gfi1 mRNA is up-regulated in Th1 and Th2 cells and down-regulated in iTreg and Th17 cells
We previously reported that loss of suppressor of cytokine signaling 1 (SOCS1) in Th cells causes defective Th17 differentiation (41). To identify candidate genes that might be involved in Th17 suppression, we compared the transcription factors expressed in wild-type (WT) and SOCS1–/– T cells using DNA microarray analysis. Among the genes up-regulated over twice as much in SOCS1–/– T cells compared with WT T cells under the Th17 differentiation conditions, we focused on Gfi1 because it has previously been shown to be involved in Th2 differentiation (33, 34).

To determine the pattern of Gfi1 mRNA expression in each Th subset, we examined the expression of Gfi1 mRNA in T cells stimulated with anti-TCR antibody in the presence of cytokines for helper T cell differentiation. As shown in Fig. 1(A), Gfi1 mRNA was highly expressed in cells exposed to the Th1 and Th2 differentiation conditions, whereas the levels were very low in cells exposed to Th17 and iTreg differentiation conditions. It has been reported that the induction of Gfi1 mRNA is dependent on STAT6 (34). Thus, we investigated Gfi1 expression in STAT6–/– T cells. In accordance with Zhu et al., in STAT6–/– T cells, increased Gfi1 mRNA expression was maintained under differentiation conditions for Th1 but not Th2 (Fig. 1B). In contrast, STAT1–/– T cells expressed high levels of Gfi1 mRNA under Th2, but not Th1, differentiation conditions (Fig. 1B). These data suggest that the induction of Gfi1 expression in Th1 and Th2 cells was dependent on STAT1 and STAT6, respectively.


Figure 1
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  		Fig. 1. The expression of Gfi1 mRNA in each Th subsets. (A) Naive T cells were cultured for 24 h with anti-TCR antibody supplemented with different combinations of antibodies to promote various differentiation pathways: anti-IFN-{gamma} antibody and anti-IL-4 antibody (Th0 differentiation); mIL-12p70 and anti-IL-4 antibody (Th1 differentiation); mIL-4 and anti-IFN-{gamma} antibody (Th2 differentiation); human TGF-β1, anti-IFN-{gamma} antibody, and anti-IL-4 antibody (iTreg differentiation) and human TGF-β1, human IL-6, anti-IFN-{gamma} antibody, and anti-IL-4 antibody (Th17 differentiation). The expression of Gfi1 mRNA was analyzed by RT–PCR. (B) The expression of Gfi1 mRNA in primary T cells from WT, STAT1–/– and STAT6–/– mice was examined after exposure to the above differentiation conditions for 24 h. (C) The HEK 293T cells were transfected with control vector or Gfi1 promoter and β-galactosidase along with CA-TβRI. After incubation, luciferase activity was measured and normalized against β-galactosidase activity. (D) The expression of Gfi1 mRNA in T cells from WT, SMAD2–/– and SMAD3–/– mice. The relative expression of Gfi1 mRNA was normalized against that of HPRT. One representative experiment out of two independent experiments is shown.

 
In contrast, Gfi1 mRNA expression decreased in the iTreg- and Th17-skewed cells (Fig. 1A). Both iTreg- and Th17-polarizing media contained TGF-β1. Thus, we speculated that Gfi1 transcription is suppressed by TGF-β1. To test this, we cloned the Gfi1 promoter and performed the reporter analysis in HEK 293T cells. The Gfi1 promoter activity was attenuated by addition of CA-TβRI, suggesting that Gfi1 promoter directly repressed by TGF-β signaling (Fig. 1C). To determine the downstream-signaling pathway of the TGF-β receptor, we examined Gfi1 expression in T cells from mice lacking Smad2 or Smad3. Unexpectedly, Gfi1 expression was suppressed in Th17 differentiation condition in both Smad2–/– and Smad3–/– T cells (Fig. 1D). It has been shown that the Smad pathway is non-essential for development of Th17 (42). Therefore, Smad-independent TGF-β-signaling pathways could be involved in both Gfi1 repression as well as ROR{gamma}t induction.

