Volume 133, Issue 1, Pages 108-123 (July 2007) Fenofibrate Represses Interleukin-17 and Interferon-γ Expression and Improves Colitis in Interleukin-10–Deficient Mice  Jimmy W. Lee, Poonam J. Bajwa, Monica J. Carson, Daniel R. Jeske, Yingzi Cong, Charles O. Elson, Christian Lytle, Daniel S. Straus  Gastroenterology  Volume 133, Issue 1, Pages 108-123 (July 2007) DOI: 10.1053/j.gastro.2007.03.113 Copyright © 2007 AGA Institute Terms and Conditions

Figure 1 Therapeutic response to fenofibrate in IL-10−/− mice. (A) IL-10−/− mice were fed a control diet or the same diet supplemented with 0.05% fenofibrate beginning at 8 weeks of age. The DAI was monitored on a weekly basis. Data were pooled from 4 different experiments. In all there were 32 mice (18 males, 14 females) fed the control diet and 34 mice (20 males, 14 females) fed the fenofibrate-supplemented diet. Analysis of the entire data set by mixed-model repeated-measures analysis indicated a significant fenofibrate treatment (main group) effect, P = .0004. *DAI significantly lower for fenofibrate-treated compared with control mice at the indicated time point by Mann–Whitney U test (P < .05). (B and C) Histopathology in the proximal colon of IL-10−/− mice fed the control diet. Sections were stained with H&E plus alcian blue, which stains the goblet cells blue. (D) Histology of the proximal colon of a mouse fed the fenofibrate-supplemented diet. (E) Histopathology scores in mice fed the control or fenofibrate-supplemented diet. Dashed line indicates the median score. (F) Mean counts of lymphocytes in coronal sections of colon. *Significantly lower than control by t test (P < .05). (G) Therapeutic response to fenofibrate in older IL-10−/− mice with established disease. Initially animals were housed in a room with limited access and given sterilized chow. Mice then were transferred to conventional caging and divided into 2 groups, one fed standard nonsterile chow (●) and the other fed the same chow supplemented with 0.05% fenofibrate (○). Each treatment group had 18 mice, paired by initial DAI, sex, and age across the 2 treatment groups. Analysis of the entire data set by mixed-model repeated-measures analysis indicated a significant fenofibrate treatment (main group) effect (P = .0001). *DAI significantly lower for fenofibrate-treated compared with control mice at the indicated time point by Mann–Whitney U test (P < .05). Gastroenterology 2007 133, 108-123DOI: (10.1053/j.gastro.2007.03.113) Copyright © 2007 AGA Institute Terms and Conditions

Figure 1 Therapeutic response to fenofibrate in IL-10−/− mice. (A) IL-10−/− mice were fed a control diet or the same diet supplemented with 0.05% fenofibrate beginning at 8 weeks of age. The DAI was monitored on a weekly basis. Data were pooled from 4 different experiments. In all there were 32 mice (18 males, 14 females) fed the control diet and 34 mice (20 males, 14 females) fed the fenofibrate-supplemented diet. Analysis of the entire data set by mixed-model repeated-measures analysis indicated a significant fenofibrate treatment (main group) effect, P = .0004. *DAI significantly lower for fenofibrate-treated compared with control mice at the indicated time point by Mann–Whitney U test (P < .05). (B and C) Histopathology in the proximal colon of IL-10−/− mice fed the control diet. Sections were stained with H&E plus alcian blue, which stains the goblet cells blue. (D) Histology of the proximal colon of a mouse fed the fenofibrate-supplemented diet. (E) Histopathology scores in mice fed the control or fenofibrate-supplemented diet. Dashed line indicates the median score. (F) Mean counts of lymphocytes in coronal sections of colon. *Significantly lower than control by t test (P < .05). (G) Therapeutic response to fenofibrate in older IL-10−/− mice with established disease. Initially animals were housed in a room with limited access and given sterilized chow. Mice then were transferred to conventional caging and divided into 2 groups, one fed standard nonsterile chow (●) and the other fed the same chow supplemented with 0.05% fenofibrate (○). Each treatment group had 18 mice, paired by initial DAI, sex, and age across the 2 treatment groups. Analysis of the entire data set by mixed-model repeated-measures analysis indicated a significant fenofibrate treatment (main group) effect (P = .0001). *DAI significantly lower for fenofibrate-treated compared with control mice at the indicated time point by Mann–Whitney U test (P < .05). Gastroenterology 2007 133, 108-123DOI: (10.1053/j.gastro.2007.03.113) Copyright © 2007 AGA Institute Terms and Conditions

