Due to its ability to inhibit cytokine production in T cells

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1 c-maf Regulates IL-10 Expression during Th17 Polarization 1 Jiangnan Xu,* Yu Yang,* Guixing Qiu, Girdhari Lal,* Zhihong Wu, David E. Levy, Jordi C. Ochando, # Jonathan S. Bromberg,* and Yaozhong Ding 2 * IL-10 production by Th17 cells is critical for limiting autoimmunity and inflammatory responses. Gene array analysis on Stat6 and T-bet double-deficient Th17 cells identified the Th2 transcription factor c-maf to be synergistically up-regulated by IL-6 plus TGF and associated with Th17 IL-10 production. Both c-maf and IL-10 induction during Th17 polarization depended on Stat3, but not Stat6 or Stat1, and mechanistically differed from IL-10 regulation by Th2 or IL-27 signals. TGF was also synergistic with IL-27 to induce c-maf, and it induced Stat1-independent IL-10 expression in contrast to IL-27 alone. Retroviral transduction of c-maf was able to induce IL-10 expression in Stat6-deficient CD4 and CD8 T cells, and c-maf directly transactivated IL-10 gene expression through binding to a MARE (Maf recognition element) motif in the IL-10 promoter. Taken together, these data reveal a novel role for c-maf in regulating T effector development, and they suggest that TGF may antagonize Th17 immunity by IL-10 production through c-maf induction. The Journal of Immunology, 2009, 182: Due to its ability to inhibit cytokine production in T cells as well as in APCs (1 6), IL-10 was originally described as cytokine synthesis inhibitory factor (1, 2). IL-10 plays a critical role in suppressing autoimmunity and inflammatory responses, as IL-10- or IL-10R2-deficient mice develop severe colitis (7, 8), and IL-10 suppresses inflammatory bowel disease in vivo and Crohn s colitis in humans (9, 10). Recent studies demonstrated that IL-10 produced by Th17 cells restrains the pathologic effects of Th17 (11, 12), further expanding the role of IL-10 in limiting inflammatory responses. Most analyses of transcriptional regulation of IL-10 have come from studies in macrophages and monocytes. IL-10 gene regulation in these cells has been shown to involve Sp1 (13) activated by p38 MAPK (14), Stat3 (15), camp (16 18), and c-maf (19). In T cells, IL-10 was shown to be regulated by Jun proteins (20), and chromatin structural differences within the IL-10 gene in differentiated Th1 and Th2 cells have been observed so that committed Th1 cells develop repressive histone modifications at the IL-10 promoter, whereas committed Th2 cells develop an open chromatin configuration (21), supporting the notion that IL-10 may be a Th2 cytokine. Recent studies demonstrated that IL-10 regulation in adaptive T cell response is more complex, with multiple Th2-independent pathways being documented. IL-6 combined with TGF Th17-polarizing signals induce Stat3-dependent IL-10 production (11). IL-27 promotes Stat1-dependent IL-10 production in both Th1- and Th2-polarizing conditions (11, 22). The Notch pathway mediates Stat4-dependent IL-10 production both in developing and established Th1 cells (23). *Department of Gene and Cell Medicine and Department of Surgery, Immunology Institute, and The Recanati/Miller Transplantation Institute, Mount Sinai School of Medicine, New York, NY 10029; Department of Surgery and Orthopaedics, Peking Union Medical College Hospital, Beijing, China; Pathology and Microbiology, New York University School of Medicine, New York, NY 10016; and # Unidad de Immunología de Trasplantes, Centro Nacional de Microbiologia, Instituto de Salud Carlos III, Madrid, Spain Received for publication January 14, Accepted for publication March 12, The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by National Institutes of Health Grants R01 AI (to Y.D.) and AI (to J.S.B). 2 Address correspondence and reprint requests to Dr. Yaozhong Ding, Department of Gene and Cell Medicine, Box 1496, Mount Sinai School of Medicine, 1 Gustave L. Levy Place, New York, NY address: yaozhong.ding@mssm.edu c-maf, the cellular homolog of the avian viral oncogene v-maf, was the first Th2-specific transcriptional factor identified. It belongs to the AP-1 family of basic region/leucine zipper factors and binds to a consensus site (MARE (Maf recognition element)) in the proximal IL-4 promoter, and it directly activates IL-4 gene transcription (24). Forced expression of c-maf results in IL-4 production, IFN- inhibition, and attenuation of Th1 development (25). Although it has been showed that c-maf deficiency did not affect IL-10 production under Th2 polarization (26), studies in macrophages showed that c-maf played a critical role in regulating IL-10 production (19). The role of c-maf in T cell IL-10 regulation under other polarizing conditions has not been