IL-21 Is Produced by Th17 Cells and Drives IL-17 Production in a STAT3-dependent Manner
CD4+ helper T cells can differentiate into several possible fates including: Th1, Th2, T regulatory, and Th17 cells. Although, cytokine production by non-T cells is an important factor in helper T cell differentiation, a characteristic feature of both Th1 and Th2 lineages is their ability to secrete cytokines that promote their respective differentiation. However, cytokines produced by T cells that help to sustain Th17 cells have not yet been identified. Here we show that IL-21 is a product of Th17 cells, which is induced in a Stat3-dependent manner. Additionally, Stat3 can directly bind the Il21 promoter. IL-21 also induces IL-17 production and expression of the transcription factor, RORγt. Furthermore, generation of Th17 cells in the conventional manner is attenuated by blocking IL-21. IL-21 is known to activate Stat3 and its ability to induce Th17 differentiation is abrogated in the absence of Stat3. These data argue that IL-21 serves as an autocrine factor secreted by Th17 cells that promotes or sustains Th17 lineage commitment. CD4+ helper T cells can differentiate into several possible fates including: Th1, Th2, T regulatory, and Th17 cells. Although, cytokine production by non-T cells is an important factor in helper T cell differentiation, a characteristic feature of both Th1 and Th2 lineages is their ability to secrete cytokines that promote their respective differentiation. However, cytokines produced by T cells that help to sustain Th17 cells have not yet been identified. Here we show that IL-21 is a product of Th17 cells, which is induced in a Stat3-dependent manner. Additionally, Stat3 can directly bind the Il21 promoter. IL-21 also induces IL-17 production and expression of the transcription factor, RORγt. Furthermore, generation of Th17 cells in the conventional manner is attenuated by blocking IL-21. IL-21 is known to activate Stat3 and its ability to induce Th17 differentiation is abrogated in the absence of Stat3. These data argue that IL-21 serves as an autocrine factor secreted by Th17 cells that promotes or sustains Th17 lineage commitment. CD4+ helper T (Th) 3The abbreviations used are: Th, helper T cells; IL, interleukin; Stat, signal transducer and activator of transcription; IFN, interferon; RORγt, RAR-related orphan receptor γ t; TGFβ-1, transforming growth factor β-1; ChIP, chromatin immunoprecipitation; ELISA, enzyme-linked immunosorbent assay. cells shape immune responses by differentiating into discrete subsets that secrete distinctive cytokines. These patterns are thought to determine the success of the immune system, protect against a particular pathogen, and limit damage to host tissues (1O'Garra A. Vieira P. Nat. Med. 2004; 10: 801-805Crossref PubMed Scopus (678) Google Scholar). Until recently, differentiating Th cells were thought to have one of two possible fates defined by their cytokine secretion, namely Th1 and Th2 (2Murphy K.M. Ouyang W. Farrar J.D. Yang J. Ranganath S. Asnagli H. Afkarian M. Murphy T.L. Annu. Rev. Immunol. 2000; 18: 451-494Crossref PubMed Scopus (547) Google Scholar). Intracellular pathogens promote the production of IL-12 by dendritic cells. In concert, with antigen stimulation, IL-12 acting via the transcription factor Stat4, induces the development of a Th1 cell that produces the signature cytokine IFN-γ. Furthermore, IFN-γ acting via Stat1 also promotes the expression of the transcription factor T-box expressed in T cells (T-bet) which is important for Th1 differentiation (3Szabo S.J. Sullivan B.M. Peng S.L. Glimcher L.H. Annu. Rev. Immunol. 