Inflammatory bowel diseases (IBDs: Crohn s disease,

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1 GASTROENTEROLOGY 2009;136: ROR -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, and MARKUS F. NEURATH*,# *Institute of Molecular Medicine, University of Mainz, Mainz, Germany; Howard Hughes Medical Institute and The Kimmel Center for Biology and Medicine at the Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, New York; Merck Serono International, Geneva, Switzerland; Regeneron Pharmaceuticals, Tarrytown, New York; Neuroimmunology Unit, Department of Neurology, University Hospital Zurich, Zurich, Switzerland; and # I. Medical Clinic, University of Mainz, Mainz, Germany Background and Aims: IL-17 producing CD4 T- helper cells (Th17) contribute to chronic autoimmune inflammation in the brain, and levels of Th17-derived cytokines increase in patients with colitis, suggesting a role in pathogenesis. We analyzed the roles of Th17 cells and the transcription factor retinoic acid receptor-related organ receptor (ROR), which regulates Th17 differentiation, in chronic intestinal inflammation. Methods: Using an adoptive transfer model of colitis, we compared the colitogenic potential of wildtype, interleukin-17a (IL-17A), IL-17F, IL-22, and ROR -deficient CD4 CD25 T cells in RAG1-null mice. Results: Adoptive transfer of IL-17A, IL-17F, or IL-22 deficient T lymphocytes into RAG1-null mice caused severe colitis that was indistinguishable from that caused by wild-type cells. In contrast, transfer of ROR -null T cells failed to increase mucosal IL-17 cytokine levels and did not induce colitis. Treatment with IL-17A was able to restore colitis after transfer of ROR -null T cells, indicating a crucial role for Th17 cells in pathogenesis. Treatment of RAG1 mice that received IL-17F null (but not wild-type) T cells with a neutralizing anti IL-17A antibody significantly suppressed disease, indicating redundant biological effects of IL-17A and IL-17F. Conclusions: We have identified a crucial role of ROR -expressing Th17 cells in chronic intestinal inflammation. ROR controls IL-17A and IL-17F production, and these cytokines have a redundant but highly pathogenic role in gut inflammation. Reagents that target ROR or a combination of anti IL-17A and anti IL-17F might be developed as therapeutics for chronic colitis. Inflammatory bowel diseases (IBDs: Crohn s disease, ulcerative colitis) are characterized by chronic relapsing inflammations of the gastrointestinal tract that are not caused by specific pathogens. Although the precise etiology of IBDs remains unclear, recent studies have suggested a key pathogenic role of barrier dysfunction with consecutive immune dysregulation and enhanced mucosal T-cell responses. 1 While Crohn s disease has been associated with a T-helper 1 (Th1) T-cell cytokine profile, T cells in ulcerative colitis produce some Th2- type cytokines such as interleukin-5 (IL-5) and IL Previous concepts on the role of T-helper cells in chronic inflammatory and autoimmune diseases have been challenged by the description of a novel T-cell subset characterized by the production of IL These Th17 cells have been described as producers of a distinct cytokine pattern, especially IL-17A, IL-17F, IL-22, and, to a lesser extent, tumor necrosis factor and IL-6. 4 The differentiation program of Th17 cells in vitro is controlled by synergistic effects of transforming growth factor (TGF ) and IL-6 mediated IL-21 expression, whereas stabilization of the Th17 phenotype has been shown to be achieved via IL-23 receptor (IL-23R) signaling. 5,6 Importantly, retinoic acid receptor-related organ receptor (ROR) t has been recently identified as the master transcription factor guiding Th17 differentiation, while at the same time synergizing with other transcription factors such as signal transducer and activator of transcription 3, ROR, and interferon regulatory factor Interestingly, Th17 cells are closely linked to Foxp3 T-regulatory cells, as it has been shown that both cell types require TGF for in vitro differentiation and that Foxp3 controls Th17 differentiation by direct interaction with ROR t. 11 Single nucleotide polymorphisms in the IL23r gene modulate the susceptibility to IBDs. 12 In addition, Th17- derived cytokines are augmented in IBD patients, indicating a potential role of these cells in disease pathogenesis. 13,14 However, the role of Th17 cells in chronic mucosal inflammation is still incompletely understood. Abbreviations used in this paper: ELISA, enzyme-linked immunosorbent assay; FACS, fluorescence-activated cell sorter; IBD, inflammatory bowel disease; IFN, interferon; IL, interleukin; MPO, myeloperoxidase; ROR, retinoic acid receptor-related orphan receptor; TGF, transforming growth factor; Th, T helper by the AGA Institute /09/$36.00 doi: /j.gastro