examined. In this study, we took advantage of the characteristic of Stat6 and T-bet double-deficient cells in which IL-6 plus TGF differed from IL-6 plus IL-23 for IL-10 production despite similar IL-17 production (27). We identified that c-maf plays an important regulatory role in IL-10 expression during Th17 polarization. In contrast to IL-4, c-maf-regulated IL-10 expression was not strictly dependent of the Stat6/GATA-3 Th2 signal cascade. We identified a MARE motif in mouse IL-10 promoter and provided a molecular basis of IL-10 regulation by c-maf. Our finding that TGF is synergic with both IL-6 and IL-27 for c-maf expression and consequent IL-10 production further supports the important regulatory role for c-maf in TGF -mediated antiinflammatory activities. Materials and Methods Mice and reagents BALB/c, C57BL/6, Stat6 knockout (KO) 3 (BALB/c background), T-bet KO (BALB/c background), and IFN- KO (BALB/c background) mice were purchased from The Jackson Laboratory. Stat1 KO (129Sv/Ev background) and 129Sv/Ev control mice were purchased from Taconic Transgenics. Stat6/T-bet double-deficient (DKO, BALB/c background) and STAT3 / (CD4 Cre:STAT3 f/f) cells were generated as previously described (27, 28). Recombinant mouse IL-2, IL-6, IFN-, and PE-anti-IL-17A mabs were purchased from BD Pharmingen. Recombinant TGF was purchased from R&D Systems. Recombinant mouse IL-23, IL-27, anti-cd3, anti-cd28, anti-ifn-, anti-il-4, allophycocyanin-anti- CD4, allophycocyanin-anti-il-10, FITC-anti-IL-10, PE-anti-IL-4, 3 Abbreviations used in this paper: KO, knockout; ChIP, chromatin immunoprecipitation; IL-10mt, mutated IL-10 promoter; IL-10wt, wild-type IL-10 promoter; MARE, Maf recognition element; ROR, retinoic acid-related orphan nuclear receptor. Copyright 2009 by The American Association of Immunologists, Inc /09/$2.00

2 6227 PE-anti-IFN-, and FITC-anti-Foxp3 mabs, intracellular staining kit, and monesin solution were all purchased from ebioscience. CD4, CD8, and CD25 microbeads were purchased from Miltenyi Biotec. Anti-c-Maf was purchased from Santa Cruz Biotechnology. PMA, ionomycin, and mitomycin C were purchased from Sigma-Aldrich. T cell purification, activation, retroviral infection, intracellular staining, and ELISA CD4 CD25 and CD8 T cells were selected by using microbeads or flow cytometry sorting and were seeded in 24-well flat bottom plates. For APCdependent assays, cells were cultured with mitomycin C-treated T-depleted splenocytes ( ) plus IL-2 (1 ng/ml) and soluble anti-cd3 (1 g/ml), combined with various cytokines as indicated. For APC-independent assays, cells were stimulated with plate-bound anti-cd3 (5 g/ml) and soluble anti-cd28 (1 g/ml), along with various cytokines. ELISA for IL-10 was performed using the mouse IL-10 ELISA kit (ebioscience) according to the manufacturer s protocol. Retroviral supernatants were generated by transfecting bicistronic MigR1-GFP or MigR1-c-Maf-GFP into EcoPack2 293 cells (Clontech Laboratories). After stimulating with anti-cd3 (5 g/ml) and anti-cd28 (1 g/ml) mabs in combination with IL-2 (5 ng/ml) for h, CD4 CD25 or CD8 T cells were infected with retroviral supernatant by spin infection. A second spin infection was performed on the following day. GFP cells were purified by cell sorting 48 h after the first infection. Purified GFP cells were rested in complete medium with IL-2 (5 ng/ml) for 2 days, washed, restimulated with platebound anti-cd3 (5 g/ml) and soluble anti-cd28 (1 g/ml) for 48 h, and intracellular staining and real-time RT-PCR were performed. Intracellular staining for IL-10, IL-4, IFN-, IL-17, and Foxp3 were performed according to the manufacturer s protocols (ebioscience). Gene array analysis and real-time RT-PCR Affymetrix mouse genome arrays were used to probe the global gene expression profiles in the Stat6 and T-bet double-deficient mouse CD4 CD25 T cells after Th17 polarization. Forty-eight hours after stimulation, total RNAs were isolated using RNeasy Protect Mini Kit (Qiagen) and gene arrays were performed at the Mount Sinai Microarray Shared Research Facility. Data were normalized and analyzed using GeneChip operating software (Affymetrix) and presented as expression of indicated molecules relative to expression of internal control probe sets. The primers for real-time RT-PCR are: cyclophilin A, AGG GTG GTG ACT TTA CAC GC and ATC CAG CCA TTC AGT CTT GG; c-maf, AGC AGT TGG TGA CCA TGT CG and TGG AGA TCT CCT GCT TGA GG; IL-10, AAC TGC ACC CAC TTC CCA GTC and CAT TAA GGA GTC GGT TAG CAG; IL-4, GAA GCC CTA CAG ACG AGC TCA and ACA GGA GAA GGG ACG CCAT; IFN-, TGG CTC TGC AGG ATT TTC ATG and TCA AGT GGC ATA GAT GTG GAA GAA; GATA-3, GAA GGC ATC CAG ACC CGA AAC and ACC CAT GGC GGT GAC CAT GC; T-bet, CGG AGC GGA CCA ACA GCA TCG TTT C and CAG GGT AGC CAT CCA CGG