Forced expression of Gfi1 suppresses IL-17A expression in HEK 293T and EL4 cells
To investigate the effect of Gfi1 on IL-17A expression, we examined the levels of IL-17A in EL4 cells using RT–PCR. We have previously reported that EL4 cells express IL-17A in response to anti-TCR stimulation because of constitutive expression of ROR{gamma}t (15). Interestingly, forced expression of Gfi1 significantly reduced the expression levels of IL-17A (Fig. 2A). However, the expression of ROR{gamma}t was not affected by Gfi1 over-expression. Suppression was not observed when Gfi1III, N-terminal-truncated Gfi1 lacking transcriptional repressor activity, was introduced (data not shown). Thus, these data suggest that Gfi1 specifically inhibits the IL-17A expression.


Figure 2
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  		Fig. 2. Gfi1 suppresses ROR{gamma}t-mediated IL-17A expression. (A) The expression of various mRNAs was determined by RT–PCR (left) or by real-time PCR (right) in EL4 cells transfected with control GFP or Gfi1-GFP. After sorting with GFP, EL4 cells were cultured with or without anti-CD3 for 24 h and then the total RNA was isolated. The relative expression of IL-17A mRNA was normalized against that of HPRT. (B) HEK 293T (left) and EL4 (right) cells were transfected with IL-17A promoter, ROR{gamma}t and β-galactosidase along with the various quantities of Gfi1 cDNA. After incubation, luciferase activity was measured and normalized against β-galactosidase activity. The means ± SDs of triplicate samples of one representative experiment out of three independent experiments are shown. (C) HEK 293T cells were transfected with Smad-binding element (SBE) promoter and CA-TβRI and β-galactosidase along with the various quantities of Gfi1 cDNA. After incubation, luciferase activity was measured and normalized against β-galactosidase activity. The means ± SDs of triplicate samples of one representative experiment out of three independent experiments are shown. (D) Left, a schematic of the mouse IL-17A promoter and the series of truncations from the 5' end of the IL-17A promoter. The closed circle and closed oblong depict the potential Gfi1 and ROR{gamma}t binding sites, respectively. The arrows indicate the positions of the primers used for ChIP analysis in Fig. 2(E) and Fig. 3(C). Right, reporter activity of truncated IL-17A promoter was assessed in the HEK 293T cells. The results are expressed as the x-fold increase in luciferase activity relative to control. The means ± SDs of triplicate samples of one representative experiment out of three independent experiments are shown. (E) The ChIP assay of ROR{gamma}t and Gfi1 on the IL-17A promoter region was performed in HEK 293T cells transfected with the IL-17A promoter and T7-ROR{gamma}t and/or FLAG-Gfi1 cDNA. Cell lysates were immunoprecipitated with anti-T7, anti-FLAG or control IgG. (F) The effect of TSA on suppression of IL-17A promoter activity by Gfi1 was assessed in the HEK 293T cells. The results are expressed as the x-fold increase in luciferase activity relative to control. The means ± SDs of triplicate samples of one representative experiment out of three independent experiments are shown.

 
To determine the suppressive mechanism of IL-17A expression by Gfi1, we investigated whether Gfi1 expression affects ROR{gamma}t-mediated IL-17A transcription using a reporter system described previously (15). As expected, Gfi1, but not Gfi1III, strongly suppressed ROR{gamma}t-mediated IL-17A promoter activity in a dose-dependent manner in HEK 293T and EL4 cells (Fig. 2B). On the other hand, Gfi1 did not affect TGF-β-mediated Smad transcriptional activity (Fig. 2C). Thus, Gfi1 suggested to directly regulate ROR{gamma}t but not Smads.

It has been shown that Gfi1 is a potent transcriptional repressor and recognizes the consensus-binding site, which is defined as AATC (43). As shown in Fig. 2(D), there are two Gfi1-binding sites within the IL-17A promoter. Thus, we used a reporter assay and a series of truncation mutants of the IL-17A promoter to examine whether both of the Gfi1-binding sites are essential for the suppression of ROR{gamma}t-mediated IL-17A reporter activity by Gfi1. Unexpectedly, Gfi1 still intensively inhibited the ROR{gamma}t-mediated reporter activity of mIL17p(-115)-Luc, which did not contain any Gfi1-binding sites (Fig. 2D). To investigate further, we used a ChIP assay to examine whether Gfi1 binds to the Gfi1-binding sites within IL-17A promoter. We did not observe any direct binding of Gfi1 to the IL-17A promoter, although the binding of ROR{gamma}t to this promoter was clearly observed with the ChIP assay (Fig. 2E) (15).