2 258 LEPPKES ET AL GASTROENTEROLOGY Vol. 136, No. 1 Therefore, we analyzed in this study the role of Th17 cells in the pathogenesis of chronic intestinal inflammation in vivo. Materials and Methods Mice IL-17A /, IL-22 /, and IL-17F / mice were provided by Regeneron Pharmaceuticals (Tarrytown, NY) and Merck Serono (Geneva, Switzerland), respectively. All mice were on a C57BL/6 background. Wild-type littermates were used as controls. RAG1 / mice, aged 6 to 12 weeks, were obtained from the animal facility of the University of Mainz. All mice were kept under specific pathogen-free conditions, and all experiments were performed in agreement with local regulations. Isolation and Culture of Primary Cells CD4 CD25 T cells were isolated from murine splenocytes by MACS separation (CD4 T Cell Isolation Kit, CD25-PE Kit, Miltenyi Biotec, Bergisch Gladbach, Germany) to a purity degree 95%, as evaluated by fluorescence-activated cell sorter (FACS) analysis. RPMI 1640 (Gibco Invitrogen, Karlsruhe, Germany) supplemented with 10% fetal bovine serum (Perbio Science, Bonn, Germany) and penicillin/streptomycin (Biochrom, Berlin, Germany) was used for all cell isolation procedures. Adoptive Transfer Colitis Freshly isolated CD4 CD25 T cells ( ) from different mouse strains (v.s.) were administered intraperitoneally into RAG / mice in 200 L of phosphate-buffered saline (PBS; Gibco Invitrogen). The COLOVIEW high-resolution mouse video endoscopic system (Karl Storz, Tuttlingen, Germany) was used as previously described. 15 In some experiments, IL-17A was neutralized by intraperitoneal administration of 100 g of a monoclonal antibody (clone 50104, R&D Systems, Wiesbaden, Germany) every other day. 16 Recombinant murine IL-17A (ImmunoTools, Friesoythe, Germany) was injected intraperitoneally 3 times a week at a dose of 0.5 g per mouse. Isolation of Lamina Propria Mononuclear Cells Murine lamina propria mononuclear cells were isolated as previously described. 17,18 In brief, colonic tissue was mechanically dissected, and intestinal epithelial cells were removed by incubation in EDTA. Remaining tissue was digested in collagenase D (Roche Diagnostics, Mannheim, Germany), DNase I (Sigma Aldrich, Munich, Germany), and dispase II (Roche Diagnostics). Digested tissue was passed through a 40- m cell strainer, and the remaining cellular content was separated from debris using a 40/80 Percoll gradient (Sigma Aldrich). Flow Cytometry Analysis With the use of specific antibodies, cells were stained for CD4, CD25, CD62L, and CD69 (BD Biosciences, Heidelberg, Germany) expression, according to the manufacturer s instructions. Intracellular labeling for IL-17 (clone TC11 18H10.1, BioLegend, San Diego, CA) and interferon (IFN ; clone XMG1.2, Caltag Invitrogen, Karlsruhe, Germany) was performed with phycoerythrin- or fluorescein isothiocyanate conjugated rat antimouse antibodies. Cells were stimulated with phorbol myristate acetate (50 ng/ml; Calbiochem Merck, Darmstadt, Germany), ionomycin (500 ng/ml; Sigma Aldrich), and brefeldin A (5 g/ml; Sigma Aldrich) before staining, and Fix and Perm Cell Permeabilization reagents (Caltag Invitrogen) were used according to the manufacturer s instructions after staining of surface antigens. Cells were analyzed by means of a FACSCalibur Flow Cytometer (BD Biosciences). Cytokine Quantification Supernatants of reisolated CD4 T cells cultured for 48 hours and stimulated with plate-bound anti-cd3 (10 g/ml, clone 145-2C11) and soluble anti-cd28 (2 g/ml, clone 37.51) antibodies were analyzed for cytokines by Flow- Cytomix assays according to the manufacturer s protocol (Bender MedSystems, Vienna, Austria). IL-17A and IL-17F were measured using enzyme-linked immunosorbent assay kits (ELISA Kits; ebioscience, San Diego, CA). Galactosidase Assay The Galactosidase Reporter Gene Assay Kit (Roche Applied Science, Mannheim, Germany) was used according to the manufacturer s protocol. Immunohistochemistry Immunofluorescence of cryosections was performed using the TSA Cy3 system (PerkinElmer, Waltham, MA) and a fluorescence microscope (Olympus, Hamburg, Germany) using primary antibodies specific for CD4 (Santa Cruz Biotechnology, Heidelberg, Germany), CD11c, and myeloperoxidase (MPO, BD Biosciences). Before examination, the nuclei were counterstained with Hoechst 3342 (Invitrogen Molecular Probes, Karlsruhe, Germany). Statistical Analysis Data were analyzed by the unpaired Student t test using Microsoft Excel (Microsoft, Redmond, WA) or an analysis of variance with post hoc Tukey HSD tests, as indicated, using SPSS software (SPSS Inc, Chicago, IL). Results T Cell Dependent Experimental Colitis Is Not Affected by IL-17A Deficiency To address the role of Th17 cells in the pathogenesis of colitis, we took advantage of an adoptive transfer

