1. Volume 136, Issue 1, Pages 257-267 (January 2009)
RORγ-Expressing Th17 Cells Induce Murine Chronic Intestinal Inflammation via Redundant Effects of IL-17A and IL-17F 
Moritz Leppkes, Christoph Becker, Ivaylo I. Ivanov, Sebastian Hirth, Stefan Wirtz, Clemens Neufert, Sandrine Pouly, Andrew J. Murphy, David M. Valenzuela, George D. Yancopoulos, Burkhard Becher, Dan R. Littman, Markus F. Neurath 
Gastroenterology 
Volume 136, Issue 1, Pages (January 2009)
DOI: /j.gastro
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2. Figure 1
T cell–dependent experimental colitis is not affected by IL-17A deficiency. (A) To induce T cell–dependent colitis, splenic CD4+CD25− T cells were adoptively transferred into immunocompromised RAG1−/− mice. WT, wild-type; KO, knockout. (B) βgalactosidase assays on colonic tissue from T cell–reconstituted mice based on the activity of the LacZ cassette controlled by the IL-17A promoter in transferred IL-17AlacZ/lacZ donor T cells. Mice reconstituted with wild-type T cells served as negative control. Whole colonic tissue lysates from reconstituted RAG−/− mice were used (n = 4 per group). Mean values ± SD are shown. Induction of transfer colitis resulted in increased IL-17A promoter activity in the colon. Analysis was performed at day 28 posttransfer. (C) Adoptive transfer of CD4+CD25− T cells from spleens of wild-type and IL-17A−/− mice into immunodeficient RAG1−/− hosts led to severe colitis in both experimental groups, as shown by high-resolution mini-endoscopy. Severity of disease was assessed by stool consistency, fibrin covering, translucency, granularity, and vascular architecture of the bowel wall. Data represent mean values ± SEM (5 mice per group). One representative experiment of three is shown. (D) H&E staining and histologic scoring showed no differences between the wild-type and IL-17A−/− groups. (E) Mesenteric lymph node cells were isolated from colitic RAG1−/− mice and subsequently stimulated for 48 hours with plate-bound anti-CD3 (10 μg/mL) and soluble anti-CD28 (2 μg/mL) antibodies. ELISA was performed using supernatants from 106 cells/mL. Data represent mean values ± SEM (5 mice per group).
Gastroenterology  , DOI: ( /j.gastro )
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3. Figure 2
RORγ expression in CD4+CD25− T cells is essential for the onset of experimental colitis. (A) To assess the function of the Th17 transcription factor RORγ for colitis activity in vivo, adoptive transfer of CD4+CD25− T cells from spleens of wild-type (WT) and RORγ−/− mice into RAG1−/− hosts was performed. Whereas transfer of wild-type T cells led to severe colitis and weight loss, RORγ−/− recipients were protected from disease onset. Data represent mean values of the body weight ± SEM (n = 5 per group). One representative experiment of five is shown. KO, knockout. (B and C) High-resolution mini-endoscopy and histology of the colon in both groups. Significant differences between scores in both groups are indicated (Student t test, *P < .05; **P < .01). Scoring was performed as described above.
Gastroenterology  , DOI: ( /j.gastro )
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4. Figure 3
RORγ−/− T-cell transfer leads to diminished infiltration of dendritic cells and neutrophils into the lamina propria and results in differential cytokine expression. (A) Adoptive transfer of CD4+CD25− T cells from spleens of wild-type (WT) and RORγ−/− mice into RAG1−/− hosts was performed. Subsequently, mesenteric lymph node cells and splenocytes isolated from reconstituted RAG−/− mice were used for FACS analysis to determine surface CD4+ expression. Both groups showed a similar fraction of transferred CD4+ T cells in the spleen and at the site of inflammation. KO, knockout. (B) Immunohistochemical staining analysis of the cellular expression of CD4, CD11c, and MPO using colonic cryosections from RAG1−/− hosts (RORγ−/− vs wild-type reconstituted groups, phosphate-buffered saline (PBS)-injected unreconstituted controls). RORγ−/− CD4+ cells could be easily detected, suggesting migration of these cells to the bowel wall. However, markedly diminished numbers of CD11c+ dendritic cells and MPO-expressing neutrophils were noted in the RORγ−/− group as compared with the wild-type group. (C) Cytokine production of adoptively transferred CD4+ T cells reisolated from the spleens of RAG1−/− hosts and consecutively stimulated with anti-CD3/anti-CD28 antibodies under serum-free conditions for 48 hours. Data are derived from a bead-based ELISA assay and represent mean values ± SEM. Supernatants of 5 mice per group were assessed. One representative experiment of two is shown. Statistically significant differences are indicated (Student t test, *P < .05; **P < .01). GM-CSF, granulocyte-macrophage colony–stimulating factor; TNF, tumor necrosis factor.
