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:

Itch inhibits IL-17-mediated colon inflammation and tumorigenesis by ROR-γt ubiquitination

Abstract

Dysregulated expression of interleukin 17 (IL-17) in the colonic mucosa is associated with colonic inflammation and cancer. However, the cell-intrinsic molecular mechanisms by which IL-17 expression is regulated remain unclear. We found that deficiency in the ubiquitin ligase Itch led to spontaneous colitis and increased susceptibility to colon cancer. Itch deficiency in the TH17 subset of helper T cells, innate lymphoid cells and γδ T cells resulted in the production of elevated amounts of IL-17 in the colonic mucosa. Mechanistically, Itch bound to the transcription factor ROR-γt and targeted ROR-γt for ubiquitination. Inhibition or genetic inactivation of ROR-γt attenuated IL-17 expression and reduced spontaneous colonic inflammation in Itch−/− mice. Thus, we have identified a previously unknown role for Itch in regulating IL-17-mediated colonic inflammation and carcinogenesis.

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

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

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

Figure 1: Spontaneous colitis and increased colitis-associated colorectal cancer in Itch−/− mice.
Figure 2: Elevated levels of IL-17 and ROR-γt in the colonic mucosa and tumors of Itch−/− mice.
Figure 3: Itch binds directly to ROR-γt.
Figure 4: Itch targets ROR-γt for predominantly K48-linked ubiquitination and promotes its degradation.
Figure 5: Inhibition of ROR-γt attenuates IL-17 expression in Itch−/− cells.
Figure 6: The ligase activity of Itch and the Itch-ROR-γt interaction are essential for inhibition of IL-17 expression.
Figure 7: Itch−/− CD45RBhi cells induce increased colitis in Rag1−/− mice.
Figure 8: Itch−/−Rorc−/− mice show reduced colon inflammation.

Similar content being viewed by others

Accession codes

Primary accessions

Gene Expression Omnibus

References

  1. Elinav, E. et al. Inflammation-induced cancer: crosstalk between tumors, immune cells and microorganisms. Nat. Rev. Cancer 13, 759–771 (2013).

    Article  CAS  PubMed  Google Scholar 

  2. Siegel, R.L., Miller, K.D. & Jemal, A. Cancer statistics, 2015. CA Cancer J. Clin. 65, 5–29 (2015).

    Article  PubMed  Google Scholar 

  3. Terzic, J., Grivennikov, S., Karin, E. & Karin, M. Inflammation and colon cancer. Gastroenterology 138, 2101–2114.e5 (2010).

    Article  CAS  PubMed  Google Scholar 

  4. Grivennikov, S.I. et al. Adenoma-linked barrier defects and microbial products drive IL-23/IL-17-mediated tumour growth. Nature 491, 254–258 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Hyun, Y.S. et al. Role of IL-17A in the development of colitis-associated cancer. Carcinogenesis 33, 931–936 (2012).

    Article  CAS  PubMed  Google Scholar 

  6. Wu, S. et al. A human colonic commensal promotes colon tumorigenesis via activation of T helper type 17 T cell responses. Nat. Med. 15, 1016–1022 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Wu, P. et al. γδT17 cells promote the accumulation and expansion of myeloid-derived suppressor cells in human colorectal cancer. Immunity 40, 785–800 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Ivanov, I.I. et al. Induction of intestinal Th17 cells by segmented filamentous bacteria. Cell 139, 485–498 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Pearson, C., Uhlig, H.H. & Powrie, F. Lymphoid microenvironments and innate lymphoid cells in the gut. Trends Immunol. 33, 289–296 (2012).

    Article  CAS  PubMed  Google Scholar 

  10. Dong, C. TH17 cells in development: an updated view of their molecular identity and genetic programming. Nat. Rev. Immunol. 8, 337–348 (2008).

    Article  CAS  PubMed  Google Scholar 

  11. Honda, K. & Littman, D.R. The microbiome in infectious disease and inflammation. Annu. Rev. Immunol. 30, 759–795 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Zhang, F., Meng, G. & Strober, W. Interactions among the transcription factors Runx1, RORγt and Foxp3 regulate the differentiation of interleukin-17-producing T cells. Nat. Immunol. 9, 1297–1306 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Korn, T., Bettelli, E., Oukka, M. & Kuchroo, V.K. IL-17 and Th17 Cells. Annu. Rev. Immunol. 27, 485–517 (2009).