3 January 2009 ROR AND TH17 CELLS IN MURINE COLITIS 259 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) galactosidase 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-17A lacz/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 g/ml) and soluble anti-cd28 (2 g/ml) antibodies. ELISA was performed using supernatants from 10 6 cells/ml. Data represent mean values SEM (5 mice per group). model, in which CD4 CD25 T cells are introduced into T cell and B cell deficient RAG1 / hosts (Figure 1A). The adoptive transfer model of colitis is characterized by a severe T cell dependent colitis with mucosal infiltration of immune cells, chronic diarrhea, and weight loss. 19 We hypothesized that Th17 cells and their hallmark cytokine IL-17A might play an important role in the pathogenesis of such T cell dependent colitis, as T-cell

4 260 LEPPKES ET AL GASTROENTEROLOGY Vol. 136, No. 1 transfer from IL-17A LacZ/LacZ reporter mice revealed IL- 17A promoter activity at the site of chronic colitis (Figure 1B). To further explore this hypothesis, we compared the colitogenic potential of wild-type and IL-17A deficient CD4 CD25 T cells. Surprisingly, adoptive transfer of IL-17A / CD4 CD25 T cells induced severe colitis in RAG1 / mice that was indistinguishable from mice given wild-type control T cells. Both experimental groups were prone to chronic diarrhea and displayed similar endoscopic and histologic features of disease activity (Figure 1C and D). Mesenteric lymph node cultures of transferred RAG1 / mice consistently showed a lack of IL-17A production in the IL-17A deficient T-cell transfer group. However, while the level of IFN was comparable in both groups, we noticed an unexpected increase of IL-17F production in mesenteric lymph node cultures of IL-17A / T cell reconstituted RAG1 / mice (Figure 1E). Thus, although IL-17A deficiency may play a protective role in other experimental models of chronic inflammation such as in experimental autoimmune encephalomyelitis, 20 our data indicate that IL-17A production is not essential for the colitogenic potential of CD4 CD25 T cells. Expression of the Transcription Factor ROR in CD4 CD25 T Cells Is Essential for the Onset of Experimental Colitis To investigate whether Th17 cells rather than their effector cytokine IL-17A may play a role in inducing T cell dependent colitis, we took advantage of ROR / mice, which are defective for generation of Th17 cells while showing normal Th1 T-cell differentiation. 8 Remarkably, CD4 CD25 T cells isolated from ROR -deficient mice did not induce significant colitis activity upon transfer in RAG1 / mice. In fact, RAG1 / mice given wild-type T cells developed wasting disease (Figure 2A) and endoscopic signs of colitis (Figure 2B), whereas mice reconstituted with ROR / T cells showed no diarrhea, gained weight, and had a normal vascular architecture of the mucosal surface. Histopathologic assessment revealed a decreased inflammatory cell infiltration and tissue destruction after ROR / T-cell transfer as compared with wild-type T-cell transfer (Figure 2C). Thus, ROR, the key transcriptional regulator for Th17 cell differentiation, is essential for the pathogenesis of colitis induced by CD4 CD25 T cell transfer. ROR / T-Cell Transfer Leads to Diminished Infiltration of Dendritic Cells and Neutrophils Into the Lamina Propria and Results in Differential Cytokine Expression ROR / CD4 cells repopulated RAG1 / mice as efficiently as wild-type cells and were able to migrate to the mesenteric lymph node in a comparable manner, as shown by FACS analysis of reisolated T cells (Figure 3A). Immunohistochemical staining demonstrated a strong Figure 2. ROR expression in CD4 CD25 T cells is essential for the onset of experimental colitis. (A) To assess the function of the Th17 transcription factor ROR for colitis activity in vivo, adoptive transfer of CD4 CD25 T cells from spleens of wild-type (WT) and ROR / mice into RAG1 / hosts was performed. Whereas transfer of wild-type T cells led to severe colitis and weight loss, ROR / 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.