GCG GGT; IL-17A, CTC CAG AAG GCC CTC AGA CTA and AGC TTT CCC TCC GCA TTG ACA; retinoic acidrelated orphan nuclear receptor (ROR) T, ACC TCT TTT CAC GGG AGG A and TCC CAC ATC TCC CAC ATT G. Reactions were performed with the LightCycler system (Roche) and the SYBR Green PCR kit (Qiagen). All experiments were performed at least three separate times. Western blotting Cells were harvested and washed twice with PBS. Cell extracts were isolated with M-PER mammalian protein extraction reagents (Pierce). Protein concentration was determined by a Micro BCA protein assay kit (Pierce). Proteins were separated by SDS-polyacrylamide, transferred to polyvinylidene difluoride membranes, probed with antiphosphorylated Stat3 or anti- Stat3, incubated with HRP-conjugated anti-rabbit IgG, and detected by the ECL system (Amersham). EMSA Purified CD4 CD25 T cells were stimulated with anti-cd3 and anti- CD28 mabs, with or without IL-6 plus TGF for 2 days. Cells extracts were isolated and incubated with biotin-labeled probes containing c-mafbinding sequence element (MARE) from IL-10 or IL-4 promoter. For supershift assays, 1 l of anti-c-maf Abs was added to the binding reactions mixture. For competition assays, 200-fold molar excess of unlabeled probe was used. Reactions were electrophoresed, transferred, and detected according to the instructions of LightShift chemiluminescent EMSA kit (Pierce). The probes used are as follows: IL-10 MARE, CTA AGG TGA CTT CCG AGT CAG CAA GAA ATA TCG and CGA TAT TTC TTG CTG ACT CGG AAG TCA CCT TAG; IL-4 MARE, CTC ATT TTC CCT TGG TTT CAG CAA CTT TAA CTC and GTG TTA AAG TTG CTG AAA CCA AGG GAA AAT GAG. Chromatin immunoprecipitation (ChIP) ChIP assays were performed according to the manufacture s protocol (Upstate Biotechnology). Briefly, cells were treated with anti-cd3 and anti- CD28 mabs, with or without IL-6 combined with TGF, cross-linked with formaldehyde at a final concentration of 1%, lysed, and sonicated to shear DNA. After immunoprecipitation with anti-c-maf at 4 C overnight, Ab/ DNA complexes were eluted and crosslinking was reversed by incubating at 65 C for 4hin5MNaCl, and the DNA was then recovered by QIAquick PCR purification kit (Qiagen). Real-time PCR was then used to quantify the precipitated DNA sequences. All experiments were performed at least tjree separate times. The primers used are as follows: IL-4 MARE, GAA TAA CTG ACA ATC TGG TGT and TAT ATA TAG AGT TAA AGT TGC; IL-10 MARE, CTC TCC TCT GAC CAA CTG CC and TGG GTT GAA CGT CCG ATA TT; IL-10 distant, CAG TCA GGA GAG AGG GCA GTG A and TTT CCA ACA GCA GAA GCA AC. Luciferase reporter assay The wild-type IL-10 promoter (IL-10wt) containing MARE binding motif were amplified from BALB/c mouse genomic DNA by PCR with primers carrying restriction sites for NheI and BglII (5 -TTG CTA GCT CCT AAC TTC TCA TGC TGG GAT-3 and 5 -CCA GAT CTA AAG AAC TGG TCG GAA TGA ACT-3 ) and cloned into the TransLucent gene promoter reporter vector (Panomics) as previously described. The mutated IL-10 promoter (IL-10mt) was generated by replacing the TGCTGA MARE binding site with an EcoRI (GAATTC) site. Transient transfection experiments were performed in 6-well plates using 2 g each of IL-10wt or IL-10mt vectors, in combination with 2 g GFP or c-maf-gfp plasmids. For dose-response experiments, IL-10wt or IL-10mt was used at 2 g, along with different doses of c-maf-gfp from 0 to 3 g. Twenty-four hours after transfection, luciferase activity was measured with the luciferase assay kit (Promega) according to the manufacturer s protocol. Results TGF is synergic with IL-6 for c-maf and IL-10 induction and requires Stat3 Differentiation of Th17 cells is driven by IL-6 plus TGF. Our recent studies showed that IL-6 combined with IL-23 differed from IL-6 combined with TGF for IL-10 production and pathogenic activities in CD4 T cells deficient in Stat6 and T-bet, despite similar IL-17 production (27). We performed gene array analysis on Stat6 and T-bet double-deficient cells. We rationalized that by comparing gene expression in cells treated with IL-6 plus TGF vs TGF alone, we would be able to identify genes specific for standard Th17 polarization and responsible for both IL-17 and IL-10 expression. By next comparing gene expression in cells treated with IL-6 plus TGF vs IL-6 plus IL-23, we could eliminate molecules involved solely in IL-17 regulation and obtain genes specifically responsible for IL-10 regulation (full microarray data sets are available at Gene Expression Omnibus: gov/geo, accession no. GSE15029). We focused only on genes that increased at least 4-fold in cells treated with IL-6 combined with TGF vs TGF alone or vs IL-6 plus IL-23. We reasoned that to have more potential biological relevance, genes needed to be expressed at relatively high levels (minimal