    Article  CAS  PubMed  Google Scholar 

  14. Hershko, A. & Ciechanover, A. The ubiquitin system. Annu. Rev. Biochem. 67, 425–479 (1998).

    Article  CAS  PubMed  Google Scholar 

  15. Venuprasad, K., Zeng, M., Baughan, S.L. & Massoumi, R. Multifaceted role of the ubiquitin ligase Itch in immune regulation. Immunol. Cell Biol. 93, 452–460 (2015).

    Article  CAS  PubMed  Google Scholar 

  16. Lohr, N.J. et al. Human ITCH E3 ubiquitin ligase deficiency causes syndromic multisystem autoimmune disease. Am. J. Hum. Genet. 86, 447–453 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Garrett, W.S. et al. Communicable ulcerative colitis induced by T-bet deficiency in the innate immune system. Cell 131, 33–45 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Zaki, M.H. et al. The NOD-like receptor NLRP12 attenuates colon inflammation and tumorigenesis. Cancer Cell 20, 649–660 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Fang, D. et al. Dysregulation of T lymphocyte function in itchy mice: a role for Itch in TH2 differentiation. Nat. Immunol. 3, 281–287 (2002).

    Article  CAS  PubMed  Google Scholar 

  20. Ivanov, I.I. et al. The orphan nuclear receptor RORγt directs the differentiation program of proinflammatory IL-17+ T helper cells. Cell 126, 1121–1133 (2006).

    Article  CAS  PubMed  Google Scholar 

  21. Powell, N. et al. The transcription factor T-bet regulates intestinal inflammation mediated by interleukin-7 receptor+ innate lymphoid cells. Immunity 37, 674–684 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Beal, A.M., Ramos-Hernández, N., Riling, C.R., Nowelsky, E.A. & Oliver, P.M. TGF-β induces the expression of the adaptor Ndfip1 to silence IL-4 production during iTreg cell differentiation. Nat. Immunol. 13, 77–85 (2012).

    Article  CAS  Google Scholar 

  23. Venuprasad, K. et al. The E3 ubiquitin ligase Itch regulates expression of transcription factor Foxp3 and airway inflammation by enhancing the function of transcription factor TIEG1. Nat. Immunol. 9, 245–253 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Theivanthiran, B. et al. The E3 ubiquitin ligase Itch inhibits p38α signaling and skin inflammation through the ubiquitylation of Tab1. Sci. Signal. 8, ra22 (2015).

    Article  CAS  PubMed  Google Scholar 

  25. Tosolini, M. et al. Clinical impact of different classes of infiltrating T cytotoxic and helper cells (Th1, Th2, Treg, Th17) in patients with colorectal cancer. Cancer Res. 71, 1263–1271 (2011).

    Article  CAS  PubMed  Google Scholar 

  26. Bettelli, E. et al. Reciprocal developmental pathways for the generation of pathogenic effector TH17 and regulatory T cells. Nature 441, 235–238 (2006).

    Article  CAS  PubMed  Google Scholar 

  27. Wang, X. et al. Transcription of Il17 and Il17f is controlled by conserved noncoding sequence 2. Immunity 36, 23–31 (2012).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  28. Ahmed, N. et al. The E3 ligase Itch and deubiquitinase Cyld act together to regulate Tak1 and inflammation. Nat. Immunol. 12, 1176–1183 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Solt, L.A. et al. Suppression of TH17 differentiation and autoimmunity by a synthetic ROR ligand. Nature 472, 491–494 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Sarra, M., Pallone, F., Macdonald, T.T. & Monteleone, G. IL-23/IL-17 axis in IBD. Inflamm. Bowel Dis. 16, 1808–1813 (2010).

    Article  PubMed  Google Scholar 

  31. Medzhitov, R. Recognition of microorganisms and activation of the immune response. Nature 449, 819–826 (2007).

    Article  CAS  PubMed  Google Scholar 

  32. Littman, D.R. & Rudensky, A.Y. Th17 and regulatory T cells in mediating and restraining inflammation. Cell 140, 845–858 (2010).