In a prior study, we determined that interaction of Foxp3 with ROR{gamma}t resulted in the suppression of the IL-17A expression by interfering with binding of ROR{gamma}t to the IL-17A promoter (15). As expected, the ChIP assay showed that forced expression of Gfi1 reduced the binding of ROR{gamma}t to the IL-17A promoter (Fig. 2E). However, we did not observe a direct association between Gfi1 and ROR{gamma}t by co-immunoprecipitation, whereas the interaction between Foxp3 and ROR{gamma}t was clearly seen (Data not shown).

It has been reported that Gfi1 coordinates epigenetic repression by recruitmenting histone deacetylase to target gene promoters (44). To test whether histone deacetylase are involved in the suppression of IL-17A transcription by Gfi1, we assayed the ability of Gfi1 to repress transcription in the absence or presence of the histone deacetylase inhibitor TSA. Expectedly, the suppressive effect of Gfi1 over-expression on ROR{gamma}t was abrogated by treatment of TSA in dose-dependent manner (Fig. 2F).

Taken together, our results indicate that Gfi1 inhibits the ROR{gamma}t-mediated IL-17A transcription by reducing ROR{gamma}t binding to the IL-17A promoter through the recruitment of histone deacetylase, although the detailed molecular mechanism for attenuation of ROR{gamma}t binding by Gfi1 remained to be clarified.

Gfi1 regulates the expression of IL-17A in primary T cells
To determine the function of Gfi1 in IL-17A expression in primary T cells, we first investigated whether Gfi1 expression inhibits Th17 differentiation in these cells. We used retroviral transduction to deliver Gfi1 to CD4+CD25–CD62LhighCD44– T cells stimulated under various polarizing conditions. As expected, over-expression of Gfi1 resulted in the repression of IL-17A expression in the transduced (GFP+) T cells (Fig. 3A). In contrast, no suppression of IL-17A was observed in cells infected with IRES-GFP control vectors. Furthermore, analysis by real-time PCR confirmed that the forced expression of Gfi1 resulted in reduction of IL-17A transcription (Fig. 3B). Then, we estimated the acetylated state of histone H4 on the IL-17A promoter in Gfi1-over-expressing primary Th17 cells. As expected, over-expression of Gfi1 significantly reduced the acetylation of histone H4 in primary T cells compared with the control vector (Fig. 3C).


Figure 3
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  		Fig. 3. The effect of Gfi1 on IL-17A production in primary T cells. (A) One day after activation under Th0-polarizing conditions, naive T cells were transduced with retroviral vectors encoding for IRES-GFP (MIG) and Gfi1-IRES-GFP (Gfi1). The cells were analyzed after a total of 4 or 5 days in Th17-skewed culture. The panels are gated on GFP-positive cells. The production of IL-17A by the transduced (GFP-positive) cells was examined by intracellular staining. (B) Relative IL-17A, ROR{gamma}t and Gfi1 mRNA expression levels in T cells cultured under the above conditions. The expression levels were monitored by real-time PCR and the data were normalized to HPRT expression. The means ± SDs of three independent experiments are shown, *P < 0.05. (C) Relative acetylated histone H4 levels in T cells cultured under the above conditions. The acetylation levels were estimated by ChIP assay. GFP-positive cell lysates were immunoprecipitated with anti-acetyl-Histone H4 or control IgG. The means ± SDs of three independent experiments are shown, *P < 0.05. (D and E) Naive T cells derived from WT or Gfi1–/– mice were cultured under Th17-polarizing conditions for 3 days and then restimulated with phorbol myristate acetate (PMA)/ionomycin for 5 h. Cytokine production was assessed by intracellular staining (D) or by real-time PCR (E). The numbers represent the percentages of cells that stained positive for IL-17A (D). The IL-17A mRNA expression is calculated as fold induction compared with WT control (= 1.0) (E). The means ± SDs of triplicate samples of one representative experiment out of two independent experiments are shown.

