Volume 135, Issue 6, Pages 2003-2013.e7 (December 2008) IL-13 Signaling via IL-13Rα2 Induces Major Downstream Fibrogenic Factors Mediating Fibrosis in Chronic TNBS Colitis  Stefan Fichtner–Feigl, Cheryl A. Young, Atsushi Kitani, Edward K. Geissler, Hans–Jürgen Schlitt, Warren Strober  Gastroenterology  Volume 135, Issue 6, Pages 2003-2013.e7 (December 2008) DOI: 10.1053/j.gastro.2008.08.055 Copyright © 2008 AGA Institute Terms and Conditions

Figure 1 Blockade of IL-13 or TGF-β1 signaling results in inhibition of tissue fibrosis. (A) Masson's trichrome staining of representative colon cross sections on day 49 of chronic TNBS-induced colitis following administration of AP-1 decoy oligonucleotides, Smad3 decoy oligonucleotides, or scrambled control oligonucleotides (administration on days 35 and 42) (original magnification 5×). (B) Collagen content of the colon on day 49 of chronic TNBS-induced colitis after administration of AP-1 decoy oligonucleotides, Smad3 decoy oligonucleotides, or scrambled control oligonucleotides (administration on days 35 and 42). Collagen content was determined during chronic TNBS-induced colitis by a Sircol assay. Data shown are mean values ± SD and are derived from at least 5 mice per group; *P < .05. (C) Body weight as a percent of starting weight after administration of decoy oligonucleotides (administration on days 35 and 42). Data shown are mean values ± SD and derived from at least 5 mice per group. (D) Histologic scores after administration of decoy oligonucleotides (administration on days 35 and 42). Scores shown are mean values ± SD from at least 5 mice per group. Gastroenterology 2008 135, 2003-2013.e7DOI: (10.1053/j.gastro.2008.08.055) Copyright © 2008 AGA Institute Terms and Conditions

Figure 2 Colonic IGF-I expression in chronic TNBS-induced colitis. (A) In situ cytokine production in the colon of mice with chronic TNBS-induced colitis measured at weekly time intervals. Total colonic protein was extracted from mice in the various groups and subjected to cytokine-specific ELISAs or total RNA was extracted from colonic tissue and subjected to quantitative polymerase chain reaction analysis. Data shown are representative of 2 independent experiments each containing at least 5 mice per group. Data shown are mean values ± SD and are derived from at least 5 mice per group; *P < .05 and **P < .01. (B) In situ cytokine production in the colon of mice with chronic TNBS-induced colitis on day 44 of chronic TNBS-induced colitis after administration of IL-13Rα2 siRNA, control siRNA (administration every other day starting on day 35), AP-1 decoy oligonucleotides, Smad3 decoy oligonucleotides, Egr-1 decoy oligonucleotides, scrambled control oligonucleotides (administration on days 35 and 42), or Z-VAD-fmk (administration every other day starting on day 35). Total colonic protein was extracted from mice in the various groups and subjected to cytokine-specific ELISAs or total RNA was extracted from colonic tissue and subjected to quantitative polymerase chain reaction analysis. Data shown are mean values ± SD and are derived from at least 5 mice per group; **P < .01. Gastroenterology 2008 135, 2003-2013.e7DOI: (10.1053/j.gastro.2008.08.055) Copyright © 2008 AGA Institute Terms and Conditions

Figure 3 Colonic Egr-1 expression in chronic TNBS-induced colitis. (A) Egr-1 expression in total colonic protein lysates at weekly intervals during the course of chronic TNBS-induced colitis as determined by Western blot analysis. (B) Egr-1 expression in colonic protein extracts as determined by Western blot analysis of mice on day 49 of chronic TNBS-induced colitis after administration of IL-13Rα2 siRNA, control siRNA (administration every other day starting on day 35), AP-1 decoy oligonucleotides, Smad3 decoy oligonucleotides, or scrambled control oligonucleotides (administration on days 35 and 42). Gastroenterology 2008 135, 2003-2013.e7DOI: (10.1053/j.gastro.2008.08.055) Copyright © 2008 AGA Institute Terms and Conditions