    Article  CAS  PubMed  Google Scholar 

  33. Chae, W.J. et al. Ablation of IL-17A abrogates progression of spontaneous intestinal tumorigenesis. Proc. Natl. Acad. Sci. USA 107, 5540–5544 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Wang, K. et al. Interleukin-17 receptor a signaling in transformed enterocytes promotes early colorectal tumorigenesis. Immunity 41, 1052–1063 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Paul, S., Kashyap, A.K., Jia, W., He, Y.W. & Schaefer, B.C. Selective autophagy of the adaptor protein Bcl10 modulates T cell receptor activation of NF-κB. Immunity 36, 947–958 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Linares, J.F. et al. K63 polyubiquitination and activation of mTOR by the p62-TRAF6 complex in nutrient-activated cells. Mol. Cell 51, 283–296 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Deng, L. et al. The ubiquitination of rag A GTPase by RNF152 negatively regulates mTORC1 activation. Mol. Cell 58, 804–818 (2015).

    Article  CAS  PubMed  Google Scholar 

  38. Huang, F. et al. Lysine 63-linked polyubiquitination is required for EGF receptor degradation. Proc. Natl. Acad. Sci. USA 110, 15722–15727 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Ben-Saadon, R., Zaaroor, D., Ziv, T. & Ciechanover, A. The polycomb protein Ring1B generates self atypical mixed ubiquitin chains required for its in vitro histone H2A ligase activity. Mol. Cell 24, 701–711 (2006).

    Article  CAS  PubMed  Google Scholar 

  40. Nakasone, M.A., Livnat-Levanon, N., Glickman, M.H., Cohen, R.E. & Fushman, D. Mixed-linkage ubiquitin chains send mixed messages. Structure 21, 727–740 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Rutz, S. et al. Deubiquitinase DUBA is a post-translational brake on interleukin-17 production in T cells. Nature 518, 417–421 (2015).

    Article  CAS  PubMed  Google Scholar 

  42. Han, L. et al. The E3 deubiquitinase USP17 is a positive regulator of retinoic acid-related orphan nuclear receptor γt (RORγt) in Th17 cells. J. Biol. Chem. 289, 25546–25555 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Shembade, N. et al. The E3 ligase Itch negatively regulates inflammatory signaling pathways by controlling the function of the ubiquitin-editing enzyme A20. Nat. Immunol. 9, 254–262 (2008).

    Article  CAS  PubMed  Google Scholar 

  44. Xiao, N. et al. The E3 ubiquitin ligase Itch is required for the differentiation of follicular helper T cells. Nat. Immunol. 15, 657–666 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Jin, H.S., Park, Y., Elly, C. & Liu, Y.C. Itch expression by Treg cells controls Th2 inflammatory responses. J. Clin. Invest. 123, 4923–4934 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Song, C. et al. Unique and redundant functions of NKp46+ ILC3s in models of intestinal inflammation. J. Exp. Med. 212, 1869–1882 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank M. Ramsay, S. Oh and A. Theiss for discussions; C. Harrod for critical reading of the manuscript; and K. Kayembe for help with the analysis of flow cytometry data. Supported by the American Cancer Society (122713-RSG-12-260-01-LIB), the Cancer Prevention Research Institute of Texas (RP160577) and the Baylor Charles A. Sammons Cancer Center (K.V.).

Author information

Authors and Affiliations

Authors

Contributions

M.K., P.K. and M.Z. performed the experiments, analyzed the data and helped to prepare the manuscript. B.C. assisted with analysis of RNA-seq data. H.Z. assisted with histopathological analysis. H.U. helped to prepare the manuscript. K.V. conceived the project, designed the experiments and wrote the manuscript.

Corresponding author

Correspondence to K Venuprasad.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Spontaneous gastrointestinal inflammation in aged Itch−/− mice.

(a) Representative image of rectal prolapse in around 8-month-old Itch−/– mice. (b) Body weight. (c) Weight-to-length ratios of colons. (d) H&E-stained sections of the small intestine of Itch+/+ and Itch−/− mice. Scale bars, 100 μm. Data are from one experiment representative of three independent experiments with similar results. Data represent means ± SD. *P<0.01 (Student’s t-test (b,c)).

Supplementary Figure 2 Itch−/− mice are hyper-susceptible to DSS-induced colitis.

