Foxp3 Inhibits RORγt-mediated IL-17A mRNA Transcription through Direct Interaction with RORγt*

Abstract

The cytokine, transforming growth factor-β1 (TGF-β1), converts naive T cells into regulatory T cells that prevent autoimmunity. However, in the presence of interleukin (IL)-6, TGF-β1 has also been found to promote differentiation into IL-17-producing helper T (Th17) cells that are deeply involved in autoimmunity and inflammation. However, it has not been clarified how TGF-β1 and IL-6 determine such a distinct fate. Here we found that a master regulator for Th17, retinoic acid-related orphan receptor γt (RORγt), was rapidly induced by TGF-β1 regardless of the presence of IL-6. IL-6 reduced Foxp3 expression, and overexpression of Foxp3 in a T cell line resulted in a strong reduction of IL-17A expression. We have characterized the IL-17A promoter and found that RORγt binding is sufficient for activation of the minimum promoter in the HEK 293T cells. RORγt-mediated IL-17A promoter activation was suppressed by forced expression of Foxp3. Foxp3 directly interacted with RORγt through exon 2 region of Foxp3. The exon 2 region and forkhead (FKH) domain of Foxp3 were necessary for the suppression of RORγt-mediated IL-17A promoter activation. We propose that induction of Foxp3 is the mechanism for the suppression of Th17 and polarization into inducible Treg. The cytokine, transforming growth factor-β1 (TGF-β1), converts naive T cells into regulatory T cells that prevent autoimmunity. However, in the presence of interleukin (IL)-6, TGF-β1 has also been found to promote differentiation into IL-17-producing helper T (Th17) cells that are deeply involved in autoimmunity and inflammation. However, it has not been clarified how TGF-β1 and IL-6 determine such a distinct fate. Here we found that a master regulator for Th17, retinoic acid-related orphan receptor γt (RORγt), was rapidly induced by TGF-β1 regardless of the presence of IL-6. IL-6 reduced Foxp3 expression, and overexpression of Foxp3 in a T cell line resulted in a strong reduction of IL-17A expression. We have characterized the IL-17A promoter and found that RORγt binding is sufficient for activation of the minimum promoter in the HEK 293T cells. RORγt-mediated IL-17A promoter activation was suppressed by forced expression of Foxp3. Foxp3 directly interacted with RORγt through exon 2 region of Foxp3. The exon 2 region and forkhead (FKH) domain of Foxp3 were necessary for the suppression of RORγt-mediated IL-17A promoter activation. We propose that induction of Foxp3 is the mechanism for the suppression of Th17 and polarization into inducible Treg. T helper (Th) 3The abbreviations used are: Th, T helper; Th17, IL-17-producing helper T cell; TGF-β1, transforming growth factor-β1; CA-TβRI, constitutively activated TGF-β receptor I; Treg, regulatory T cell; iTreg, inducible Treg; IL, interleukin; RORγt, retinoic acid-related orphan receptor γt; RORE, ROR-responsive element; Foxp3, forkhead box protein 3; RT-PCR, reverse transcription-PCR; ChIP, chromatin immunoprecipitation; Ab, antibody; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; WT, wild type; NFAT, nuclear factor of activated T cells; ZnF, zinc finger; Zip, leucine zipper; FKH, forkhead domain; Luc, luciferase. cells play a pivotal role in adaptive immunity. Upon stimulation by antigens, Th cells undergo distinct developmental pathways, attaining specialized properties and effector functions. CD4+ T cells are traditionally thought to differentiate into Th1 and Th2 cell subsets (1Mosmann T.R. Coffman R.L. Annu. Rev. Immunol. 1989; 7: 145-173Crossref PubMed Scopus (6863) Google Scholar). Recently, a third subset of the polarized T cell subset Th17, 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 (2Kikly K. Liu L. Na S. Sedgwick J.D. Curr. Opin. Immunol. 2006; 18: 670-675Crossref PubMed Scopus (267) Google Scholar, 3Aujla S.J. Dubin P.J. Kolls J.K. Semin. Immunol. 2007; 19: 377-382Crossref PubMed Scopus (230) Google Scholar, 4Kottke T. Sanchez-Perez L. Diaz R.M. Thompson J. Chong H. Harrington K. Calderwood S.K. Pulido J. Georgopoulos N. Selby P. Melcher A. Vile R. Cancer Res. 2007; 67: 11970-11979Crossref PubMed Scopus (79) Google Scholar). It has been shown that TGF-β1 plays a pivotal role in the induction of Th17 (5Li M.O. Wan Y.Y. Flavell R.A. Immunity. 2007; 26: 579-591Abstract Full Text Full Text PDF PubMed Scopus (564) Google Scholar). Th17 differentiation of naive T cells is initiated by IL-6 and TGF-β1 (6Mangan P.R. Harrington L.E. O'Quinn D.B. Helms W.S. Bullard D.C. Elson C.O. Hatton R.D. Wahl S.M. Schoeb T.R. Weaver C.T. Nature. 2006; 441: 231-234Crossref PubMed Scopus (2611) Google Scholar, 7Bettelli E. Carrier Y. Gao W. Korn T. Strom T.B. Oukka M. Weiner H.L. Kuchroo V.K. Nature. 2006; 441: 235-238Crossref PubMed Scopus (5591) Google Scholar, 8Veldhoen M. Hocking R.J. Atkins C.J. Locksley R.M. Stockinger B. Immunity. 2006; 24: 179-189Abstract Full Text Full Text PDF PubMed Scopus