Fenofibrate Represses Interleukin-17 and Interferon-γ Expression and Improves Colitis in Interleukin-10–Deficient Mice

Background & Aims: Interleukin-10 knockout (IL-10−/−) mice spontaneously develop colitis characterized by T-helper cell type 1–polarized inflammation. We tested the possible therapeutic activity of the peroxisome proliferator-activated receptor α (PPARα) ligand fenofibrate, and the PPARδ ligand GW0742, in IL-10−/− mice and investigated the cellular/molecular mechanisms for fenofibrate action. Methods: The effect of fenofibrate or GW0742 on the progression of colitis in C3H.IL-10−/− mice was evaluated. Effects of fenofibrate on cytokine and chemokine gene expression were studied in cultured splenocytes, pathogenic T cells isolated from C3H/HeJBir mice, and HT-29 colorectal cancer cells. Results: Treatment of C3H.IL-10−/− mice with fenofibrate delayed the onset of colitis, decreased the colonic histopathology score, and decreased colonic expression of genes encoding the inflammatory cytokines interferon-γ and interleukin (IL)-17. The target for fenofibrate, PPARα, was expressed in lymphocytes, macrophages, and crypt and surface epithelial cells of the colon. The mean number of lymphocytes was decreased by more than 75% in colonic sections of fenofibrate-treated as compared with control IL-10−/− mice, and fenofibrate repressed interferon-γ and IL-17 expression in isolated T cells. Fenofibrate also repressed the expression of the genes encoding 3 chemokines, CXCL10, CCL2, and CCL20, and repressed CXCL10 gene promoter activity in tumor necrosis factor-α–treated HT-29 cells. In contrast to the beneficial effect of fenofibrate, the PPARδ ligand GW0742 accelerated the onset of colitis in IL-10−/− mice. Conclusions: The immunopathology observed in IL-10−/− mice resembles that seen in Crohn’s disease. The novel therapeutic activity of fenofibrate in this mouse model suggests that it may also have activity in Crohn’s disease. Background & Aims: Interleukin-10 knockout (IL-10−/−) mice spontaneously develop colitis characterized by T-helper cell type 1–polarized inflammation. We tested the possible therapeutic activity of the peroxisome proliferator-activated receptor α (PPARα) ligand fenofibrate, and the PPARδ ligand GW0742, in IL-10−/− mice and investigated the cellular/molecular mechanisms for fenofibrate action. Methods: The effect of fenofibrate or GW0742 on the progression of colitis in C3H.IL-10−/− mice was evaluated. Effects of fenofibrate on cytokine and chemokine gene expression were studied in cultured splenocytes, pathogenic T cells isolated from C3H/HeJBir mice, and HT-29 colorectal cancer cells. Results: Treatment of C3H.IL-10−/− mice with fenofibrate delayed the onset of colitis, decreased the colonic histopathology score, and decreased colonic expression of genes encoding the inflammatory cytokines interferon-γ and interleukin (IL)-17. The target for fenofibrate, PPARα, was expressed in lymphocytes, macrophages, and crypt and surface epithelial cells of the colon. The mean number of lymphocytes was decreased by more than 75% in colonic sections of fenofibrate-treated as compared with control IL-10−/− mice, and fenofibrate repressed interferon-γ and IL-17 expression in isolated T cells. Fenofibrate also repressed the expression of the genes encoding 3 chemokines, CXCL10, CCL2, and CCL20, and repressed CXCL10 gene promoter activity in tumor necrosis factor-α–treated HT-29 cells. In contrast to the beneficial effect of fenofibrate, the PPARδ ligand GW0742 accelerated the onset of colitis in IL-10−/− mice. Conclusions: The immunopathology observed in IL-10−/− mice resembles that seen in Crohn’s disease. The novel therapeutic activity of fenofibrate in this mouse model suggests that it may also have activity in Crohn’s disease. 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The remaining tissue was incubated in RPMI-1640 with 0.5% FBS, 30 U/mL collagenase I, and 10 U/mL deoxyribonuclease I for 1 hour at 37°C with gentle rocking. Suspended cells including lamina propria immune cells were filtered and pooled with previous collections, washed with PBS, and resuspended in binding buffer.40Powrie F. Leach M.W. Mauze S. Caddle L.B. Coffman R.L. Phenotypically distinct subsets of CD4+ T cells induce or protect from chronic intestinal inflammation in C.B-17 scid mice.Int Immunol. 1993; 5: 1461-1471Google Scholar, 41Li J. Kudsk K.A. Gocinski B. Dent D. Glezer J. Langkamp-Henken B. Effects of parenteral and enteral nutrition on gut-associated lymphoid tissue.J Trauma. 1995; 39: 44-52Google Scholar Suspended immune cells were incubated with conjugated antibodies for 1 hour at room temperature. Surface marker antibodies included CD4-Alexa700 (RM4-5), CD3-Pacific Blue (17A2), CD3-Alexa647 (17A2), B220-PE (RA3-6B2), and F4/80-Pacific Blue (BM8). Activation state markers included CD69-PE (H1.2F3) and CD62L-antigen-presenting cells (MEL-14). In addition, isotype controls corresponding to each antibody were used to exclude nonspecific staining. All fluorescent conjugated antibodies were purchased from BD Pharmingen (San Diego, CA) and eBioscience (San Diego, CA). After staining for surface markers, cells were fixed in 1% paraformaldehyde and permeabilized in 0.1% saponin/sodium azide.42Sander B. Andersson J. Andersson U. Assessment of cytokines by immunofluorescence and the paraformaldehyde-saponin procedure.Immunol Rev. 1991; 119: 65-93Google Scholar Intracellular staining for PPARα was performed using rabbit anti-mouse primary (PA1-822A, Affinity Bioreagents) and Alexa488 chicken anti-rabbit secondary (Invitrogen, Carlsbad, CA). Cells were washed in PBS and resuspended in fluorescence-activated cell sorter (FACS) sorting buffer (1 mmol/L EDTA, 25 mmol/L HEPES, and 1% FBS). FACS was performed on BD FACSAria (San Diego, CA) and analyzed using FloJo 7.1 software (Ashland, OR). Mouse splenocytes were isolated as described earlier. Red blood cells were lysed, and the remaining white blood cells were resuspended in RPMI-1640 supplemented with 0.5% FBS, penicillin (100 U/mL), and streptomycin (100 μg/mL). White blood cells were pooled from 6 mice, counted, and plated at 8 × 106 cells/mL in 3 separate groups. The control group received no treatment, whereas the phorbol 12-myristate 13-acetate (PMA)/ionomycin group was stimulated with 50 ng/mL PMA and 1 μg/mL ionomycin for 5 hours. The fenofibrate group was pretreated with fenofibrate for 2 hours and then stimulated with PMA/ionomycin fo