Volume 134, Issue 4, Pages 1038-1048.e2 (April 2008) Regulation of Gut Inflammation and Th17 Cell Response by Interleukin-21  Daniele Fina, Massimiliano Sarra, Massimo C. Fantini, Angelamaria Rizzo, Roberta Caruso, Flavio Caprioli, Carmine Stolfi, Iris Cardolini, Marta Dottori, Monica Boirivant, Francesco Pallone, Thomas T. MacDonald, Giovanni Monteleone  Gastroenterology  Volume 134, Issue 4, Pages 1038-1048.e2 (April 2008) DOI: 10.1053/j.gastro.2008.01.041 Copyright © 2008 AGA Institute Terms and Conditions

Figure 1 Induction of DSS-colitis in wild-type mice is associated with enhanced IL-21 synthesis. (A) RNA transcripts for IL-21 were evaluated in freshly obtained colonic specimens of wild-type mice treated with oral DSS for 7 days and controls (CTR) by real-time PCR. Data indicate mean ± SD of 3 separate experiments in which 6 mice per group were analyzed. (B) IL-21 protein was analyzed by ELISA in colonic extracts, and data are expressed as picogram/milligram total protein. Data indicate mean ± SD of 2 separate experiments. In each experiment, 6 mice per group were used. (C) IL-21-deficient mice are largely protected against the development of DSS colitis. Both wild-type (WT) and IL-21 knockout (IL-21 KO) mice were treated with oral DSS for 7 days, and body weight changes were daily recorded. Each point represents the cumulative mean weight data from 2 experiments in which 6 mice per group were considered. Bars represent SEM (*P < .03). (D) Representative macroscopic pictures of colons of wild-type and IL-21-deficient mice treated with DSS. (E) Representative H&E-stained sections of colon collected from wild-type and IL-21 knockout mice. Induction of colitis in wild-type mice is evidenced by a marked infiltration of the mucosa with inflammatory cells, reduction of goblet cells, extensive destruction of mucosal layer, and lymphoid aggregates. By contrast, IL-21-deficient mice show a mild mucosal infiltration of inflammatory cells and minimal reduction of goblet cells. Gastroenterology 2008 134, 1038-1048.e2DOI: (10.1053/j.gastro.2008.01.041) Copyright © 2008 AGA Institute Terms and Conditions

Figure 2 Protection of DSS-colitis in IL-21-deficient mice is associated with a significant decrease in the expression of Th17-associated markers. Wild-type and IL-21 knockout mice were treated with or without oral DSS and then killed at day 7. Colonic samples were analyzed for the content of the indicated molecules by real-time PCR. Data indicate mean ± SD of 2 separate experiments in which 6 mice per group were considered (*P = .05; **P = .01). Gastroenterology 2008 134, 1038-1048.e2DOI: (10.1053/j.gastro.2008.01.041) Copyright © 2008 AGA Institute Terms and Conditions

Figure 3 IL-21 is significantly increased in mice with TNBS-relapsing colitis in comparison with controls. Transcripts for IL-21 (A) were evaluated by real-time PCR. Data are normalized for β-actin RNA content and presented as mean ± SD of 2 separate experiments. IL-21 (B) protein expression was evaluated by ELISA. Data are expressed as picogram/milligram total protein and indicate mean ± SD of 2 separate experiments, in which 5 mice per group were used. Gastroenterology 2008 134, 1038-1048.e2DOI: (10.1053/j.gastro.2008.01.041) Copyright © 2008 AGA Institute Terms and Conditions

Figure 4 IL-21 knockout mice develop less inflammation after treatment with escalating doses of TNBS. (A) Photomicrograph (original magnification, 20×) of an H&E-stained paraffin section of a representative colonic sample of wild-type and IL-21 knockout mice after TNBS treatment. A moderate mucosal mononuclear cell infiltration, elongation and distortion of crypts, and few hyperplastic lymphoid follicles are seen in sections from wild-type mice, whereas only a minimal inflammatory infiltrate is noted in the colon of IL-21-deficient mice. The photomicrographs are representative of 2 experiments in which 5 mice per group were studied. Percentages of mice showing various degrees of colitis are also indicated. (B–D) TNBS-relapsing colitis in IL-21-deficient mice is associated with a significant reduction in the expression of Th17-associated molecules. Two days after the last TNBS injection, mice were killed, and RNA was extracted from colonic specimens and used for analyzing the indicated molecules by real-time PCR. Data are normalized for β-actin RNA content and are presented as mean ± SD of 2 separate experiments, in which 5 mice per group were used. Gastroenterology 2008 134, 1038-1048.e2DOI: (10.1053/j.gastro.2008.01.041) Copyright © 2008 AGA Institute Terms and Conditions