signal strength of 500 in the array assay). As shown in Table I, a total of 10 probes met these criteria, and two probes were identical for c-maf, the cellular homolog v-maf. Importantly, IL-10 was also one of the nine selected genes. Among these nine molecules, only c-maf is a transcription factor and had been shown to play a critical role in transactivating IL-4 expression and Th2 differentiation (24 26). Since IL-10 was originally considered as a Th2 cytokine, and c-maf regulated IL-10 expression in macrophages (19), we further examined c-maf expression in Th17 polarization and its potential role in Th17 IL-10 regulation. Real-time RT-PCR for c-maf and IL-10 was performed on these DKO cells to confirm the array result. As shown in Fig. 1A, IL-6

3 6228 TGF SYNERGIZES WITH IL-6 FOR c-maf AND IL-10 EXPRESSION Table I. Gene array for identifying molecules involved in IL-10 regulation a Probe Set ID Gene Symbol Gene Title TGF Signal IL-6/IL-23 Signal IL-6/TGF Signal _at Spp1 Secreted phosphoprotein , , _at Pdpn Podoplanin , _at Basp1//LOC Brain abundant, membrane-attached signal protein 1//similar ,371.1 to 22-kDa neuronal tissue-enriched acidic protein _s_at Maf Avian musculoaponeurotic fibrosarcoma (v-maf) AS42 oncogene ,237.9 homolog _at Serpinb1a Serine (or cysteine) peptidase inhibitor, clade B, member 1a , _at Il10 IL , _at Cxcr4 Chemokine (C-X-C motif) receptor _at Stard10 START domain containing _at Maf Avian musculoaponeurotic fibrosarcoma (v-maf) AS42 oncogene homolog _at Tec Cytoplasmic tyrosine kinase, Dscr28C related (Drosophila) a Purified Stat6 and T-bet double-deficient CD4 CD25 T cells were stimulated with plate-bound anti-cd3 (5 g/ml) and anti-cd28 (1 g/ml) mabs, TGF (5 ng/ml), IL-6 (10 ng/ml) plus TGF, or IL-6 plus IL-23 (10 ng/ml) for 2 days; RNAs were isolated and Affymetrix GeneChip array analysis was performed. Full microarray data sets are available at Gene Expression Omnibus ( accession no. GSE15029). alone induced some c-maf expression, consistent with our previous findings (29). TGF alone also induced some c-maf expression. Importantly, the combination of IL-6 and TGF induced c-maf at much higher levels. IL-6 plus TGF also induced c-maf expression in wild-type cells (Fig. 1A), indicating that induction was not an artifact due to Stat6 and T-bet double deficiency. Realtime RT-PCR also confirmed that IL-6 plus TGF induce higher levels of IL-10 (Fig. 1A). Since our previous studies showed that sustained Stat3 activation was required for c-maf induction (29), we examined the role of Stat3 for c-maf by using Stat3-deficient CD4 T cells. As shown in Fig. 1B, Stat3 deficiency abolished IL-6-induced c-maf, but did not affect TGF -induced c-maf. Stat3 deficiency also abolished the synergistic effect on c-maf induction by IL-6 plus TGF, further demonstrating the critical role for Stat3 in c-maf regulation. In agreement with a previous report (11), Stat3 deficiency completely abolished IL-6 plus TGF -induced IL-10 (not shown). Western blotting for Stat3 activation showed that 24 h after stimulation there was still measurable Stat3 activation in the TGF plus IL-6 but not in the IL-6 alone group (Fig. 1C), suggesting that TGF up-regulates c-maf expression by sustaining Stat3 activation. c-maf expression correlates with IL-10 production To examine the relationship of c-maf expression to IL-10 production, dose-response experiments were performed. Wild-type cells were stimulated with either a fixed concentration of IL-6 (10 ng/ ml) and variable concentrations of TGF (0 5 ng/ml), or a fixed concentration of TGF (5 ng/ml) with variable concentrations of IL-6 (0 10 ng/ml). Real-time RT-PCR for c-maf and IL-10 were performed 2 days after stimulation, and intracellular staining for IL-17, Foxp3, and ELISA for IL-10 production was performed 3 days after stimulation. As shown in Fig. 2, A and B, when the concentration of IL-6 was fixed, increasing concentrations of TGF induced higher levels of c-maf, IL-10, IL-17, and Foxp3. When the concentration of TGF was fixed, higher concentrations of IL-6 induced higher levels of c-maf, IL-10, and IL-17 and decreasing Foxp3 expression (Fig. 2, C and D). The finding that c-maf correlated with IL-17 expression suggested that c-maf might play a role in IL-17 regulation. However, since IL-6 plus IL-23 induced higher levels of IL-17 without increasing c-maf expression in Stat6 and T-bet double-deficient cells (Fig. 1A), this indicated that c-maf did not directly regulate IL-17 expression. c-maf and IL-10 expression did not correlate with Foxp3, indicating that c-maf and IL-10 induction by IL-6 plus TGF were independent of Foxp3 and regulatory T cell development. Taken together, these results showed that c-maf correlated directly with IL-10 expression during Th17 