2003; 21: 713-758Crossref PubMed Scopus (776) Google Scholar). In contrast, helminthic pathogens promote the generation Th2 cells that produce the key cytokine IL-4, which activates Stat6 (4Ansel K.M. Djuretic I. Tanasa B. Rao A. Annu. Rev. Immunol. 2006; 24: 607-656Crossref PubMed Scopus (532) Google Scholar). IL-4 has pleiotropic effects, but a key effect is the promotion of Th2 differentiation and antagonism of IFN-γ production. Thus, a classic feature of Th cells is their production of cytokines that promote their own differentiation and antagonize the differentiation to the other lineage. This simplistic dichotomotous view of T cell differentiation has recently been challenged by the discovery of a new lineage of T cells characterized by the ability to preferentially secrete a proinflammatory cytokine, IL-17 and thus designated Th17 cells (5Weaver C.T. Harrington L.E. Mangan P.R. Gavrieli M. Murphy K.M. Immunity. 2006; 24: 677-688Abstract Full Text Full Text PDF PubMed Scopus (1174) Google Scholar). These T cells have been implicated in protection from extracellular bacteria (6Fedele G. Stefanelli P. Spensieri F. Fazio C. Mastrantonio P. Ausiello C.M. Infect. Immun. 2005; 73: 1590-1597Crossref PubMed Scopus (54) Google Scholar, 7Happel K.I. Dubin P.J. Zheng M. Ghilardi N. Lockhart C. Quinton L.J. Odden A.R. Shellito J.E. Bagby G.J. Nelson S. Kolls J.K. J. Exp. Med. 2005; 202: 761-769Crossref PubMed Scopus (498) Google Scholar, 8Mangan 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) and in autoimmune diseases in animals and humans (9Langrish C.L. Chen Y. Blumenschein W.M. Mattson J. Basham B. Sedgwick J.D. McClanahan T. Kastelein R.A. Cua D.J. J. Exp. Med. 2005; 201: 233-240Crossref PubMed Scopus (3259) Google Scholar, 10Zheng Y. Danilenko D.M. Valdez P. Kasman I. Eastham-Anderson J. Wu J. Ouyang W. Nature. 2007; 445: 648-651Crossref PubMed Scopus (1567) Google Scholar). Initially, an IL-12-related cytokine also produced by dendritic cells, IL-23, was thought to be the main driver of Th17 differentiation. However, it was later recognized that IL-23 did not act on naïve CD4+ T cells to induce Th17 differentiation. Rather, other inflammatory cytokines produced by dendritic cells, IL-1, and IL-6 in conjunction with transforming growth factorβ-1 (TGFβ-1) were found to be efficient inducers of Th17 differentiation from naïve CD4+ T cell precursors. IL-23 is therefore thought to be more critical for in vivo maintenance of Th17 cells (11Weaver C.T. Hatton R.D. Mangan P.R. Harrington L.E. Annu. Rev. Immunol. 2007; 25: 821-852Crossref PubMed Scopus (1544) Google Scholar). Both IL-6 and IL-23 activate Stat3, which is found to be critical for Th17 differentiation (12Chen Z. Laurence A. Kanno Y. Pacher-Zavisin M. Zhu B.M. Tato C. Yoshimura A. Hennighausen L. O'Shea J.J. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 8137-8142Crossref PubMed Scopus (534) Google Scholar, 13Yang X.O. Panopoulos A.D. Nurieva R. Chang S.H. Wang D. Watowich S.S. Dong C. J. Biol. Chem. 2007; 282: 9358-9363Abstract Full Text Full Text PDF PubMed Scopus (1145) Google Scholar). Another key transcription factor associated with the Th17 lineage, the orphan retinoid receptor, RORγt, is critical for Th17 differentiation and is induced by the aforementioned cytokines in a Stat3-dependent manner (14Ivanov II, McKenzie B.S. Zhou L. Tadokoro C.E. Lepelley A. Lafaille J.J. Cua D.J. Littman D.R. Cell. 2006; 126: 1121-1133Abstract Full Text Full Text PDF PubMed Scopus (3988) Google Scholar). As indicated, a characteristic feature of both Th1 and Th2 cells is the production of cytokines that sustain their respective differentiation, whereas simultaneously antagonizing differentiation to the opposing lineage. However, the