Sensitivity and Resistance to Regulation by IL-4 during Th17 Maturation Laura A. Cooney, Keara Towery, Judith Endres and David A. Fox This information is current as of September 30, 2021. J Immunol 2011; 187:4440-4450; Prepublished online 26 September 2011; doi: 10.4049/jimmunol.1002860 http://www.jimmunol.org/content/187/9/4440 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2011/09/27/jimmunol.100286 Material 0.DC1 References This article cites 45 articles, 20 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/187/9/4440.full#ref-list-1 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists by guest on September 30, 2021 • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Sensitivity and Resistance to Regulation by IL-4 during Th17 Maturation Laura A. Cooney,1 Keara Towery,2 Judith Endres, and David A. Fox Th17 cells are highly pathogenic in a variety of immune-mediated diseases, and a thorough understanding of the mechanisms of cytokine-mediated suppression of Th17 cells has great therapeutic potential. In this article, we characterize the regulation of both in vitro- and in vivo-derived Th17 cells by IL-4. We demonstrate that IL-4 suppresses reactivation of committed Th17 cells, even in the presence of TGF-b, IL-6, and IL-23. Downregulation of IL-17 by IL-4 is dependent on STAT6 and mediated by inhibition of STAT3 binding at the Il17a promoter. Although Th1 cytokines were shown to induce IFN-g expression by Th17 cells, IL-4 does not induce a Th2 phenotype in Th17 cells. Suppression by IL-4 is stable and long-lived when applied to immature Th17 cells, but cells that have undergone multiple rounds of stimulation, either in vivo during a Th17-mediated inflammatory disease, or in vitro, become resistant to suppression by IL-4 and lose the ability to signal through IL-4R. Thus, although IL-4 is a potent suppressor of Downloaded from the Th17 genetic program at early stages after differentiation, prolonged stimulation renders Th17 cells impervious to regulatory cytokines. The Journal of Immunology, 2011, 187: 4440–4450. h17 cells, which express the proinflammatory cytokine shown that cytokine-regulatory networks can change as T cells IL-17, play an important role in the pathogenesis of a mature. For example, IL-27 suppresses Th17 development from + variety of autoimmune and inflammatory diseases. Naive naive CD4 T cells, but it fails to suppress reactivation of com- http://www.jimmunol.org/ T+ CD4 T cells differentiate into Th17 cells in response to TGF-b, mitted Th17 cells (15). Understanding the regulation of pre- IL-6, and IL-21 and become more pathogenic in the presence of existing activated T cells may also have therapeutic applications IL-23 (1–7). In addition, Th17 cell development depends on the in the control of chronic T cell-mediated diseases, such as rheu- transcription factors STAT3, IRF4, RORgt, and RORa (8–11). matoid arthritis. In vitro, induction of Th17 differentiation is inhibited by the Th1 Several groups recently demonstrated a high degree of plasticity cytokines IFN-g and IL-12, as well as the Th2 cytokines IL-4 and in Th17 cells, such that stimulation with IL-12 upregulates T-bet IL-13 (12–14). and IFN-g and induces a Th1-like phenotype, whereas stimula- Most of what is known about the cross-regulation of Th17 cells is tion with TGF-b upregulates Foxp3 and induces a regulatory limited to the earliest stages of differentiation, which occur within T cell-like phenotype (16, 17). One report suggested that the Th17 by guest on September 30, 2021 the first few hours to days following the initial T cell activation. phenotype is unstable, and Th17 cells will spontaneously convert However, it is also important to address the role of cytokine- to Th1 cells in lymphopenic hosts (18). However, another group mediated regulation of committed Th17 cells. Little is known demonstrated that in vitro-generated Th17 cells quickly lose IL-17 about the regulation of secondary stimulation, which may be more expression unless IL-23 is added and opposing cytokines are representative of a Th17 cell that has become activated and dif- blocked, whereas in vivo-generated Th17 cells continue to express ferentiated in a lymph node in the presence of TGF-b and IL-6, IL-17, regardless of which cytokines are added (19). Similarly, exited the lymph node, and traveled to a site of inflammation with in vitro-generated Th17 cells could convert to a Th1 or Th2 a distinct cytokine milieu, such as an