polarization. c-maf and IL-10 induction during Th17 polarization are independent of IL-4/Stat6/GATA-3 Since c-maf was the first Th2-specific transcription factor identified, we next examined if Th1 or Th2 signals affected c-maf and IL-10 expression during Th17 polarization. Intracellular staining and ELISA for IL-10 showed that IL-10 production occurred together after TCR activation of wild-type and T-bet-deficient, but not Stat6- or Stat6/T-bet-deficient, CD4 T cells (Fig. 3A), confirming the notion that IL-10 is associated with the Th2 developmental signal cascade. Addition of IL-6 plus TGF induced only IL-10 but not IL-4, regardless of whether the cells were deficient in Stat6, T-bet, or both (Fig. 3A), indicating that IL-10 production during Th17 polarization was independent of Th1 or Th2 pathways. Realtime RT-PCR for c-maf showed that IL-6 combined with TGF induced similarly high levels of c-maf in wild-type, Stat6, T-bet, or Stat6/T-bet double-deficient CD4 T cells (Fig. 3B), indicating that c-maf expression during Th17 polarization was also independent of Th1 or Th2 signals. c-maf activates IL-4 gene transcription (24); however, there was very little IL-4 expression during Th17 polarization despite high levels of c-maf (Fig. 3). GATA-3 has been demonstrated to play a master role in controlling Th2 and IL-4 expression (30, 31). There was little GATA-3 expression in cells under Th17-polarizing conditions (Fig. 3B), suggesting that TGF limits the ability of c-maf to activate IL-4 by suppressing GATA-3 expression. Taken together, these results showed that c-maf and IL-10 regulation during Th17 polarization differed from IL-4 and IL-10 regulation during Th2 polarization and was independent of the Stat6/GATA-3 Th2 signal cascade. c-maf and IL-10 induction by Th17-polarizing signals are independent of Stat1 IL-27 has recently been demonstrated to regulate IL-10 production in both Th1- and Th2-polarizing conditions in a mostly Stat1-dependent fashion (11, 22). We next investigated if c-maf is associated with IL-27-mediated IL-10 regulation. CD4 CD25 T cells from wild-type and Stat1-deficient mice were stimulated with IL- 27, IL-27 plus TGF, or IL-6 plus TGF in combination with anti-il-4 and anti-ifn- mabs. In agreement with previous reports (11, 22), real-time RT-PCR showed that IL-27 induced IL-10 expression (Fig. 4A). Stat1 deficiency abolished IL-27-mediated

4 6229 FIGURE 1. IL-6 plus TGF induce high levels of c-maf expression. A, Real-time RT-PCR for c-maf, IL-10, ROR T, and IL-17 expression in Stat6/T-bet DKO and wild-type CD4 CD25 T cells stimulated with anti-cd3 (1 g/ml), IL-2 (1 ng/ml), T-depleted wild-type splenocytes, and IL-6 (10 ng/ml), TGF (5 g/ml), IL-6 plus IL-23 (10 ng/ml), or IL-6 plus TGF for 48 h. B, Real-time RT-PCR for c-maf expression in wild-type and Stat3 / CD4 CD25 T cells after anti-cd3 plus anti-cd28 mabs stimulation, with or without IL-6, TGF, or IL-6 plus TGF for 48 h. C, Western blotting for Stat3 activation. BALB/c CD4 CD25 T cells were stimulated with anti-cd3 plus anti-cd28 mabs plus IL-6 or IL-6 plus TGF for 0.5 or 24 h. Antiphosphorylated Stat3 or anti-stat3 Abs were used for Western blotting. IL-10 expression but had little effect on IL-6 plus TGF -mediated IL-10 expression. Surprisingly, and in contrast to IL-27 alone, we found that IL-27 plus TGF also induced significant IL-10 expression in Stat1-deficient cells (Fig. 4A), showing that IL-27 and IL-27 plus TGF use different mechanisms to regulate IL-10. Measurements of c-maf expression showed that IL-27 alone induced some c-maf expression in a Stat1-dependent fashion, while IL-27 plus TGF significantly induced c-maf expression in a Stat1-independent fashion (Fig. 4A), suggesting a role for c-maf in IL-10 expression induced by IL-27 plus TGF. Stat1 deficiency also allowed IL-27 plus TGF to induce significant IL-17 production and ROR T expression (Fig. 4, B and C), suggesting that

5 6230 TGF SYNERGIZES WITH IL-6 FOR c-maf AND IL-10 EXPRESSION FIGURE 2. c-maf expression correlates with IL-10 production. A, Wild-type CD4 CD25 T cells were stimulated with anti-cd3 (1 g/ml), IL-2 (1 ng/ml), T-depleted wild-type splenocytes, and IL-6 (10 ng/ml) with variable concentrations of TGF ; real-time RT-PCR for c-maf and IL-10 expression was performed at day 2, and ELISA for IL-10 was performed at day 3. B, Same cells as in A, intracellular staining for IL-17 and Foxp3 after 3 days. C, Wild-type CD4 CD25 T cells stimulated with anti-cd3 (1 g/ml), IL-2 (1 ng/ml), T-depleted wild-type splenocytes, and TGF (5 ng/ml) with variable concentrations of IL-6; real-time RT-PCR for c-maf and IL-10 expression was performed at day 2, and ELISA for IL-10 was performed at day 3. D, Same cells as in C, intracellular staining for IL-17 and Foxp3 after 3 days.

