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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

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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.

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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.

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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.).

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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

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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.

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Correspondence to Markus F Neurath.

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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.

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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

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