Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

TH9 cells that express the transcription factor PU.1 drive T cell–mediated colitis via IL-9 receptor signaling in intestinal epithelial cells

Nature Immunology volume 15, pages 676–686 (2014)Cite this article

Subjects

A Corrigendum to this article was published on 16 January 2015

Abstract

The molecular checkpoints that drive inflammatory bowel diseases are incompletely understood. Here we found more T cells expressing the transcription factor PU.1 and interleukin 9 (IL-9) in patients with ulcerative colitis. In an animal model, citrine reporter mice had more IL-9-expressing mucosal T cells in experimental oxazolone-induced colitis. IL-9 deficiency suppressed acute and chronic colitis. Mice with PU.1 deficiency in T cells were protected from colitis, whereas treatment with antibody to IL-9 suppressed colitis. Functionally, IL-9 impaired intestinal barrier function and prevented mucosal wound healing in vivo. Thus, our findings suggest that the TH9 subset of helper T cells serves an important role in driving ulcerative colitis by regulating intestinal epithelial cells and that TH9 cells represent a likely _target for the treatment of chronic intestinal inflammation.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Expression of IL-9 by lamina propria CD4+ T cells in UC.
Figure 2: Patients with UC have higher expression of PU.1 in lamina propria cells and of IL-9R on epithelial cells.
Figure 3: TH9 cells and IL-9 regulate oxazolone-induced colitis.
Figure 4: Central regulatory role of IL-9 and PU.1 in CD4+ T cells in experimental oxazolone-induced colitis.
Figure 5: Protective mechanism of IL-9 deficiency in experimental oxazolone-induced colitis.
Figure 6: IL-9 regulates intestinal barrier function.
Figure 7: IL-9 serves a crucial role in mucosal wound healing in vivo.
Figure 8: Crucial role for IL-9 in chronic intestinal inflammation.

Similar content being viewed by others

References

  1. Danese, S. New therapies for inflammatory bowel disease: from the bench to the bedside. Gut 61, 918–932 (2012).

    CAS  PubMed  Google Scholar 

  2. Strober, W., Fuss, I. & Mannon, P. The fundamental basis of inflammatory bowel disease. J. Clin. Invest. 117, 514–521 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Macdonald, T.T. & Monteleone, G. Immunity, inflammation, and allergy in the gut. Science 307, 1920–1925 (2005).

    CAS  PubMed  Google Scholar 

  4. Danese, S. & Fiocchi, C. Ulcerative colitis. N. Engl. J. Med. 365, 1713–1725 (2011).

    CAS  PubMed  Google Scholar 

  5. Neurath, M.F., Finotto, S. & Glimcher, L.H. The role of Th1/Th2 polarization in mucosal immunity. Nat. Med. 8, 567–573 (2002).

    CAS  PubMed  Google Scholar 

  6. Neurath, M.F. et al. The transcription factor T-bet regulates mucosal T cell activation in experimental colitis and Crohn's disease. J. Exp. Med. 195, 1129–1143 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Fuss, I.J. et al. Disparate CD4+ lamina propria (LP) lymphokine secretion profiles in inflammatory bowel disease. Crohn's disease LP cells manifest increased secretion of IFN-gamma, whereas ulcerative colitis LP cells manifest increased secretion of IL-5. J. Immunol. 157, 1261–1270 (1996).

    CAS  PubMed  Google Scholar 

  8. Fuss, I.J. & Strober, W. The role of IL-13 and NK T cells in experimental and human ulcerative colitis. Mucosal Immunol. 1 (suppl. 1), S31–S33 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Kobayashi, T. et al. IL23 differentially regulates the Th1/Th17 balance in ulcerative colitis and Crohn's disease. Gut 57, 1682–1689 (2008).

    CAS  PubMed  Google Scholar 

  10. Kleinschek, M.A. et al. Circulating and gut-resident human Th17 cells express CD161 and promote intestinal inflammation. J. Exp. Med. 206, 525–534 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Ono, Y. et al. T-helper 17 and interleukin-17-producing lymphoid tissue inducer-like cells make different contributions to colitis in mice. Gastroenterology 143, 1288–1297 (2012).