5 January 2009 ROR AND TH17 CELLS IN MURINE COLITIS 261 Figure 3. ROR / 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 ROR / 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 (ROR / vs wild-type reconstituted groups, phosphate-buffered saline (PBS)-injected unreconstituted controls). ROR / 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 ROR / 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.

6 262 LEPPKES ET AL GASTROENTEROLOGY Vol. 136, No. 1 infiltration of CD4 T cells into the lamina propria in both groups, whereas a marked reduction of dendritic cells and neutrophils could be observed in ROR / T cell transferred animals as compared with mice given wild-type cells (Figure 3B). To investigate the phenotype of in vivo transferred ROR -deficient T cells in experimental colitis, we next performed ELISA and FACS analysis of wild-type and ROR -deficient T cells isolated from reconstituted RAG1 / mice. No difference was found in the expression of activation markers (CD62L, CD69, and CD25) between adoptively transferred wild-type and ROR deficient T cells (Supplementary Figure 1A; see online at suggesting that ROR deficient T cells had been stimulated in vivo but were unable to elicit colitis. Furthermore, ROR / CD4 T cells produced similar amounts of IL-2, IL-4, and IL-10 as wild-type T cells. However, IL-6, IL-17, granulocyte-macrophage colony stimulating factor, and IFN production by T cells was significantly reduced in the absence of ROR (Figure 3C). FACS analysis revealed a lack of IL- 17 producing T cells in draining mesenterial lymph nodes of ROR / T cell reconstituted mice, while a similar amount of IFN producing cells was found as compared to wild-type controls (Supplementary Figure 1B; see online at Finally, culture of lamina propria mononuclear cells and mesenteric lymph node cells revealed reduced amounts of IL-17A and IL-17F at the site of inflammation (Supplementary Figure 1C and D; see online at Because of the close relation between Th17 cells and Foxp3 T-regulatory cells, we also determined the number of these cells in both experimental groups. Although a slight increase in relative fractions of CD4 Foxp3 T cells could be observed in ROR / T cell reconstituted mice, absolute numbers did not show significant differences (Supplementary Figure 2A; see online at www. gastrojournal.org). In addition, wild-type and ROR / CD4 T cells showed similar responses to TGF -enforced Foxp3 induction and IL-6 mediated suppression in vitro (Supplementary Figure 2B; see online at www. gastrojournal.org). 21 Taken together, these data suggest that expression of ROR in colitogenic T cells controls mucosal cytokine production and is required for the development of experimental T cell dependent colitis. 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 miniendoscopy 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.

7 January 2009 ROR AND TH17 CELLS IN MURINE COLITIS 263 IL-17F or IL-22 Deficient T Cells Can Still Cause Disease Activity in Experimental Colitis The above data demonstrate that although Th17 cells are important for development of disease, IL-17A production is not. However, Th17 cells produce a number of other effector cytokines in addition to IL-17A, such as IL-17F and IL-22. We thus analyzed the effects of IL-17F in the adoptive transfer model of colitis using T cells from IL-17F deficient animals. Similar to IL-17A, IL-17F production was dispensable for the colitogenic potential of CD4 CD25 T cells, as indicated by endoscopic and histologic criteria of disease (Figure 4A and B). Consistently, no marked differences were observed between the wild-type and IL-17F / groups with regard to the infiltration of T cells, granulocytes, and dendritic cells (Supplementary Figure 3; see online at www. gastrojournal.org). A subpopulation of Th17 cells was recently shown to produce IL-22 in addition to IL-17A and IL-17F. We therefore analyzed wild-type and IL-22 / CD4 CD25 T cells in the adoptive transfer model of colitis. Again, no differences in colitis development were found between the wild-type and IL-22 / groups (Figure 4C and D). Thus, our data demonstrate that T cells cannot be inhibited from causing disease in the adoptive transfer model of colitis by single depletion of either IL-17A, IL-17F, or IL-22. 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 wildtype 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.