Figure 2 (A) Western blot confirming PPARα expression in mouse colon and HT-29 cells. Protein extracts (50 μg) from proximal colon of strain-matched PPARα+/+ and PPARα−/− mice, HT-29 cells, and MPC-11 cells were subjected to Western blot analysis using an affinity-purified rabbit polyclonal anti-mouse PPARα antibody (PA1-822A; Affinity Bioreagents). Location of molecular size markers (kilodaltons) is shown on right. (B) Immunofluorescence localization of PPARα in formalin-fixed mouse proximal (top panels) or distal (bottom panels) colon using the same PA1-822A antibody (green). Nuclei were stained blue with ToPro-3 (blue). Digital images were acquired using identical gain, background, and confocal aperture settings. Prominent labeling of cells within the epithelial layer, lamina propria, and muscularis can be seen in control mice (left panels), but not in genotype-matched PPARα knock-out mice (right panels). Gastroenterology 2007 133, 108-123DOI: (10.1053/j.gastro.2007.03.113) Copyright © 2007 AGA Institute Terms and Conditions

Figure 3 PPARα expression in lymphocytes and monocytes/macrophages. Cells were isolated from spleen and colonic tissue and labeled using immune cell–specific conjugated antibodies along with PPARα polyclonal antibody. PPARα signal was amplified using conjugated secondary antibody. Histograms from a representative mouse show PPARα signal in control (minus first antibody for PPARα), colon, and spleen samples gated on (A) CD3, (B) CD3/CD4, (C) B220, and (D) F4/80. Gastroenterology 2007 133, 108-123DOI: (10.1053/j.gastro.2007.03.113) Copyright © 2007 AGA Institute Terms and Conditions

Figure 4 Direct effect of fenofibrate on cytokine expression in splenocytes and isolated T cells. (A–C) Splenocytes were isolated from IL-10−/− mice with high disease activity and divided into 3 treatment groups. One group received no treatment, one group was stimulated with PMA and ionomycin for 5 hours, and one group was pretreated with fenofibrate for 2 hours before stimulation with PMA/ionomycin for 5 hours. RNA was extracted, reverse-transcribed, and analyzed by real-time PCR for quantification of (A) IFNγ, (B) IL-17, and (C) TNFα mRNA. The results were normalized to the abundance of β-actin mRNA, which also was quantified by real-time PCR. The mean level of normalized mRNA expression in the control mice was set at 1.00. The results were pooled from 2 different experiments. Each bar represents the mean ± standard error for 6 different cultures. (D and E) Effect of fenofibrate on IL-17 mRNA expression in splenocytes from PPARα+/+ and strain-matched PPARα−/− mice. Significantly lower than mean for cultures treated with PMA/ionomycin only (*P < .05, **P < .001). (F) Cecal bacterial (cba) Ag-specific CD4+ T cells (Bir14 T cells) were treated with fenofibrate for 2 hours. PMA and ionomycin then were added for a period 5 of hours to activate IL-17 and IFNγ synthesis. GolgiStop (BD Biosciences) was added during the last 3 hours of the incubation period. Cells then were collected, stained for IL-17 and IFNγ, and subjected to FACS analysis. Numbers within the boxed scatterplots indicate the percentage of cells expressing IL-17 (left) or IFNγ (right) at each dose of fenofibrate. Gastroenterology 2007 133, 108-123DOI: (10.1053/j.gastro.2007.03.113) Copyright © 2007 AGA Institute Terms and Conditions