(a) Itch+/+ (n=10) and Itch−/− (n=10) mice given 3% DSS solution for 7 days. Survival was monitored. (b-d) Itch+/+ (n=10) and Itch−/− (n=10) mice given 2.5% DSS solution for 5 days followed by 3 days of water. Mice were sacrificed on day 8. Body weight, FOB score and diarrhea score were scored daily. (e) Weight-to-length ratios of colons. (f-g) Size of the spleen and the MLNs of the DSS-treated Itch+/+ and Itch−/− mice. (h-i) H&E-stained sections of Itch+/+ and Itch−/− colons and histology scores. Scale bars, 100 μm. (j) Colonic LPLs were isolated from Itch+/+ and Itch−/− mice after DSS treatment and stained with antibodies against CD11b and Gr1, followed by flow cytometry analysis. Data are from one experiment representative of three independent experiments with similar results. Data represent means ± SD. *P<0.01 (Student’s t-test (b-e, i)).

Supplementary Figure 3 Colitis-associated colorectal tumorigenesis in Itch−/− mice.

(a) Itch+/+ (n=10) and Itch−/− (n=10) mice were injected with 10 mg/kg body weight. On day 10, mice were given 1.5% DSS solution for 5 days followed by 2 weeks of water. This cycle was repeated twice, and mice were sacrificed on day 85. (b) Weight change was measured during the entire course of experiment. (c) Representative gross appearance of colon tumors of Itch+/+ and Itch−/− mice at the end of the experiment. (d-e) H&E-stained sections were scored for inflammation, ulceration, hyperplasia and inflamed area. Scale bars, 100 μm. Data are from one experiment representative of three independent experiments with similar results. Data represent means ± SD. *P<0.01 (Student’s t-test (d,e)).

Supplementary Figure 4 Expression of IL-22 and frequency of TH2 and TH17 cells in peripheral organs in Itch−/− mice.

(a) RNA isolated from the colons of Itch+/+ and Itch−/−mice given DSS was assayed for expression of IL-22. (b-c) Cells from spleen and MLN of Itch+/+ and Itch−/− mice were stained with antibodies against CD4, IL-4, and IL-17 and analyzed by flow cytometry. Data are from one experiment representative of three independent experiments with similar results. NS, not significant.

Supplementary Figure 5 Increased expression of chemokines and MMPs in colonic mucosa of Itch−/− mice treated with DSS.

(a) Heat map of chemokines and MMPs from RNAseq analysis of Itch+/+ and Itch−/− mice treated with DSS. (b-c) RNA was isolated from colonic lamina propria of Itch+/+ and Itch−/− mice given DSS and assayed for expression of chemokines and MMPs. Data are from one experiment representative of three independent experiments with similar results. Data represent means ± SD., *P<0.01 (Student’s t-test (b,c)).

Supplementary Figure 6 Quantification of Foxp3+ cells and expression of Il17 and ROR-γt in Itch+/+ and Itch−/− mice.

(a) Expression of Foxp3 in CD4 T cells from cLPLs of Itch+/+ and Itch−/− mice (b) Expression of Foxp3 in CD4 T cells from spleen and MLN of Itch+/+ and Itch−/− mice. (c) Relative mRNA expression of Il17a and Il17f in Itch+/+ and Itch−/– skin. (d) Relative mRNA and protein levels of ROR-γt in CD4 T cells kept under TH17 polarizing conditions. Data are from one experiment representative of three independent experiments with similar results. Data represent means ± SD. NS, not significant.

Supplementary Figure 7 Inhibition of ROR-γt attenuates spontaneous colon inflammation in Itch−/− mice.

6-8-month-old Itch+/+ (n=6) and Itch−/− (n=6) mice were treated with SR1001 for four weeks, and the following parameters were assayed at end of the experiment. (a) Body weight. (b) Diarrhea score. (c) Weight-to-length ratio. (d) H&E-stained sections of Itch+/+ and Itch−/− colons treated with SR1001. Scale bars, 100 μm. (e) Histology score. (f) RNA isolated from cLPLs of Itch+/+ and Itch−/− mice treated with SR1001 was assayed for expression of Il17a and Il17f by real-time PCR. (g) IL-17 was measured in explant cultures of colons from Itch+/+ and Itch−/− mice treated with SR1001 by ELISA. (h-i) Expression of chemokines and MMPs. Data are from one experiment representative of three independent experiments with similar results. Data represent means ± SD. *P<0.01 (Student’s t-test (a-c,e-i)).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–7 (PDF 1456 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kathania, M., Khare, P., Zeng, M. et al. Itch inhibits IL-17-mediated colon inflammation and tumorigenesis by ROR-γt ubiquitination. Nat Immunol 17, 997–1004 (2016). https://doi.org/10.1038/ni.3488

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ni.3488

This article is cited by

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