Figure 4 Blockade of Egr-1 activity in chronic TNBS-induced colitis leads to reduced tissue fibrosis. (A) Masson's trichrome staining of representative colon cross sections on day 49 during chronic TNBS-induced colitis after administration of Egr-1 decoy oligonucleotides or scrambled control oligonucleotides (administration on days 35 and 42) (original magnification 5×). (B) Collagen content of the colon on day 49 during chronic TNBS-induced colitis after administration of Egr-1 decoy oligonucleotides or scrambled control oligonucleotides (administration on days 35 and 42). Collagen content was determined during chronic TNBS-induced colitis by a Sircol assay. Data shown are mean values ± SD and are derived from at least 5 mice per group; *P < .05. (C) Body weight during the course of chronic TNBS-induced colitis as a percent of starting weight after administration of Egr-1 decoy oligonucleotides or scrambled control oligonucleotides (administration on days 35 and 42). Data shown are mean values ± SD and derived from at least 5 mice per group. (D) Histologic scores of colonic tissue on day 49 of TNBS-induced colitis after administration of Egr-1 decoy oligonucleotides or scrambled control oligonucleotides (administration on days 35 and 42). Scores shown are mean values ± SD from at least 5 mice per group. (E) Egr-1 expression in the colon of mice on day 49 of chronic TNBS-induced colitis was determined after administration of IGF-I receptor antibody or control antibody (administration on days 35 and 42). Total colonic protein was extracted from mice in the various groups and subjected to Western blot analysis. Gastroenterology 2008 135, 2003-2013.e7DOI: (10.1053/j.gastro.2008.08.055) Copyright © 2008 AGA Institute Terms and Conditions

Figure 5 Blockade of myofibroblast apoptosis prevents the development of fibrosis in chronic TNBS-induced colitis. (A) Masson's trichrome staining of representative colon cross sections on day 49 of chronic TNBS-induced colitis after administration of Z-VAD-fmk or vehicle control (original magnification 5×) (administration every other day starting on day 35). (B) Collagen content of the colon on day 49 during chronic TNBS-induced colitis after administration of Z-VAD-fmk or vehicle (administration every other day starting on day 35). Collagen content was determined during chronic TNBS-induced colitis by a Sircol assay. Data shown are mean values ± SD and are derived from at least 5 mice per group; *P < .05. (C) Body weight of mice with TNBS-induced colitis as a percent of starting weight after administration of Z-VAD-fmk or vehicle control (administration every other day starting on day 35). Data shown are mean values ± SD and derived from at least 5 mice per group. (D) Histologic scores of mice on day 49 of chronic TNBS-induced colitis after administration of Egr-1 decoy oligonucleotides or scrambled control oligonucleotides (administration on days 35 and 42). Scores shown are mean values ± SD from at least 5 mice per group. (E) IL-13Rα2 expression in the colon of mice on day 49 with chronic TNBS-induced colitis was determined after administration of AP-1 decoy oligonucleotides, Smad3 decoy oligonucleotides, Egr-1 decoy oligonucleotides, scrambled control oligonucleotides (administration on days 35 and 42), or Z-VAD-fmk (administration every other day starting on day 35). Lysates of colonic LMPCs were extracted from mice in the various groups and subjected to Western blot analysis. Gastroenterology 2008 135, 2003-2013.e7DOI: (10.1053/j.gastro.2008.08.055) Copyright © 2008 AGA Institute Terms and Conditions

Figure 6 The fibrotic phase of chronic TNBS-induced colitis is associated with myofibroblast apoptosis. (A) Flow cytometry performed on isolated total colonic cells on day 37 (or day 44) of chronic TNBS-induced colitis after administration of AP-1 decoy oligonucleotides, Smad3 decoy oligonucleotides, Egr-1 decoy oligonucleotides, scrambled control oligonucleotides (administration on day 35), or Z-VAD-fmk (administration on day 35). Staining was performed utilizing an anti–á-SMA antibody and annexin V after fixation and permeabilization. (B) Western blot analysis for detection of caspase-3 performed on cell lysates of isolated colonic a-SMA–positive cells obtained on day 37 (or day 44) of chronic TNBS-induced colitis after administration of AP-1 decoy oligonucleotides, Smad3 decoy oligonucleotides, Egr-1 decoy oligonucleotides, scrambled control oligonucleotides (administration on day 35), or Z-VAD-fmk (administration on day 35). Gastroenterology 2008 135, 2003-2013.e7DOI: (10.1053/j.gastro.2008.08.055) Copyright © 2008 AGA Institute Terms and Conditions

Figure 7 Procollagen type 1 production by intestinal myofibroblasts during chronic TNBS-induced colitis. (A) RNA extracts of isolated colonic á-SMA–positive cells (isolated by flow sorting after permeabilization and fixation) were analyzed for procollagen type 1 mRNA expression on day 37 and day 44 of chronic TNBS-induced colitis after administration of AP-1 decoy oligonucleotides, Smad3 decoy oligonucleotides, Egr-1 decoy oligonucleotides, scrambled control oligonucleotides (administration on days 35 and 42), or Z-VAD-fmk on day (administration every other day starting on day 35). Data shown are mean values ± SD and are derived from at least 5 mice per group; **P < .01. (B) RNA extracts of isolated colonic á-SMA–positive cells were analyzed for procollagen type 1 mRNA expression on day 44 of TNBS-induced colitis after administration of IGF-I receptor antibody, IGF-I-antibody, or control antibody (administration on days 35 and 42). Data shown are mean values ± SD and are derived from at least 5 mice per group; *P < .05. Gastroenterology 2008 135, 2003-2013.e7DOI: (10.1053/j.gastro.2008.08.055) Copyright © 2008 AGA Institute Terms and Conditions