6 6231 FIGURE 3. c-maf and IL-10 induction by Th17-polarizing signals are independent of IL-4/Stat6/GATA-3. A, Intracellular staining for IL-4, IL-10, and ELISA for IL-10 in wild-type, Stat6-deficient, T-bet-deficient, or Stat6/T-bet double-deficient CD4 CD25 T cells stimulated with anti-cd3 (1 g/ml), IL-2 (1 ng/ml), and T-depleted wild-type splenocytes, with or without IL-6 (10 ng/ml) plus TGF (5 ng/ml) for 3 days. B, c-maf, IFN-, IL-4, and GATA-3 expression measured by real-time RT-PCR 2 days after stimulation.

7 6232 TGF SYNERGIZES WITH IL-6 FOR c-maf AND IL-10 EXPRESSION FIGURE 4. c-maf and IL-10 induction during Th17 polarization is independent of Stat1. A, Real-time RT-PCR for IL-10, c-maf, and GATA-3 expression in wild-type and Stat1-deficient CD4 CD25 T cells 48 h after anti-cd3 (1 g/ml), IL-2 (1 ng/ml), T-depleted wild-type splenocytes, and IL-27 (10 ng/ml), IL-27 plus TGF (5 ng/ml), or IL-6 (10 ng/ml) plus TGF, in combination with anti-il-4 (10 g/ml) plus anti-ifn- (10 g/ml) mabs. B, Flow cytometry of intracellular IL-17 expression after 3 days of stimulation. C, Real-time RT-PCR for IL-17A and ROR T expression 48 h after stimulation. D, Intracellular staining for IL-10 and IL-17 in wild-type and IFN- -deficient CD4 CD25 T cells 3 days after anti-cd3 (1 g/ml), IL-2 (1 ng/ml), and T-depleted wild-type splenocytes, with or without IL-27 plus TGF, IL-6 plus TGF, or IL-6 plus TGF plus IFN- (100 ng/ml). Stat1 activation by IL-27 negatively regulates its effects on Th17 polarization. Since IFN- activates Stat1, we next examined how IFN- may affect IL-10 and IL-17 expression in Th17 polarization. As shown in Fig. 4D, IFN- negatively regulated IL-10 and IL-17 expression during Th17 polarization, but IFN- deficiency did not allow IL-27 plus TGF to induce IL-17 expression (Fig. 4D), further showing that Stat1 activation by IL-27 directly negatively regulates IL-10 and IL-17 expression during Th17 polarization. Collectively, these results indicate that c-maf and IL-10 regulation during Th17 polarization is different than IL-27 alone-mediated IL-10 expression, and c-maf may also play an important role in IL-27 plus TGF -mediated IL-10 regulation. Retroviral c-maf induces IL-10 expression To further determine that c-maf regulates IL-10 expression, we used a bicistronic GFP retroviral vector to transduce c-maf into T

8 6233 FIGURE 5. c-maf induces IL-10 production in T cells. A, Intracellular staining for IL-10 and IL-4 expression in wild-type and Stat6-deficient CD4 CD25 or CD8 T cells infected with GFP or c-maf-gfp retrovirus. GFP cells were isolated after 2 days of stimulation, rested for 2 days, and then restimulated with anti-cd3 plus anti-cd28 mabs for an additional 2 days. B, Real-time RT-PCR for IL-10 and IL-4. C, Real-time RT-PCR for GATA-3. cells. After 2 days, cells expressing GFP alone or c-maf-gfp were purified by flow cytometric sorting, rested for 2 days, and then restimulated with anti-cd3 plus anti-cd28 mabs for an additional 2 days. Intracellular staining and real-time RT-PCR showed that c-maf significantly increased both IL-4 and IL-10 expression in wild-type CD4 T cells (Fig. 5, A and B). Stat6 deficiency abolished c-maf-induced IL-4 expression, confirming that IL-4/Stat6/ GATA-3 signals were pivotal in controlling IL-4 expression and Th2 differentiation, and that high levels of c-maf expression alone were not sufficient to efficiently initiate IL-4 transcription. In contrast, there was low but significant IL-10 induction by c-maf in Stat6-deficient CD4 T cells, indicating that c-maf regulated IL-10 expression could occur in an IL-4/Stat6/GATA-3-independent fashion. c-maf induced high levels of IL-10 but no IL-4 in

9 6234 TGF SYNERGIZES WITH IL-6 FOR c-maf AND IL-10 EXPRESSION FIGURE 6. c-maf binds and transactivates the IL-10 promoter. A, MARE motifs in mouse IL-4 and IL-10 promoters. B, EMSA assay for IL-10 MARE or IL-4 MARE. Cell extracts were prepared from wild-type CD4 CD25 T cells stimulated with anti-cd3 and anti-cd28 mabs, with or without IL-6 plus TGF for 2 days. Competition was performed with 200-fold unlabeled oligonucleotide probes. Anti-c-Maf Abs were used for supershift. C, ChIP assay for IL-4 MARE, IL-10 MARE, or an IL-10 distant promoter region. Purified CD4 T cells were stimulated with anti-cd3 and anti-cd28 mabs, with or without IL-6 plus TGF for 2 days, and anti-c-maf Abs were used for immunoprecipitation. Data expressed as the ratio of the enrichment of the promoter region target in cells treated with TCR stimulation in combination with IL-6 plus TGF are compared with cells treated with TCR stimulation alone. D, c-maf activated the IL-10 promoter through the MARE motif. Luciferase reporter constructs (2 g each) containing wild-type IL-10 promoter with MARE motif (IL-10wt) or mutated IL-10 promoter without MARE binding motif (IL-10mt), along with GFP or c-maf-gfp encoding plasmids were transiently transfected to HeLa cells. Luciferase activity measured 24 h after transfection. E, Dose response to c-maf for activation of the IL-10 promoter. Cells transfected with wild-type or mutated IL-10 promoter luciferase constructs (2 g) in combination with variable amounts of c-maf-gfp plasmid as indicated.