potential cytokine that might play a similar sustaining role in Th17 cells has not been identified. In the present study, we investigated cytokines produced by Th17 cells with the goal of identifying candidates that would promote/sustain Th17 differentiation. We found that Th17 cells selectively produce IL-21. Additionally, IL-21 induces expression of RORγt, IL-17A, and IL-17F. Thus IL-21 appears to serve as an autocrine regulator of IL-17 production and serves to promote/sustain Th17 lineage commitment. Mice—Mice bearing loxP-flanked conditional (fl/fl) alleles of STAT3 on a C57BL/6J inbred background were kindly provided by David Levy (NYU, New York). Stat3fl/fl mice were bred with mice expressing Cre under the control of either the CD4 promoter (CD4-Cre) to produce Stat3fl/fl; CD4-Cre mice or the MMTV-LTR (MMTV-Cre) to produce Stat3fl/fl; MMTV-Cre mice. Animals were handled and housed in accordance with the guidelines of the NIH Animal Care and Use Committee. Isolation of Cells and Cell Culture—Splenocytes were obtained by disrupting organs of 10- to 13-week-old mice. Unless stated otherwise, all cell cultures were performed in RPMI 1640 supplemented with 10% fetal calf serum, 2 mm glutamine, 100 IU/ml penicillin, 0.1 mg/ml streptomycin (Invitrogen), and 2 mm β-mercaptoethanol. T cells were enriched with a mouse CD4+ T cell kit using AutoMacs isolator (Miltenyi Biotec, Bergisch Gladbach, Germany). Naive T cells were obtained by surface staining with anti-CD62L, anti-CD44, and anti-CD25. The CD62L+CD44–CD25– population was isolated by flow cytometry cell sorting with a Mo-Flo cell sorter (Dako, Carpinteria, CA). Cells were activated by plate-bound anti-CD3 (5 μg/ml) and anti-CD28 (5 μg/ml) (BD Pharmingen) for 3 days. Th0 indicates neutral conditions (no exogenous cytokines and anti-cytokine antibodies). Th1 conditions indicate addition of IL-12 (10 ng/ml) and anti-IL-4 (10 μg/ml). Th2 conditions indicate addition of IL-4 (10 ng/ml) and anti-IL-12 (10 μg/ml). Th17 conditions indicate addition of IL-6 (10 ng/ml), TGFβ-1 (5 ng/ml), anti-IFN-γ (10 μg/ml), and anti-IL-4 (10 μg/ml). Where indicated, IL-21 (100 ng/ml) and anti-IL-21 antibody (4 μg/ml) were added to the cell cultures. All cytokines were from R&D Systems (Minneapolis, MN), except for IL-21, which was from PeproTech (Rocky Hill, NJ). All anti-cytokine antibodies were from BD Pharmingen, except for anti-IL-21 antibody, which was from R&D Systems. Measurement of Cytokines—Peripheral naïve CD4+ lymphocytes were isolated as described above. Detection of IFN-γ- and IL-17-producing cells was determined by intracellular cytokine staining with anti-IFN-γ-fluorescein isothiocyanate and anti-IL-17-phycoerythrin (BD Biosciences). Briefly, cells were stimulated for 4 h with phorbol 12-myristate 13-acetate and ionomycin. GolgiStop (BD Biosciences) was added after 2 h, and cell was fixed in 4% formyl saline. Fixed cells were stained with fluorescent antibodies in 0.1% saponin permeabilization buffer and analyzed on a FACSCalibur (BD Biosciences). Events were collected and analyzed using FLOWJO software (Tree Star, Ashland, OR). Cytokine production in cell culture supernatants was analyzed using mouse IL-17A Quantikine assay kits (R&D Systems), IL-17F DuoSet assay kit (R&D Systems), and mouse IL-21 DuoSet assay kit (R&D Systems) according to the manufacturer’s instructions. RNA Preparation and Microarray Analysis—Total cellular RNA from cells cultured under Th0 and optimal Th17 conditions was extracted with TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Approximately 10 μg of RNA was labeled and hybridized to GeneChip mouse genome 430 2.0 arrays (Affymetrix, Santa Clara, CA) according