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9 January 2009 ROR AND TH17 CELLS IN MURINE COLITIS 265 Redundant Pathologic Effects of IL-17A and IL-17F in Experimental Colitis Since none of the Th17-related cytokines alone were essential for colitis development, we hypothesized that redundant cytokine effects might account for the pathogenicity of ROR -expressing Th17 cells in colitis. As IL-17A and IL-17F have been shown to signal via the same heteromeric receptor complex and exert similar effector functions in vitro, 22 we investigated whether these cytokines might play a redundant role in the induction of colitis in vivo. Consistently, increased amounts of IL-17F were found in mesenteric lymph nodes of IL-17A / T cell reconstituted mice (Figure 1E), suggesting an induction of IL-17F production in the absence of IL-17A. To suppress the activity of IL-17A in the absence of IL-17F, we injected a neutralizing IL-17A antibody into RAG1 / mice given IL-17F / T cells. Significant protection was observed after this treatment as demonstrated by histopathologic and endoscopic analysis (Figure 5A and B). Immunohistochemical staining of the colon wall revealed a marked infiltration with CD4 T cells in both groups, whereas diminished numbers of dendritic cells and neutrophils were noted in the colon of anti IL-17A antibody-treated, IL-17F / T cell reconstituted mice as compared with control mice (Figure 5C). Importantly, neutralizing IL-17A function in mice given wild-type T cells had no effects on colitis activity, suggesting that the combined absence of IL-17A and IL-17F is required to achieve protection in T cell dependent experimental colitis. Administration of Recombinant IL-17A Induces Colitis After ROR / T-Cell Transfer The above data suggested that ROR -expressing Th17 cells can infiltrate into the colon upon T-cell transfer but lack the capacity to induce colitis because of deficient IL-17A/F production. To further explore the pathogenic function of these cytokines in vivo, we tested whether the colitogenicity of ROR -deficient T cells could be restored by exogenous IL-17A. Remarkably, administration of recombinant murine IL-17A alone was able to induce colitis in ROR / T cell transferred RAG1 / hosts. In contrast to saline-treated ROR / T-cell recipients, IL-17A treated ROR / T-cell recipients developed marked colitis, as determined by endoscopic and histopathologic criteria (Figure 6A and B), and the severity of such colitis was comparable to that seen after reconstitution of RAG1 / mice with wild-type T cells. Finally, immunohistochemical staining revealed that exogenous IL-17A was able to restore infiltration of inflammatory cells and inflammation-induced tissue destruction in experimental colitis in vivo (Figure 6C). Discussion The role of Th17 cells in chronic intestinal inflammation has recently been under debate While the potential relevance of Th17 cells has been highlighted by the finding that Th17 cells accumulate in the mesenteric lymph nodes and colonic lamina propria in T-cell transfer colitis, 24 recent studies demonstrated that IL-17A production by T cells is not essential to induce such chronic T cell dependent colitis. 23,28 However, IL-23, a heterodimeric cytokine that drives Th17 cell activation, has been shown to play a pathogenic role in chronic intestinal inflammation. 25 Specifically, Yen and colleagues 26 described that monoclonal anti IL-23p19 antibodies protect mice from experimental colitis, and this finding was associated with a suppression of both mucosal IL-17 and IL-6 production. Despite these observations, there are few data investigating directly the role of Th17 cell differentiation and T cell derived IL-17 and IL-22 in chronic gut pathology. Although previous studies used IL-23p19 deficient mice to study the role of Th17 cells in models of chronic inflammation, recent data clearly demonstrate an important T cell independent role of IL-23 in driving intestinal inflammation. 25,29 In fact, IL-23p19 / mice were protected from intestinal inflammation in Helicobacter hepaticus infected RAG / mice in the complete absence of T and B cells. Moreover, it was nicely shown that IL-23 restrains T-regulatory cells in chronic colitis, thereby challenging the simplistic view of an IL-23/Th17 axis. 23,29 Taken together, these data did not provide unequivocal evidence for a pathogenic role of Th17 cells in chronic intestinal inflammation. In addition, IL-17 and IL-22 can be produced by non T cells, and analysis of IL-17/IL-22 cytokine knockout mice therefore can not be used to directly determine the role of Th17 cells for colitis development in vivo. Based on the above findings, we aimed in this study at the characterization of the functional role of Th17 cells 4 Figure 6. Recombinant IL-17A induces colitis after ROR / T-cell transfer. (A) Mini-endoscopy of the colonic mucosa was performed in RAG1 / mice reconstituted with wild-type (WT) or ROR -deficient cells followed by treatment of some mice with recombinant IL-17A, as indicated. Endoscopic analysis revealed the onset of colitis in ROR / 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 ROR / T-cell hosts were noted. Data represent mean values SD and are representative of 2 independent experiments (n 4 mice per group).