    CAS  PubMed  Google Scholar 

  12. Ahern, P.P. et al. Interleukin-23 drives intestinal inflammation through direct activity on T cells. Immunity 33, 279–288 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Leppkes, M. et al. RORγ-expressing Th17 cells induce murine chronic intestinal inflammation via redundant effects of IL-17A and IL-17F. Gastroenterology 136, 257–267 (2009).

    CAS  PubMed  Google Scholar 

  14. Heller, F., Fuss, I.J., Nieuwenhuis, E.E., Blumberg, R.S. & Strober, W. Oxazolone colitis, a Th2 colitis model resembling ulcerative colitis, is mediated by IL-13-producing NK-T cells. Immunity 17, 629–638 (2002).

    CAS  PubMed  Google Scholar 

  15. Dardalhon, V. et al. IL-4 inhibits TGF-β-induced Foxp3+ T cells and, together with TGF-β, generates IL-9+IL-10+Foxp3− effector T cells. Nat. Immunol. 9, 1347–1355 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Veldhoen, M. et al. Transforming growth factor-β 'reprograms' the differentiation of T helper 2 cells and promotes an interleukin 9-producing subset. Nat. Immunol. 9, 1341–1346 (2008).

    CAS  PubMed  Google Scholar 

  17. Gessner, A., Blum, H. & Rollinghoff, M. Differential regulation of IL-9-expression after infection with Leishmania major in susceptible and resistant mice. Immunobiology 189, 419–435 (1993).

    CAS  PubMed  Google Scholar 

  18. Licona-Limón, P. et al. Th9 cells drive host immunity against gastrointestinal worm infection. Immunity 39, 744–757 (2013).

    PubMed  Google Scholar 

  19. Wilhelm, C. et al. An IL-9 fate reporter demonstrates the induction of an innate IL-9 response in lung inflammation. Nat. Immunol. 12, 1071–1077 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Turner, J.E. et al. IL-9-mediated survival of type 2 innate lymphoid cells promotes damage control in helminth-induced lung inflammation. J. Exp. Med. 210, 2951–2965 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. Angkasekwinai, P., Chang, S.H., Thapa, M., Watarai, H. & Dong, C. Regulation of IL-9 expression by IL-25 signaling. Nat. Immunol. 11, 250–256 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Chang, H.C. et al. The transcription factor PU.1 is required for the development of IL-9-producing T cells and allergic inflammation. Nat. Immunol. 11, 527–534 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Staudt, V. et al. Interferon-regulatory factor 4 is essential for the developmental program of T helper 9 cells. Immunity 33, 192–202 (2010).

    CAS  PubMed  Google Scholar 

  24. Jabeen, R. et al. Th9 cell development requires a BATF-regulated transcriptional network. J. Clin. Invest. 123, 4641–4653 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Goswami, R. & Kaplan, M.H. Gcn5 is required for PU.1-dependent IL-9 induction in Th9 cells. J. Immunol. 189, 3026–3033 (2012).

    CAS  PubMed  Google Scholar 

  26. Temann, U.A., Ray, P. & Flavell, R.A. Pulmonary overexpression of IL-9 induces Th2 cytokine expression, leading to immune pathology. J. Clin. Invest. 109, 29–39 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Jabeen, R. & Kaplan, M.H. The symphony of the ninth: the development and function of Th9 cells. Curr. Opin. Immunol. 24, 303–307 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Devos, S. et al. Allergen-induced interleukin-9 production in vitro: correlation with atopy in human adults and comparison with interleukin-5 and interleukin-13. Clin. Exp. Allergy 36, 174–182 (2006).

    CAS  PubMed  Google Scholar 

  29. Schulzke, J.D. et al. Epithelial tight junctions in intestinal inflammation. Ann. NY Acad. Sci. 1165, 294–300 (2009).

    PubMed  Google Scholar 

  30. Lejeune, D., Demoulin, J.B. & Renauld, J.C. Interleukin 9 induces expression of three cytokine signal inhibitors: cytokine-inducible SH2-containing protein, suppressor of cytokine signalling (SOCS)-2 and SOCS-3, but only SOCS-3 overexpression suppresses interleukin 9 signalling. Biochem. J. 353, 109–116 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Kaplan, M.H. Th9 cells: differentiation and disease. Immunol. Rev. 252, 104–115 (2013).