to the manufacturer's protocols. Expression values were determined using GeneChip Operating Software (GCOS) v1.1.1. All data analysis was performed using GeneSpring software GX 7.3.1 (Agilent Technologies, Santa Clara, CA). Gene expression values were normalized as described previously (15Pfeffer L.M. Kim J.G. Pfeffer S.R. Carrigan D.J. Baker D.P. Wei L. Homayouni R. J. Biol. Chem. 2004; 279: 31304-31311Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Fold induction of individual gene between Th0 and Th17 cells is represented by the ratio of normalized gene expression value in Th17 cells to value in Th0 cells. Quantitative Real-time PCR—Total RNA was extracted by RNeasy kit (Qiagen, Valencia, CA). cDNA was synthesized with Reverse Transcription kit (Applied Biosystems, Foster City, CA) using random hexamers as primers according to the manufacturer's instruction. Actin was used as endogenous control. TaqMan primers and probes for murine IL-21, IL-17A, IL-17F, RORγt, and actin were purchased from Applied Biosystems, and samples were analyzed using the ABI PRISM 7500 Sequence Detection System (Applied Biosystems). Chromatin Immunoprecipitation (ChIP)—ChIP experiments were performed using the ChIP-IT™ Chromatin Immunoprecipitation kit (Active Motif, Carlsbad, CA) according to the manufacturer's instructions. CD4+ T cells were polarized under neutral condition (Th0) or Th17 conditions for 72 h. Anti-Stat3 antibody (sc7179; Santa Cruz Biotechnology, Santa Cruz, CA) was used. ChIP samples were analyzed by quantitative real-time PCR using primers targeting the Il21 promoter as shown: 5′-TGCCGCTGCTTTACTCATTG-3′ and 5′-GCACCGTCAGCTTTCAGAGA-3′. Selective Production of IL-21 by Th17 Cells—To survey cytokines potentially produced by Th17 cells, we initially employed microarray analysis, comparing differentiated Th17 cells with Th0 cells. Among the genes selectively expressed by Th17 cells were Rorc (RORγt), Rora, Ccr6, Ccl20, and Il1r1, genes now known to be expressed by Th17 cells (Table 1). In addition, we noted that the gene encoding IL-21 was also selectively expressed in Th17 cells. To verify and quantify IL-21 production by Th17 cells, we sorted naïve CD4+CD62L+CD44–CD25– peripheral T cells from wild-type mice and cultured the cells under optimal Th0, Th1, Th2, and Th17 conditions for 72 h. As shown in Fig. 1, IL-21 mRNA (A) and protein (B) were expressed at low levels in Th0, Th1, and Th2 cells. By comparison, very high levels of IL-21 were generated under Th17 conditions. These results suggest that IL-21 is a product of Th17 cells, which produce much greater levels than other differentiated T cell subsets.TABLE 1Selected genes induced in Th17 cellsGene symbolFold inductionDescriptionCytokinesIl17244Interleukin 17Il216Interleukin 21ChemokinesCcl2033Chemokine (C-C motif) ligand 20Xcl133Chemokine (C motif) ligand 1Ccl98Chemokine (C-C motif) ligand 9Ccl63Chemokine (C-C motif) ligand 6Cell surface receptorsIl1r1129Interleukin 1 receptor, type ITnfrsf12a56Tumor necrosis factor receptor superfamily, member 12aGpr1549G protein-coupled receptor 15Itgae28Integrin, alpha E, epithelial-associatedStab127Stabilin 1Fcgr2b23Fc receptor, IgG, low affinity IIbCcr623Chemokine (C-C motif) receptor 6Cx3cr119Chemokine (C-X3-C) receptor 1Lgr416Leucine-rich repeat-containing G protein-coupled receptor 4Nuclear receptorsRorc999RAR-related orphan receptor γRora45RAR-related orphan receptor αAhr13Aryl-hydrocarbon receptorVdr7Vitamin D receptorThra5Thyroid hormone receptor α Open table in a new tab Regulation of IL-21 by Stat3—We and others have previously shown that IL-17 production is Stat3-dependent (12Chen Z. Laurence A. Kanno Y. Pacher-Zavisin M. Zhu B.M. Tato C. Yoshimura A. Hennighausen L. O'Shea J.J. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 8137-8142Crossref PubMed Scopus (534) Google Scholar, 13Yang X.O. Panopoulos A.D. Nurieva R. Chang S.H. Wang D. Watowich S.S. Dong C. J. Biol. Chem. 2007; 282: 9358-9363Abstract Full Text Full Text PDF PubMed Scopus (1145) Google Scholar). To determine whether IL-21 production might also be dependent upon this transcription factor, we analyzed IL-21 production in Stat3-deficient CD4+ T cells. Indeed, Stat3-deficient T cells cultured under standard Th17 conditions failed to produce IL-17 mRNA or protein (see below). Additionally, IL-21 mRNA (Fig. 1C) and protein (D) were also undetectable in Stat3-deficient cells even though wild-type cells cultured under the same conditions produced comparatively high levels of this cytokine. To determine whether Stat3 might play a direct role in regulating IL-21 in Th17 cells, we sought to determine whether Stat3 can bind the Il21 promoter using chromatin immunoprecipitation assays. As shown in Fig. 1E, Stat3 binding to the Il21 promoter was significantly enriched under Th17 culture conditions compared with Th0 conditions. Taken together, these results argue that Th17 cells selectively produce IL-21, which appears to be induced directly in a Stat3-dependent manner. IL-21 Promotes IL-17 Production by Naïve CD4+ T Helper Cells—Given the critical role of Stat3 in Th17 differentiation and given that IL-21 has been reported to selectively induce phosphorylation of Stat3 (16Zeng R. R. Zhu W. Levy 2007; PubMed Scopus Google we whether IL-21 promote Th17 differentiation. naïve CD4+ T cells were cultured under optimal Th17 conditions or under conditions in which IL-6 was and with IL-21. As shown in Fig. and optimal Th17 conditions TGFβ-1 induced high levels of IL-17A, IL-17F and as as high of cells of IL-6 with IL-21 was a very of IL-17-producing cells. IL-6 IL-21 in the absence of TGFβ-1 ability to induce Th17 differentiation. The transcription factor is for Th17 differentiation (14Ivanov II, McKenzie B.S. Zhou L. Tadokoro C.E. Lepelley A. Lafaille J.J. Cua D.J. Littman D.R. Cell. 2006; 126: 1121-1133Abstract Full Text Full Text PDF PubMed Scopus (3988) Google Scholar). of naïve CD4+ T cells under standard Th17 conditions expression of this transcription factor (Fig. under conditions in which IL-21 expression was As IL-21 is a activator of Stat3. We therefore whether the ability of IL-21 to induce Th17 differentiation was also naïve CD4+ T cells from Stat3-deficient mice were cultured under optimal Th17 conditions or under conditions in which IL-6 was and with IL-21. As shown in Fig. and very low IL-17A and IL-17F mRNA protein were under all culture conditions. In addition, very cells were found in Stat3-deficient mice These results indicate that induction of IL-17 by IL-21 and TGFβ-1 on Stat3, which is to in the of TGFβ-1 and of IL-21 with the of Th17 data that Th17 cells produce of IL-21, and IL-21 can promote Th17 lineage differentiation. We therefore that with the conventional of Th17 cells, autocrine IL-21 was an important but previously We that IL-21 was an important factor, IL-21 produced by T cells would the of Th17 cells. Indeed, antibody of IL-21 in the Th17 cultures IL-17A and IL-17F mRNA expression (Fig. as as protein production by Intracellular staining of IL-17A also that of IL-21 differentiation of Th17 cells (Fig. These results indicate that autocrine IL-21 production to the optimal in differentiation of Th17 cells. In this study, we that IL-21 is a important product of Th17 cells; and compared with other T cells Th17 cells selectively produce this cytokine. The induction of IL-21 is and Stat3 