10 wild-type and Stat6-deficient CD8 T cells (Fig. 5, A and B), likely due to highly restricted IL-4 promoter accessibility in CD8 T cells that strongly favor type 1 polarization (32, 33). Low levels of GATA-3 expression in Stat6-deficient CD4, wild-type CD8, and Stat6-deficient CD8 T cells further confirmed that IL-10 regulation by c-maf is less strictly dependent on GATA-3 (Fig. 5C). Taken together, these data demonstrated that c-maf directly up-regulates IL-10 expression, independent of the IL-4/Stat6/GATA-3 Th2 development cascade. c-maf binds and transactivates the IL-10 promoter Sequence analysis of the mouse IL-10 promoter identified a half MARE TCAGCA binding motif, similar to one present in the mouse IL-4 promoter (24), located 500 bp upstream of the transcription initiation site (Fig. 6A), within a region previously shown to have high degree of histone H3 phosphorylation after stimulation (34). To examine if this MARE motif bound c-maf, cell lystes were isolated from CD4 CD25 T cells treated with anti-cd3 plus anti-cd28 mabs, with or without IL-6 plus TGF. As shown in Fig. 6B, similar to IL-4 MARE, IL-10 MARE bound lysates from cells activated by IL-6 plus TGF but not TCR alone. Antic-Maf Abs supershifted the protein/dna complex, and IL-4 MARE oligonucleotides competed IL-10 MARE binding, while IL-10 MARE competed IL-4 MARE binding. To determine whether c-maf bound to the IL-10 promoter in vivo following IL-6 plus TGF stimulation, wild-type CD4 CD25 T cells were stimulated with anti-cd3 and anti-cd28 mabs, with or without IL-6 plus TGF, and 48 h after stimulation a ChIP assay for IL-10 and IL-4 MARE sequences was performed using anti-c-maf for immunoprecipitation. As shown Fig. 6C, anti-c-maf enriched for IL-10 MARE in IL-6 plus TGF -treated cells by 6-fold compared with TCR activation alone, while there was no enrichment for an IL-10 distant promoter region 1000 bp upstream of MARE. In contrast to IL-10 MARE, there was much less enrichment for IL-4 MARE, demonstrating that IL-6 plus TGF induced c-maf differentially bound to the IL-10 and IL-4 promoters, correlating with differential effects on IL-10 and IL-4 gene expression. Stat6 deficiency did not affect c-maf binding to IL-10 promoter, further indicating that c-maf binding to IL-10 promoter could occur independently of IL-4/Stat6/GATA-3 Th2 signal cascades. To further prove that c-maf transactivates the IL-10 promoter, 500-bp regions of the IL-10 promoter containing MARE motif with or without specific mutations were cloned into a vector containing a minimal TA promoter and the TATA box from the herpes simplex virus thymidine kinase promoter. HeLa cells were transiently transfected with wild-type and mutated reporter constructs, along with control GFP or c-maf-gfp expressing vectors. As shown in Fig. 6, D and E, transfection of GFP alone did not transactivate reporter gene expression, while c-maf-gfp induced a strong reporter gene response, and mutating the MARE binding region abolished the significant response. Taken together, these experiments demonstrate that IL-6/TGF induced c-maf transactivates the IL-10 promoter to regulate IL-10 production during Th17 polarization. Discussion Th17 cells play a central role in T cell-driven autoimmune diseases (35), and whether Th17 cells express IL-10 is crucial in determining their protective or pathological consequences (11, 12). Most studies for IL-10 gene regulation have been on innate immune cells, and little is known about its transcriptional regulation in adaptive T cell responses. We showed there are at least three different signaling pathways that regulate IL-10 production in T cells: a Stat6-dependent Th2 development-coupled pathway, an IL-27/ 6235 Stat1 pathway, and a TGF in combination with IL-6- or IL-27- mediated Stat3/c-Maf pathway. We uncovered a novel role for c-maf in modulating T effector responses through regulating IL-10 expression, and provided new insights for understanding TGF mediated antiinflammatory responses. Consistent with the original notion that IL-10 could be a Th2 cytokine, we showed that IL-10 production is associated with IL-4 production in Th2-polarizing conditions in a Stat6-dependent fashion (Fig. 3A). c-maf transactivated both IL-10 and IL-4 gene transcription (Fig. 6 and Ref. 24), but