 
Next, we examined the expression of IL-17A with CD4+ T cells from Gfi1–/– mice. CD4+ T cells from Gfi1+/+ or Gfi1–/– mice were cultured for 3 days with anti-CD3 antibody and anti-CD28 antibody under Th17-polarizing conditions. As shown in Fig. 3(D), Gfi1–/– T cells produced an abnormally high levels of IL-17A protein compared with WT T cells. Under Th17-polarizing conditions, 52.5% of the differentiated WT T cells stained positive for IL-17A, whereas 84.3% of Gfi1–/– T cells produced IL-17A (Fig. 3D). Consistent with the flow cytometry data, high levels of IL-17A expression were observed in Gfi1–/– T cells by real-time PCR (Fig. 3E). Together, these data demonstrate that Gfi1 is a potent negative regulator for Th17 differentiation in primary T cells.

Gfi1 partially reduces the expression of Foxp3 in iTregs
To define the role of Gfi1 on iTreg differentiation, we first measured the levels of Foxp3 mRNA in EL4 cells transfected with Gfi1 using RT–PCR. Forced expression of Gfi1 only partially reduced the expression of Foxp3 (Fig. 4A). Gfi1 slightly suppressed CREB-mediated Foxp3 promoter activity in a dose-dependent manner in HEK 293T (Fig. 4B). These data suggest that Gfi1 weakly inhibits the Foxp3 expression.


Figure 4
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  		Fig. 4. The effect of Gfi1 on Foxp3 production. (A) The expression of various mRNAs was determined by RT–PCR in EL4 cells transfected with control GFP or Gfi1-GFP. After sorting with GFP, EL4 cells were cultured with or without phorbol myristate acetate (PMA)/ionomycin for 6 h and then the total RNA was isolated. (B) HEK 293T cells were transfected with Foxp3 promoter, CREB and β-galactosidase along with the various quantities of Gfi1 cDNA. After incubation, luciferase activity was measured and normalized against β-galactosidase activity. The means ± SDs of triplicate samples of one representative experiment out of three independent experiments are shown. (C) One day after activation under Th0-polarizing conditions, naive T cell were transduced with retroviral vectors encoding for IRES-GFP (MIG) and Gfi1-IRES-GFP (Gfi1). The cells were analyzed after a total of 4 days in iTreg-skewed culture. The panels are gated on GFP-positive cells. The production of Foxp3 by the transduced (GFP-positive) cells was examined by intracellular staining. (D) Naive T cells derived from WT or Gfi1–/– mice were cultured under iTreg-polarizing conditions for 24 h. Cytokine production was assessed by real-time PCR. The Foxp3 mRNA expression is calculated as fold induction compared with WT control (= 1.0). The means ± SDs of triplicate samples of one representative experiment out of two independent experiments are shown.

 
Next, we investigated the effect of forced expression or deletion of Gfi1 on primary T cells. Similar to EL4 cells, over-expression of Gfi1 resulted in a slight reduction of Foxp3 expression in the transduced (GFP+) T cells compared with control cells (Fig. 4C). Then, CD4+ T cells from Gfi1–/– mice were cultured for 24 h with anti-CD3 antibody and anti-CD28 antibody under iTreg-polarizing conditions. As shown in Fig. 4(D), Foxp3 mRNA levels in Gfi1–/– T cells were higher than those in WT T cells (Fig. 4D). Together, these data indicate that Gfi1 could suppress the iTreg differentiation; however, its effect on iTreg was not as drastic as in Th17.


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
Funding
 References
 
In this study, we found that Gfi1 negatively regulates Th17 differentiation by repressing ROR{gamma}t activity. Since Gfi1 expression is induced by the IFN-{gamma}/STAT1 or IL-4/STAT6-signaling pathways, Gfi1 could provide a mechanism to suppress IL-17A expression through the presence of IFN-{gamma} and IL-4. Indeed, we observed that the addition of IFN-{gamma} or IL-4 to Th17 differentiation conditions (media supplemented with TGF-β1 and IL-6) increased the expression of Gfi1 in whole cultured cells (data not shown).