    PubMed  PubMed Central  Google Scholar 

  32. Blom, L., Poulsen, B.C., Jensen, B.M., Hansen, A. & Poulsen, L.K. IL-33 induces IL-9 production in human CD4+ T cells and basophils. PLoS ONE 6, e21695 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Uyttenhove, C., Brombacher, F. & Van Snick, J. TGF-beta interactions with IL-1 family members trigger IL-4-independent IL-9 production by mouse CD4+ T cells. Eur. J. Immunol. 40, 2230–2235 (2010).

    CAS  PubMed  Google Scholar 

  34. Townsend, J.M. et al. IL-9-deficient mice establish fundamental roles for IL-9 in pulmonary mastocytosis and goblet cell hyperplasia but not T cell development. Immunity 13, 573–583 (2000).

    CAS  PubMed  Google Scholar 

  35. Zhou, M. & Ouyang, W. The function role of GATA-3 in Th1 and Th2 differentiation. Immunol. Res. 28, 25–37 (2003).

    CAS  PubMed  Google Scholar 

  36. Moussata, D. et al. Confocal laser endomicroscopy is a new imaging modality for recognition of intramucosal bacteria in inflammatory bowel disease in vivo. Gut 60, 26–33 (2011).

    PubMed  Google Scholar 

  37. Sato, T. & Clevers, H. Growing self-organizing mini-guts from a single intestinal stem cell: mechanism and applications. Science 340, 1190–1194 (2013).

    CAS  PubMed  Google Scholar 

  38. Pickert, G. et al. STAT3 links IL-22 signaling in intestinal epithelial cells to mucosal wound healing. J. Exp. Med. 206, 1465–1472 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Monteleone, I., Sarra, M., Pallone, F. & Monteleone, G. Th17-related cytokines in inflammatory bowel diseases: friends or foes? Curr. Mol. Med. 12, 592–597 (2012).

    CAS  PubMed  Google Scholar 

  40. Mudter, J. et al. The transcription factor IFN regulatory factor-4 controls experimental colitis in mice via T cell-derived IL-6. J. Clin. Invest. 118, 2415–2426 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Pastorelli, L. et al. Epithelial-derived IL-33 and its receptor ST2 are dysregulated in ulcerative colitis and in experimental Th1/Th2 driven enteritis. Proc. Natl. Acad. Sci. USA 107, 8017–8022 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. Kobori, A. et al. Interleukin-33 expression is specifically enhanced in inflamed mucosa of ulcerative colitis. J. Gastroenterol. 45, 999–1007 (2010).

    CAS  PubMed  Google Scholar 

  43. Elyaman, W. et al. IL-9 induces differentiation of TH17 cells and enhances function of FoxP3+ natural regulatory T cells. Proc. Natl. Acad. Sci. USA 106, 12885–12890 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Forbes, E.E. et al. IL-9- and mast cell-mediated intestinal permeability predisposes to oral antigen hypersensitivity. J. Exp. Med. 205, 897–913 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Heller, F. et al. Interleukin-13 is the key effector Th2 cytokine in ulcerative colitis that affects epithelial tight junctions, apoptosis, and cell restitution. Gastroenterology 129, 550–564 (2005).

    CAS  PubMed  Google Scholar 

  46. Kim, H.S. & Chung, D.H. IL-9-producing invariant NKT cells protect against DSS-induced colitis in an IL-4-dependent manner. Mucosal Immunol. 6, 347–357 (2013).

    CAS  PubMed  Google Scholar 

  47. Hardman, C.S., Panova, V. & McKenzie, A.N. IL-33 citrine reporter mice reveal the temporal and spatial expression of IL-33 during allergic lung inflammation. Eur. J. Immunol. 43, 488–498 (2013).

    CAS  PubMed  Google Scholar 

  48. Kulkarni, M.S. & Yielding, K.L. DNA damage and repair in epithelial (mucous) cells and crypt cells from isolated colon. Chem. Biol. Interact. 52, 311–318 (1985).