these cytokines differed in requirements for Stat6/GATA-3 Th2 developmental signals. IL-4 gene regulation was strictly dependent on GATA-3 and Th2 polarization, and TGF signals switched c-maf from transactivating IL-4 to IL-10 by suppressing GATA-3 (Figs. 3B and 6C). It has been reported that chromatin remodeling of the IL-10 promoter is associated with chromatin remodeling of Th2 loci (21). However, the finding here that c-maf bound to the IL-10 promoter in Stat6- deficient cells (Fig. 6C) suggests that chromatin remodeling of the IL-10 gene locus is also regulated by other signals, which allows c-maf to regulate IL-10 in a Stat6/Th2 signal cascade-independent fashion. The IL-10 MARE is in a region previously shown to have high degree of histone H3 phosphorylation after stimulation (34), supporting its active role in IL-10 gene transcription. Studies on human IL-10 promoter demonstrated a MARE motif located to nucleotides 196/ 184 relative to the transcription initiation site (19), indicating that the c-maf/il-10 signal pathway is conserved. c-maf-deficient mice are neonatal lethal (25, 36). We are not able to dissect roles of c-maf, Stat3, or TGF signals in IL-10 regulation during Th17 polarization. Since retroviral-transduced c-maf readily induced IL-10 expression (Fig. 5), this suggests that Stat3 or TGF signals may not be directly involved in IL-10 gene transcription by c-maf. IFN- and Stat1 signals negatively regulated IL-10 (Fig. 4D), and retroviral c-maf induced much less IL-10 expression in CD4 T cells deficient in Stat6 (Fig. 5A), indicating that optimal IL-10 production by c-maf required suppression of IFN- and Th1 signals. Retroviral c-maf induced higher levels IL-10 expression in wild-type CD8, Stat6-deficient CD8 compared with Stat6-deficient CD4 T cells, indicating differential regulation in CD4 and CD8 T cells. Despite high levels of IFN- expression, CD8 T cells rapidly lost responsiveness to IFN- (37), and thus the differential IL-10 production by retroviral c-maf in Stat6-deficient CD4 and CD8 T cells may be due to their differential responses to IFN-. TGF has been shown to inhibit IFN- production and Th1 differentiation while promote IL-10 expression (38, 39), so that it may promote IL-10 production during Th17 polarization both by inducing c-maf and by suppressing IFN-. IL-27 has recently been demonstrated to regulate IL-10 expression in both Th1- and Th2-polarizing conditions (11, 22), and our data further supported its role in IL-10 regulation. IL-27 alone induced IL-10 expression in a Stat1-dependent fashion, but in combination with TGF it was able to regulate IL-10 in a Stat1- independent fashion (Fig. 4). Similar to IL-6, IL-27 activates both Stat3 and Stat1; however, IL-6-mediated Stat3 activation is stronger and has a longer duration (11). Stat1 deficiency enabled IL-27 to act similar to IL-6 so that Stat3 activation in combination with TGF signals induced c-maf, IL-10, and IL-17. IL-27 alone induced some c-maf expression, but Stat1 deficiency decreased c-maf expression. It is possible that IL-27 activated Stat1 and Stat3 to form Stat1/Stat3 heterodimers that were capable of inducing c-maf. Stat1 deficiency may allow IL-27 to form only fewer numbers of Stat3 homodimers, resulting in less c-maf induction. Since IL-27 promotes IL-10 expression in Th1-polarizing conditions (11, 22), c-maf is expressed in Th2 but not in Th1 cells (24), and since

11 6236 TGF SYNERGIZES WITH IL-6 FOR c-maf AND IL-10 EXPRESSION IFN- /Stat1/Th1 antagonizes c-maf, it is unlikely that c-maf plays a significant role in IL-27/Stat1-mediated IL-10 regulation. TGF has long been associated with antiinflammatory functions. It induces Foxp3 expression and regulatory T cell development to control immune response (40 42). Regulatory T cells have been suggested to be one of the main sources for TGF in Th17 development, and their TGF production is critical for suppressing autoimmune disease development (43). Here, we showed a novel pathway for TGF to attenuate inflammatory immunity by modulating IL-6 and IL-27 signals to synergistically induce c-maf expression and consequently up-regulate IL-10 production. Overexpression of c-maf suppressed Th1-mediated experimental colitis (44). 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