Alternatively, Gfi1 may ensure low levels of IL-17A expression in Th1 or Th2 cells. We found that the expression of Gfi1 mRNA is mediated by the TGF-β1 stimulation. Thus, Gfi1 repression by TGF-β1 could be essential for the expression of IL-17A in Th17 cells. Th17 cells express not only IL-17A but also IL-17F and IL-22, and Gfi1 may affect their expression as well. Further study is necessary to ascertain the role of Gfi1 in regulation of Th17-related genes.

Previously, we found that Th17 development was severely impaired in SOCS1-deficient T cells. We showed that this is due to strong activation of STAT1, which leads to suppression of STAT3 and activation of Smad (41). We noticed high levels of expression of Gfi1 in SOCS1–/– T cells, which can be explained by increased STAT1 activity or decreased Smad activity.

It has been shown that Gfi1 acts as a transcriptional repressor through the interaction of a unique N-terminal SNAG domain with various proteins (45). Thus, we hypothesized that Gfi1 inhibits the ROR{gamma}t function by physical interaction with ROR{gamma}t at the N-terminal region. To test this hypothesis, we investigated whether Gfi1 interferes with ROR{gamma}t binding at the ROR{gamma}t-binding site within the IL-17A promoter. As expected, Gfi1 inhibits the ROR{gamma}t-mediated IL-17A transcription by reducing ROR{gamma}t binding to the IL-17A promoter. Unexpectedly, we did not observe a direct association between Gfi1 and ROR{gamma}t by co-immunoprecipitation (Data not shown). However, we could not rule out the possibility that Gfi1 weakly associates with ROR{gamma}t or binds indirectly through other factors. We found that the suppressive function of Gfi1 was abrogated by treatment of TSA (Fig. 2F) and that Gfi1 over-expression suppressed histone acetylation on the IL-17A promoter in primary T cells (Fig. 3C). It has been reported that Gfi1 recruits histone deacetylases to target gene promoters (44). Thus, Gfi1 is most likely to repress ROR{gamma}t-mediated IL-17A transcription by enhancing histone deacetylation, although detailed molecular mechanism for attenuation of ROR{gamma}t binding by Gfi1 remains to be clarified.

Alternatively, Ets1 may be involved in Gfi1-mediated suppression. Previous studies have demonstrated that Ets1 is a negative regulator of Th17 differentiation (46). In addition, it has been shown that the interaction between Ets1 and Gfi1 transcription factors cooperatively facilitates the repression of their target gene expression (47). Identifying the target genes or protein interactions of Gfi1 will help us understand this observation.

Recently, Zhu et al. (48) reported about the role of Gfi1 in regulating the differentiation of Th17 and iTreg differentiation. They demonstrated that Gfi1 significantly inhibited IL-17A production, although it had a minimal effect on the expression of Foxp3 and ROR{gamma}t. Our data are consistent with the results by Zhu et al. In addition, we found that Gfi1 suppressed the ROR{gamma}t-mediated IL-17A transcription by reducing ROR{gamma}t binding to the IL-17A promoter through the recruitment of histone deacetylase.

In conclusion, we propose that Gfi1 is a negative regulator of Th17 differentiation, which provides a novel mechanism for regulation of Th cell development. This suggests that Gfi1 could be a promising therapeutic target for autoimmune disease and allergy.


   Funding
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Funding
References
 
Ministry of Education, Culture, Sports, Science and Technology of Japan; Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (07-4); Astellas Foundation for Research on Metabolic Disorders.


   Acknowledgements
 
We thank M. Ohtsu, M. Asakawa and N. Shiino for their technical assistance, as well as N. Soma for manuscript preparation.


   Abbreviations
 
CA-TβRI, constitutively active transforming growth factorβ receptor I
cDNA, complementary DNA
ChIP, chromatin immunoprecipitation
Gfi1, growth factor independent 1
iTreg, inducible regulatory T
nTreg, naturally occurring regulatory T
ROR{gamma}t, retinoid-related orphan receptor {gamma}t
RT, reverse transcription
SOCS1, suppressor of cytokine signaling 1
TGF-β1, transforming growth factor-β1
Treg, regulatory T
WT, wild-type

   Notes
 
Transmitting editor: T. Watanabe

Received 29 January 2009, accepted 13 May 2009.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Funding
 References
 

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