    CAS  PubMed  Google Scholar 

  49. Nguyen, H.T. et al. CD98 expression modulates intestinal homeostasis, inflammation, and colitis-associated cancer in mice. J. Clin. Invest. 121, 1733–1747 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Wong, S.H. et al. Transcription factor RORα is critical for nuocyte development. Nat. Immunol. 13, 229–236 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

Supported by the Clinical Research Group CEDER of the German Research Council (B.W., R.A. and M.F.N.), Deutsche Forschungsgemeinschaft (WE 4656/1-1 for support of B.W., R.A. and M.F.N.), Interdisziplinares Zentrum fur Klinische Forschung (B.W., R.A. and M.F.N.), the Emerging Field Initiative (B.W., R.A. and M.F.N.), the ELAN programs of the University Erlangen-Nürnberg (B.W., R.A. and M.F.N.), Deutsche Forschungsgemeinschaft Collaborative Research Centers 643 and 796 (H.D., S.W. and M.F.N.), the American Asthma Foundation, the UK Medical Research Council, the Wellcome Trust (100963 to A.N.J.M.) and the Agency for Science Technology and Research (Y.Y.H.).

Author information

Author notes

  1. Benno Weigmann and Markus F Neurath: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Medicine 1, University of Erlangen-Nuremberg, Kussmaul Campus for Medical Research, Erlangen, Germany

    Katharina Gerlach, Raja Atreya, Heike Dornhoff, Stefanie Steiner, Stefan Wirtz, Benno Weigmann & Markus F Neurath

  2. MRC Laboratory of Molecular Biology, Cambridge, UK

    YouYi Hwang & Andrew N J McKenzie

  3. Institute of Molecular Medicine, University Medical Center Mainz, Mainz, Germany

    Alexej Nikolaev & Ari Waisman

  4. Institute of Pathology, Campus Bodensee, Friedrichshafen, Germany

    Hans-Anton Lehr

  5. Institute of Pathology, Bayreuth Clinic, Bayreuth, Germany

    Michael Vieth

  6. Laboratory of Molecular Stem Cell Biology, University of Münster, Münster, Germany

    Frank Rosenbauer

Authors
  1. Katharina Gerlach
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. YouYi Hwang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Alexej Nikolaev
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Raja Atreya
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Heike Dornhoff
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Stefanie Steiner
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Hans-Anton Lehr
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Stefan Wirtz
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Michael Vieth
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Ari Waisman
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Frank Rosenbauer
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Andrew N J McKenzie
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Benno Weigmann
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Markus F Neurath
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

K.G. did experiments together with Y.Y.H., A.N., S.S. and B.W.; R.A., H.D., S.W., F.R., A.N.J.M., B.W. and M.F.N. provided clinical samples, protocols or mice or designed experiments; K.G., Y.Y.H., A.N., S.S., H.-A.L., M.V., A.N.J.M. and B.W. analyzed data; K.G., H.-A.L., A.W., F.R., A.N.J.M., B.W. and M.F.N. discussed and interpreted findings; and K.G., Y.Y.H., B.W. and M.F.N. wrote the manuscript.

Corresponding author

Correspondence to Markus F Neurath.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Analysis of claudin expression in human colonic samples

Total mRNA was isolated from 24-35 human samples of human control tissue or IBD tissue. Expression of PIAS1, SOCS3, CLDN1 and CLDN2 mRNA in human colonic tissue of IBD patients was analyzed. SOCS3 and CLDN2 mRNA levels were up regulated in active UC patient samples. Significant differences are indicated.

