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