inflamed joint. It was also phenotype when stimulated under Th1- or Th2-skewing condi- tions, whereas in vivo-generated Th17 cells were refractory to Division of Rheumatology, Department of Internal Medicine and Rheumatic Disease Th1- and Th2-polarizing signals. Research Core Center, University of Michigan, Ann Arbor, MI 48109 Some Th17-to-Th1 conversion may not be surprising, consid- 1Current address: Malaria Program, Seattle Biomedical Research Institute, Seattle, ering the evidence for the close relationship between these two WA. lineages. Th17 cells and Th1 cells were thought to share a com- 2Current address: Department of Microbiology, Michigan State University, East mon lineage precursor (20), and IL-17/IFN-g double-positive cells Lansing, MI. are quite common in humans (21, 22). Similarly, the shared de- Received for publication August 23, 2010. Accepted for publication August 18, 2011. pendence on TGF-b implies some regulatory T cell–Th17 com- This work was supported by a grant from the Arthritis Foundation and by National monality. However, there is much less evidence of any Th2–Th17 Institutes of Health Grants AR38477 and P30 AR048310. L.A.C. was supported by a National Institutes of Health immunopathology training grant, a National Institutes commonality. Unlike the frequent occurrence of IL-17/IFN-g of Health regenerative sciences training grant, and a University of Michigan Rack- double-positive cells, IL-17/IL-4 double-positive cells have only ham postdoctoral fellowship. rarely been encountered (23), suggesting that the relationship Address correspondence and reprint requests to Dr. David A. Fox, University of between Th1 and Th17 may be very different from the relationship Michigan, 3918 Taubman Center, 1500 East Medical Center Drive, SPC 5358, Ann Arbor, MI 48109-5358. E-mail address: [email protected] between Th2 and Th17. The online version of this article contains supplemental material. Although IL-4 was shown to inhibit Th17 differentiation and IL- Abbreviations used in this article: BM-DC, bone marrow-derived dendritic cell; ChIP, 17 expression, nothing is known about the molecular mechanisms chromatin immunoprecipitation; CIA, collagen-induced arthritis; ICS, intracellular of this suppression or about the effects of IL-4 on other Th17- cytokine staining; i.d., intradermally; IRS, insulin receptor substrate; KLH, keyhole family genes, such as RORgt and IL-22. Similarly, the effects of limpet hemocyanin; SOCS, suppressor of cytokine signaling. IL-4 on developing versus committed Th17 cells have not been Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 explored. In this study, we demonstrate that IL-4 suppresses www.jimmunol.org/cgi/doi/10.4049/jimmunol.1002860 The Journal of Immunology 4441 a subset of Th17 genes distinct from IFN-g. Suppression of IL-17 with streptavidin HRP (BioLegend). The plates were developed with depends on STAT6, but not GATA-3, and correlates with a loss of OptEIA TMB substrate (BD) and absorbance at 450 nm was quantitated STAT3 binding at the Il17a promoter. IL-4 induces a stable, ir- with a Bio-Rad (Hercules, CA) plate reader using KC4 software (Biotek, Winooski, VT). reversible loss of IL-17 expression without inducing conversion to a Th2 phenotype. However, repeated stimulation renders both Flow cytometry in vivo- and in vitro-derived Th17 cells refractory to suppression For intracellular cytokine staining (ICS), cells were stimulated for 6 h with by IL-4. 5 ng/ml PMA and 500 ng/ml ionomycin, with 10 mg/ml brefeldin A added for the last 5 h (all chemicals were purchased from Sigma). Cells were then treated with mouse Fc Block anti-CD16/32, stained with FITC- or Materials and Methods PE-conjugated anti-CD4 (clone GK1.5), and fixed overnight. The next Mice day, cells were permeabilized with saponin and stained with fluorescently For in vitro Th17 differentiation and in vivo keyhole limpet hemocyanin labeled anti–IL-17 (clone TC11-18H10.1), anti–IFN-g (clone XMG 1.2), (KLH) immunization, male 6–8-wk-old BALB/c mice were obtained from and anti–IL-4 (clone 11B11) or the appropriate isotype control (all Abs Jackson Laboratories. For cII/CFA immunization, male 8–10 wk-old from BioLegend). Staining was measured with a FACSCalibur, and data DBA1 mice were obtained from Jackson Laboratories. STAT6-deficient were analyzed using Cell Quest software (BD). and IL-4R mutant mice on a BALB/c background were obtained from Real-time PCR Jackson Laboratories.