Supplementary Figure 2 Generation and analysis of IL-9–citrine reporter mice

(a) Generation of II9Cit reporter mice. The _targeting strategy to insert the citrine reporter gene at the start codon of the II9 gene is shown. neo, neomycin cassette; TK, thymidine kinase cassette; e1, II9 exon 1. The black rectangle represents the 5' probe for Southern blot screening. (b) Southern hybridization for II9Cit-_targeted clones. A positive colony of an II9Cit embryonic stem cell clone using the 5' probe is shown. Wild-type allele, 7.0 kb; _targeted allele, 6.0 kb. (c) Detection of II9 in CD4+ T cells differentiated in vitro using citrine reporter (citrine - top panels) or intracellular staining (ICS – lower panels). Before FACS analysis CD4+ T cells were purified from naïve spleen cells and plated under the following conditions for 72 hours. TH0: plate bound anti-CD3 and anti-CD28 antibodies. TH1: plate bound anti-CD3 antibodies, anti-CD28 antibodies, IL2, IL12, anti-IL4 antibodies. TH2: plate bound anti-CD3 antibodies, anti-CD28 antibodies, IL2, IL4, anti-IFN-γ antibodies. TH9: plate bound anti-CD3 antibodies, anti-CD28 antibodies, IL4, TGF-β. (d) Detection of II9Cit and IL9 ICS in mice following rIL33 administration. Mice were treated with three daily doses of IL33. Mesenteric lymph nodes (MLN) were harvested and cells were analyzed for citrine expression or IL9 ICS. Representative of three biological replicates are shown. (e) Detection of Il9Cit following Nippostrongylus brasiliensis infection. Six days post-infection MLN were harvested and analyzed for citrine expression. Only ILC2 in the mesenteric lymph nodes on day 6 expressed citrine. T cells and other cell types in other organs and other time points did not express detectable citrine at this time point (not shown). Representative images of three biological replicates are shown.

Supplementary Figure 3 IL33 induces IL9 production

Quantitative RT-PCR analysis of mucosal II33 mRNA levels in murine oxazolone-induced colitis and normal colon (n= 22). Relative expression of II33 was normalized to the house keeping gene 18sRNA. Significant differences are indicated (left panel). Spleens were taken from seven animals. Isolated murine spleen CD4+ T cells were stimulated with different cytokines to analyze IL9 production. Supernatants were taken after 48 hours and analyzed for IL9 content by ELISA. Significant differences are indicated (middle panel). Isolated T cell-enriched lamina propria cells from seven wild-type mice were stimulated with different cytokines and IL9 production was analyzed in the supernatant after 48h by ELISA (right panel). A second independent experiment gave similar results. FACS analysis for expression of cytokine receptors was performed in splenic and lamina propria CD3+ T cells. Representative stainings from three independent experiments are shown (lower panel).

Supplementary Figure 4 TH9 transcription factor analysis in wild-type and IL-9-deficient mice

(a) Expression of pro-inflammatory cytokines was analyzed using quantitative RT-PCR. Total mRNA was isolated from the inflamed colon of wild-type and II9-deficient mice (n= 8-21) followed by qPCR analysis. Data were normalized to the house keeping gene 18sRNA. Significant differences are indicated. (b) Expression of T cell transcription factors was analyzed using quantitative RT-PCR. Data were normalized to the house keeping gene 18sRNA. Levels of Gata3, Irf4, Tbet, Foxp3, Rorγt and Spi1 were analyzed in II9 knockout mice as compared to wild-type animals (n= 14-18). (c) Immunofluorescence staining of PU.1 was performed on cryosections from colonic tissue of wildtype and II9 knockout mice. Cell nuclei were counterstained with HOECHST dye. Representative stainings are shown. In addition, double staining with CD4 was performed and significant changes are indicated.

Supplementary Figure 5 A role for IL9 in adoptive T cell transfer

Syngenic SCID mice were reconstituted with wild-type or II9-deficient T cells followed by analysis of colitis activity. High resolution endoscopy was performed at day 42 to analyze colitis activity (n=9). Representative histological pictures at the end of the experiment are shown as well as histological scoring of colitis activity. Significant differences are indicated. In addition, representative stainings of in vivo imaging with IVIS analysis are shown. Data are representative of two independent experiments.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–5 and Supplementary Table 1 (PDF 769 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Gerlach, K., Hwang, Y., Nikolaev, A. et al. TH9 cells that express the transcription factor PU.1 drive T cell–mediated colitis via IL-9 receptor signaling in intestinal epithelial cells. Nat Immunol 15, 676–686 (2014). https://doi.org/10.1038/ni.2920

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ni.2920

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing
  NODES
design 1
eth 6
News 3
orte 8
see 1
twitter 1