directly the Il21 that it be a direct of antigen cells, IL-6 and IL-23, are important for and Th17 cells, antibody blocking experiments indicate that autocrine production of IL-21 is previously factor that to Th17 differentiation. IL-21 is a member of the γ of which to a receptor of IL-21 receptor and the γ receptor Immunol. Rev. 2004; 202: PubMed Scopus Google Scholar). other of the γ IL-21 is dependent upon 3 other γ cytokines and are of IL-21 Stat3 (16Zeng R. R. Zhu W. Levy 2007; PubMed Scopus Google Scholar). This is both IL-6 and IL-23 activate Stat3 in T cells, and it is now that Stat3 is a critical factor in Th17 differentiation. of Stat3 Th17 differentiation X.O. Panopoulos A.D. Nurieva R. Chang S.H. Wang D. Watowich S.S. Dong C. J. Biol. Chem. 2007; 282: 9358-9363Abstract Full Text Full Text PDF PubMed Scopus (1145) Google whereas of of cytokine which Stat3 promotes Th17 differentiation (12Chen Z. Laurence A. Kanno Y. Pacher-Zavisin M. Zhu B.M. Tato C. Yoshimura A. Hennighausen L. O'Shea J.J. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 8137-8142Crossref PubMed Scopus (534) Google Scholar). The is associated with autoimmune characterized by of IL-17 and expression of IL-17 (12Chen Z. Laurence A. Kanno Y. Pacher-Zavisin M. Zhu B.M. Tato C. Yoshimura A. Hennighausen L. O'Shea J.J. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 8137-8142Crossref PubMed Scopus (534) Google Scholar). results that IL-21 induction in Th17 cells was also dependent upon Stat3. IL-21 activates Stat3 to promote Th17 differentiation, more that Stat3 a critical role in Th17 differentiation. to the of the it be noted that results that blocking IL-21 but did not IL-17 production. Additionally, one of IL-17 is to induce IL-6 production in cells Z. S.L. Immunity. Full Text PDF PubMed Scopus Google therefore the of IL-23 IL-21 in Th17 lineage differentiation to be in the cytokine mice. IL-21 is known to be a product of activated CD4+ T cells J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). in vivo effect of IL-21 is the of cells, the of cells to cells R. J. A. C. PubMed Scopus Google Scholar, R. R. R. J. Immunol. 2005; PubMed Scopus Google Scholar). IL-21 has also been reported to be important for the growth of T cells R. R. S. J. Exp. Med. 2005; 201: PubMed Scopus Google Scholar). In the IL-21 has been reported to be preferentially produced by Th2 cells, IL-21 protein is not A.R. M. J. Exp. Med. PubMed Scopus Google Scholar, J. M. T.R. M. J. 2006; PubMed Scopus Google Scholar, A. I. M. M. 2007; PubMed Scopus Google Scholar). to the present study, the production of IL-21 in Th1 and Th2 cells was not compared with Th17 cells. We now that Th1 and Th2 cells IL-21 compared with Th17 cells. IL-21 promotes Th17 differentiation and to to the of Th1 and Th2 cytokines on their respective IL-21 is of acting on Th17 cells, in an autocrine in to antigen Thus IL-21 serves as a factor that is produced by Th17 cells to promote or sustain their differentiation. has that of naïve CD4+ T cells to IL-21 Th1 differentiation and IFN-γ production A. S.L. J. Immunol. 2006; PubMed Scopus Google Scholar). IL-21 production by Th17 cells also be to other cytokines and that not promote their respective lineage but also antagonize differentiation to other cell receptor has been reported to in murine D. S. M. J. Immunol. 2007; PubMed Scopus Google Scholar) and M. L.M. L. M. Wu B. Y. C. M. 2007; PubMed Scopus Google Scholar) that IL-21 promotes Th17 differentiation the of the of this be of to determine the of IL-21 in Th17 differentiation and to whether blocking of IL-21 is in diseases associated with of We David Levy for the Stat3fl/fl and Kanno for critical of the and for