Figure 5 T cells from IL-21 knockout mice fail to differentiate into Th17 cells. CD4+CD25− T cells were sorted from splenocytes of wild-type (WT) and IL-21 knockout (KO) mice and activated with anti-CD3 and anti-CD28 in the presence of Th1-, Th2-, or Th17-polarizing stimuli for 4 days. Transcripts for IL-17 (A) and ROR-γt (B) were analyzed by real-time PCR. Data indicate mean ± SD of triplicates of a single assay and are representative of 3 separate experiments in which similar results were obtained. (C) Representative dot plots showing IL-17-positive cells in CD4+CD25− T cells sorted from splenocytes of wild-type (WT) and IL-21 knockout (IL-21 KO) mice and activated with anti-CD3 and anti-CD28 in the presence of Th17-polarizing stimuli for 4 days. Numbers in quadrants indicate the percentages of cells in the designated gates. One of 3 representative experiments is shown. Panel D shows the level of IL-17 in culture supernatants of Th17 cells generated from wild-type and IL-21-deficient CD4+CD25− T cells as measured by ELISA. Data indicate mean ± SD of 3 separate experiments. (E) CD4+CD25− T cells were sorted from splenocytes of wild-type mice and activated with anti-CD3 and anti-CD28 in the presence of Th17-polarizing stimuli, with the initial addition of an antagonistic IL-21R/Fc or control IgG/Fc for 4 days. Levels of IL-17 in culture supernatants were measured by ELISA. Gastroenterology 2008 134, 1038-1048.e2DOI: (10.1053/j.gastro.2008.01.041) Copyright © 2008 AGA Institute Terms and Conditions

Figure 6 (A) IL-6 but not TGF-β1 induces IL-21 in CD4+ T cells. CD4+ CD25− T cells were sorted from splenocytes of wild-type mice and activated with anti-CD3 and anti-CD28 in the presence or absence of IL-6 and/or TGF-β1 for 4 days, and IL-21 RNA was analyzed by real-time PCR. Data indicate mean ± SD of triplicates of a single assay and are representative of 4 separate experiments in which similar results were obtained. (B) IL-21 promotes IL-17 induction in CD4+ T cells activated in the presence of TGF-β1. CD4+ CD25− T cells were sorted from splenocytes of wild-type mice and activated with anti-CD3 and anti-CD28 in the presence or absence of TGF-β1 and/or IL-21 with or without IL-6 for 4 days. IL-17 RNA transcripts were analyzed by real-time PCR. Data indicate mean ± SD of triplicates of a single assay and are representative of 3 separate experiments in which similar results were obtained. Gastroenterology 2008 134, 1038-1048.e2DOI: (10.1053/j.gastro.2008.01.041) Copyright © 2008 AGA Institute Terms and Conditions

Figure 7 Administration of IL-21R/Fc attenuates the inflammation in mice with established DSS colitis. DSS colitis was induced in wild-type mice as indicated in the Materials and Methods section, and IL-21R/Fc or control Fc was intraperitoneally administered the day after. Mice were killed at day 7. (A) Representative macroscopic pictures of colons of mice treated with control Fc (control) or IL-21R/Fc. (B) Representative H&E-stained sections of colon collected from such mice. Gastroenterology 2008 134, 1038-1048.e2DOI: (10.1053/j.gastro.2008.01.041) Copyright © 2008 AGA Institute Terms and Conditions

Figure 8 (A) IL-21 and IL-17 RNA transcripts are increased in CD tissue. IL-21 and IL-17 transcripts were evaluated by real-time PCR in biopsy samples taken from 8 patients with Crohn’s disease (CD) and 8 healthy controls (HC). Data are normalized for β-actin RNA content and are presented as mean ± SD of all samples. (B) Blockade of IL-21 reduces IL-17 secretion in CD CD3+ T-LPL. CD3+ T-LPL were isolated from CD colonic specimens and cultured in the presence of anti-CD3 with a neutralizing IL-21 (a-IL-21) or control IgG antibody for 48 hours. Supernatants were analyzed for IL-17 content by ELISA. Data indicate mean ± SD of all experiments. Gastroenterology 2008 134, 1038-1048.e2DOI: (10.1053/j.gastro.2008.01.041) Copyright © 2008 AGA Institute Terms and Conditions

Supplementary Figure 1 A. Representative endoscopic pictures of colons of both wild-type (WT) and IL-21-knockout (IL-21 KO) mice receiving 7% DSS orally for 7 days. Multiple erosions and easily bleeding mucosa were evident in wild-type mice, while IL-21-knockout mice exhibited essentially no inflammation. B. The endoscopic score in IL-21-knockout mice is significantly lower than that in wild-type mice (P = .03). Gastroenterology 2008 134, 1038-1048.e2DOI: (10.1053/j.gastro.2008.01.041) Copyright © 2008 AGA Institute Terms and Conditions

Supplementary Figure 2 IL-21-knockout mice show less severe TNBS-relapsing colitis. Colitis was monitored by endoscopy of mice at week 2 and 4 of the TNBS experiments. The mean values of the endoscopic score in both groups of mice are indicated. Gastroenterology 2008 134, 1038-1048.e2DOI: (10.1053/j.gastro.2008.01.041) Copyright © 2008 AGA Institute Terms and Conditions

Supplementary Figure 3 T cells from IL-21-knockout mice differentiate normally into Th1 or Th2 cells. CD4+CD25− T cells were sorted from splenocytes of wild-type (WT) and IL-21 knockout (KO) mice and activatd with anti-CD3 + anti-CD28 in the presence of polarizing stimuli (IL-12 + anti-IL-4 for TH1; IL-4 + anti-IFN-γ for Th2; TGF-β1 + IL-6 + anti-IL-4 + anti-IFN-γ for Th17; anti-IL-4 + anti-IFN-γ for Th-null) for 4 days. Cells were used for RNA extraction, and analysis of transcripts for IFN-γ (A) and IL-4 (B) was performed by real-time PCR. Data indicate mean ± SD of triplicates of a single assay and are representative of 3 separate experiments in which similar results were obtained. Gastroenterology 2008 134, 1038-1048.e2DOI: (10.1053/j.gastro.2008.01.041) Copyright © 2008 AGA Institute Terms and Conditions