Gastroenterology  , DOI: ( /j.gastro )
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5. Figure 4
Effects of IL-17F and IL-22 deficiency of T cells in adoptive transfer colitis. (A and C) Adoptive transfer of CD4+CD25− T cells from spleens of wild-type (WT), IL-17F−/−, and IL-22−/− mice into immunodeficient RAG1−/− hosts led to severe colitis in all experimental groups. Endoscopic scoring and high-resolution mini-endoscopy of the colon are shown. Scoring was performed as specified above. Data represent mean values ± SEM (5 mice per group). One representative experiment of two is shown. KO, knockout. (B and D) Histologic analysis showed severe colitis in all 3 groups of mice after T-cell transfer.
Gastroenterology  , DOI: ( /j.gastro )
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6. Figure 5
Neutralization of IL-17A in mice given IL-17F−/− T cells protects from transfer colitis. (A and B) Adoptive transfer of CD4+CD25− T cells from spleens of wild-type (WT) and IL-17F−/− mice into RAG1−/− hosts was performed, and some mice were given neutralizing anti–IL-17A antibodies as indicated. In contrast to mock-treated animals and wild-type T cell–reconstituted mice given anti–IL-17A antibodies, IL-17A–treated RAG1−/− mice reconstituted with IL-17F−/− T cells were significantly protected from colitis, as shown by high-resolution endoscopy (A) and histologic scoring (B). Scoring was performed as described above. Data represent mean values ± SEM (4 mice per group; analysis of variance, Tukey HSD test, *P < .05; **P < .01). A second independent experiment gave similar results. KO, knockout. (C) Immunohistochemical staining analysis of the cellular expression of CD4, CD11c, and MPO using colon cryosections from RAG1−/− mice of indicated groups. IL-17A–treated RAG1−/− mice reconstituted with IL-17F−/− T cells showed a marked reduction in the number of colonic Dendritic Cells expressing CD11c and MPO+ granulocytes.
Gastroenterology  , DOI: ( /j.gastro )
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7. Figure 6
Recombinant IL-17A induces colitis after RORγ−/− T-cell transfer. (A) Mini-endoscopy of the colonic mucosa was performed in RAG1−/− mice reconstituted with wild-type (WT) or RORγ-deficient cells followed by treatment of some mice with recombinant IL-17A, as indicated. Endoscopic analysis revealed the onset of colitis in RORγ−/− T cell–recipient RAG1−/− hosts after treatment with rmIL-17A, whereas saline-treated controls were still protected from adoptive transfer colitis. Control mice given recombinant IL-17A showed no colitis. KO, knockout. (B) Histopathologic scoring underlined the macroscopic effects seen by endoscopy. Scoring was performed as described above (analysis of variance, Tukey HSD test, *P < .05; **P < .01). (C) Immunohistochemical staining analysis of the cellular expression of CD4, CD11c, and MPO using colonic cryosections from adoptively transferred RAG1−/− mice. The diminished infiltration of CD11c+ dendritic cells and MPO+ granulocytes could be reversed by administration of recombinant murine IL-17A. No differences between wild-type and IL-17A–treated RORγ−/− T-cell hosts were noted. Data represent mean values ± SD and are representative of 2 independent experiments (n = 4 mice per group).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2009 AGA Institute Terms and Conditions