Figure 8 Summary diagram of the fibrogenic pathway. Fibrosis occurring in experimental colitis is uniquely dependent on IL-13 signaling via the IL-13Rα2 receptor that leads in turn to secretion of TGF-β1 and IGF-I as well as increased expression of Egr-1. These factors then act in a coordinated fashion to cause fibrosis through the induction of myofibroblast apoptosis and generation of myofibroblasts producing collagen. Gastroenterology 2008 135, 2003-2013.e7DOI: (10.1053/j.gastro.2008.08.055) Copyright © 2008 AGA Institute Terms and Conditions

Supplementary Figure 1 LPMC cytokine production in mice after administration of decoy oligonucleotides (administration on days 35 and 42). Cells were extracted from the lamina propria and cultured for 48 hours under stimulation as described in Materials and Methods. Cytokine concentration was determined in culture supernatants by ELISA. Data shown are mean values ± SD from individual cultures of cells derived from mice in 2 separate experiments, each containing at least 5 mice per group. Gastroenterology 2008 135, 2003-2013.e7DOI: (10.1053/j.gastro.2008.08.055) Copyright © 2008 AGA Institute Terms and Conditions

Supplementary Figure 2 LPMC cytokine production of mice on day 49 of chronic TNBS-induced colitis after administration of Egr-1 decoy oligonucleotides or scrambled control oligonucleotides (administration on days 35 and 42). Cells were extracted from the lamina propria and cultured for 48 hours under stimulation as described in Materials and Methods. Cytokine concentrations were determined in culture supernatants by ELISA. Data shown are mean values ± SD from individual cultures of cells derived from mice in 2 separate experiments, each containing at least 5 mice per group. Gastroenterology 2008 135, 2003-2013.e7DOI: (10.1053/j.gastro.2008.08.055) Copyright © 2008 AGA Institute Terms and Conditions

Supplementary Figure 3 LPMC cytokine production of mice on day 49 of chronic TNBS-induced colitis after administration of Z-VAD-fmk or vehicle control (administration every other day starting on day 35). Cells were extracted from the lamina propria and cultured for 48 hours under stimulation as described in Materials and Methods. Data shown are mean values ± SD from individual cultures of cells derived from mice in 2 separate experiments, each containing at least 5 mice per group. Gastroenterology 2008 135, 2003-2013.e7DOI: (10.1053/j.gastro.2008.08.055) Copyright © 2008 AGA Institute Terms and Conditions

Supplementary Figure 4 Lamina propria cells secrete high levels of active TGF-β1 on day 49 of chronic TNBS-induced colitis. (A) LPMCs obtained from mice on day 49 of TNBS-induced colitis after administration of Egr-1 decoy oligonucleotides, scrambled control oligonucleotides (administration on days 35 and 42), or Z-VAD-fmk (administration every other day starting on day 35) were cultured for 48 hours under stimulation as described in Materials and Methods. Cytokine concentrations were determined in culture supernatants by ELISA. Data shown are mean values ± SD from individual cultures of cells derived from mice in 2 separate experiments, each containing at least 5 mice per group; *P < .05. (B) uPA expression in the colon of mice on day 49 of chronic TNBS-induced colitis was determined after administration of Egr-1 decoy oligonucleotides, scrambled control oligonucleotides (administration on days 35 and 42), or Z-VAD-fmk (administration every other day starting on day 35). Total colonic protein was extracted from mice in the various groups and subjected to Western blot analysis. Gastroenterology 2008 135, 2003-2013.e7DOI: (10.1053/j.gastro.2008.08.055) Copyright © 2008 AGA Institute Terms and Conditions

Supplementary Figure 5 Transfection of colonic α-SMA–positive myofibroblasts with fluorochrome-conjugated oligonucleotides. Flow cytometry performed on isolated total colonic cells on day 36 of chronic TNBS-induced colitis after administration of Cy-5 conjugated (or unconjugated) AP-1 decoy oligonucleotides (fluorochrome conjugation of oligonucleotides was performed using Label IT Nucleic Acid Labeling Reagents; Mirus Bio Corp, Madison, WI). Transfection using the HVJ-E transfection method was performed on day 35 of chronic TNBS-induced colitis. Staining of myofibroblasts was performed utilizing a fluorescein isothiocyanate anti–α-SMA antibody after fixation and permeabilization. The transfection efficiency for colonic α-SMA–positive myofibroblasts achieved was 81.3% (±7.1% SD). Data shown are representative of 3 independent experiments. Gastroenterology 2008 135, 2003-2013.e7DOI: (10.1053/j.gastro.2008.08.055) Copyright © 2008 AGA Institute Terms and Conditions