Figure 4 Direct effect of fenofibrate on cytokine expression in splenocytes and isolated T cells. (A–C) Splenocytes were isolated from IL-10−/− mice with high disease activity and divided into 3 treatment groups. One group received no treatment, one group was stimulated with PMA and ionomycin for 5 hours, and one group was pretreated with fenofibrate for 2 hours before stimulation with PMA/ionomycin for 5 hours. RNA was extracted, reverse-transcribed, and analyzed by real-time PCR for quantification of (A) IFNγ, (B) IL-17, and (C) TNFα mRNA. The results were normalized to the abundance of β-actin mRNA, which also was quantified by real-time PCR. The mean level of normalized mRNA expression in the control mice was set at 1.00. The results were pooled from 2 different experiments. Each bar represents the mean ± standard error for 6 different cultures. (D and E) Effect of fenofibrate on IL-17 mRNA expression in splenocytes from PPARα+/+ and strain-matched PPARα−/− mice. Significantly lower than mean for cultures treated with PMA/ionomycin only (*P < .05, **P < .001). (F) Cecal bacterial (cba) Ag-specific CD4+ T cells (Bir14 T cells) were treated with fenofibrate for 2 hours. PMA and ionomycin then were added for a period 5 of hours to activate IL-17 and IFNγ synthesis. GolgiStop (BD Biosciences) was added during the last 3 hours of the incubation period. Cells then were collected, stained for IL-17 and IFNγ, and subjected to FACS analysis. Numbers within the boxed scatterplots indicate the percentage of cells expressing IL-17 (left) or IFNγ (right) at each dose of fenofibrate. Gastroenterology 2007 133, 108-123DOI: (10.1053/j.gastro.2007.03.113) Copyright © 2007 AGA Institute Terms and Conditions

Figure 5 (A) Effect of fenofibrate on CXCL10 mRNA expression in HT-29 cells, as measured by conventional PCR. Cells were treated with fenofibrate (100 μmol/L) or dimethyl sulfoxide vehicle for 2 hours. TNFα (25 ng/mL) then was added to some cultures and incubation was continued for another 2 hours. RNA was extracted, reverse-transcribed, and subjected to PCR for 35 cycles (CXCL10) or 30 cycles (β-actin). Each lane represents RNA from a different culture. (B) Dose-response data showing relative potency of fenofibrate and GW7647 in repressing TNFα-stimulated CXCL10 mRNA expression. All bars are the means of 3 different cultures except the zero treatment bar, which represents a single culture. *Significantly lower than mean for cultures treated with TNFα only (P < .05). (C and D) Fenofibrate and GW7647 repress TNFα-activated CXCL10 promoter activity. HT-29 cells, cultured in 3.5-cm cell culture wells, were transfected with the TGL-IP-10 CXCL10/luciferase reporter (1 μg/well), pCMV-βgal (1 μg/well), and CMX-mPPARα (100 ng/well). Transfected cells were treated with TNFα (25 ng/mL) or TNFα + increasing doses of (C) fenofibrate or (D) GW7647. Cell extracts were assayed for luciferase and β-galactosidase activity, and luciferase activity then was normalized to β-galactosidase activity. Each bar represents the mean ± standard error of 4 transfected wells. *Significantly lower than mean for cultures treated with TNFα only (P < .05). (E) Fenofibrate represses IFNγ-stimulated CXCL10 promoter activity. Transfected cells were treated with IFNγ (50 ng/mL) or IFNγ + increasing doses of fenofibrate, as indicated. *Significantly lower than mean for cultures treated with IFNγ only (P < .05). Gastroenterology 2007 133, 108-123DOI: (10.1053/j.gastro.2007.03.113) Copyright © 2007 AGA Institute Terms and Conditions

Figure 6 (A) Structure of PPARδ ligands. GW0742, R=F. GW501516, R=H. (B) IL-10−/− mice were fed a control diet (●) or the same diet supplemented with GW0742 (○), formulated to deliver 5 mg/kg/day, beginning at a mean age of 14 weeks. Data from experiments 1 and 2 were pooled. In all there were 14 mice (6 males, 8 females) fed the control diet and 14 mice (5 males, 9 females) fed the GW0742-supplemented diet. Analysis of the entire data set by mixed-model repeated-measures analysis indicated a significant GW0742 treatment (main group) effect (P = .004). (C) Weight loss as a function of time. Data were pooled from experiments 1 and 2. (D and E) H&E-stained sections of proximal colon from IL-10−/− mice fed the GW0742-supplemented diet. (F) Histopathology scores for IL-10−/− mice fed the control or GW0742-supplemented diet. Data are from experiment 1. Dashed lines indicate median histopathology score. Gastroenterology 2007 133, 108-123DOI: (10.1053/j.gastro.2007.03.113) Copyright © 2007 AGA Institute Terms and Conditions