10 266 LEPPKES ET AL GASTROENTEROLOGY Vol. 136, No. 1 in experimental colitis in vivo. In these studies, we took advantage of an adoptive transfer model of colitis in which donor CD4 CD25 T cells were derived from Th17-related knockout mice in order to directly investigate the role of these factors for the colitogenic potential of T cells. 19 Furthermore, we used T cells from ROR deficient mice to investigate the functional relevance of the Th17 subset as a whole. As ROR -deficient T cells have been shown to display normal Th1 differentiation but profound defects in Th17 differentiation and cytokine production, 8 such cells provided an ideal tool to study the in vivo relevance of Th17 cells in T-cell transfer colitis in B cell and T cell deficient RAG1 / mice. We found that ROR -deficient T cells migrated to the colon but failed to induce mucosal IL-17 production and colitis development upon T-cell transfer in RAG1 / mice. This finding was associated with diminished numbers of both dendritic cells and granulocytes at the site of inflammation in the absence of ROR T-cell signaling potentially due to regulation of chemokine expression by Th17 cells. 27 In any case, our data demonstrate the importance of T cell ROR signaling in chronic colitis in vivo. In additional studies, we observed that neither of the Th17 cell derived cytokines IL-17A and IL-17F alone is required for the development of adoptive transfer colitis. Furthermore, although recombinant IL-22 has been recently shown to protect mice from a Th2 model of colitis in mice resembling human ulcerative colitis, 30 we found that IL-22 deficient T cells are fully capable to induce T cell mediated colitis in the adoptive transfer model, yet not excluding the physiologic importance of this cytokine. The IL-17 cytokine family consists of several members, of which IL-17A and IL-17F are expressed by T cells and display the most homology. 27 We next assessed a potential functional redundancy of both cytokines in experimental colitis. In these studies, we found that functional deficiency of both IL-17A and T cell derived IL-17F by neutralization of IL-17A after IL-17F / T-cell transfer significantly reduces colitis severity. These findings suggested redundant effects of IL-17A and IL-17F in vivo. Although a contribution of the blockade of non T cell IL-17A in this experimental setup cannot be fully excluded, a substantial influence of the neutralizing antibody after wild-type T-cell transfer was not observed. Consistently, administration of recombinant IL-17A alone was sufficient to induce colitis in RAG1 / mice upon transfer of ROR -deficient T cells. Thus, our results indicate a crucial pathogenic but redundant role of IL- 17A and IL-17F in chronic T cell dependent colitis in vivo. Our findings are consistent with data from a recent study by Zhang et al 31 in which IL-17 receptor deficient animals were protected from acute trinitrobenzene sulfonic acid induced colitis. Taken together, these findings suggest a pathogenic role of IL-17 signaling in both acute and chronic T cell dependent colitis. In contrast, neutralization of IL-17A using antibodies in dextran sodium sulfate colitis has been shown to aggravate mucosal inflammation, 16 indicating a potential protective role of IL-17 in this T cell independent colitis model. In summary, we demonstrate a crucial role of ROR expressing Th17 cells in T cell dependent gut inflammation. ROR emerges as a master regulator of Th17 cytokine production in the inflamed gut and appears to play an important role in controlling chronic mucosal inflammation. ROR controls mucosal IL-17A and IL-17F production, and these cytokines play a redundant but highly pathogenic role in chronic intestinal inflammation. As IBDs in humans are characterized by marked T-cell activation and Th17 cytokine production, 13,14,32,33 our data suggest the potential therapeutic utility of combined targeting of IL-17A and IL-17F or of inhibiting ROR activity in chronic colitis such as that observed in Crohn s disease. Supplementary Data Note: To access the supplementary material accompanying this article, visit the online version of Gastroenterology at and at doi: /j.gastro References 1. Strober W, Fuss I, Mannon P. The fundamental basis of inflammatory bowel disease. J Clin Invest 2007;117: Fuss IJ, Heller F, Boirivant M, et al. Nonclassical CD1d-restricted NK T cells that produce IL-13 characterize an atypical Th2 response in ulcerative colitis. J Clin Invest 2004;113: Cua DJ, Sherlock J, Chen Y, et al. Interleukin-23 rather than interleukin-12 is the critical cytokine for autoimmune inflammation of the brain. Nature 2003;421: Liang SC, Tan XY, Luxenberg DP, et al. 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