Volume 136, Issue 1, Pages 268-277.e3 (January 2009) Degeneration of the Pericryptal Myofibroblast Sheath by Proinflammatory Cytokines in Inflammatory Bowel Diseases  Caroline Francoeur, Yamina Bouatrouss, Amira Seltana, Iryna V. Pinchuk, Pierre H. Vachon, Don W. Powell, Basem Sawan, Ernest G. Seidman, Jean–François Beaulieu  Gastroenterology  Volume 136, Issue 1, Pages 268-277.e3 (January 2009) DOI: 10.1053/j.gastro.2008.10.014 Copyright © 2009 AGA Institute Terms and Conditions

Figure 1 Characterization of the pericryptal myofibroblastic sheath in the small intestine from controls and patients with CD. Indirect immunofluorescence micrographs from representative fields of cryosections from (A and B) control specimens vs (C and D) uninflamed and (E and F) inflamed paired regions of CD specimens stained for the detection of (A, C, and E) αSMA using 1A4, (B, D, and F) desmin using 2P3M, or both. In (A) control and (C) uninflamed CD mucosa, αSMA was detected in the (A and C, arrows and inserts) pericryptal myofibroblastic layer, the isolated smooth muscle cells in the lamina propria, and the muscularis mucosa (mm), whereas (B and D, arrowheads and inserts) desmin was detected only in isolated smooth muscle cells of the lamina propria and of the mm. In the inflamed mucosa, the staining for (E) αSMA showed an irregular and thicker pattern around some crypts (arrows) whereas many other crypts (asterisks) were negative for αSMA. (F) Colocalization of desmin showed that most pericryptal structures stained for αSMA also were desmin positive (arrows and arrowheads in E/F and E/F') in contrast to uninflamed regions (C/D'). The staining for desmin in the smooth muscle cells of the (F) lamina propria and the mm of the inflamed mucosa remained similar to the (D) uninflamed mucosa and (B) control. Magnifications: A–F, 209×; A and B inserts, 469×; C and F inserts, 294×. Gastroenterology 2009 136, 268-277.e3DOI: (10.1053/j.gastro.2008.10.014) Copyright © 2009 AGA Institute Terms and Conditions

Figure 2 Analysis of Tn-C expression and distribution in the small intestine from controls and patients with CD. Indirect immunofluorescence micrographs from representative fields of cryosections from (A and B) control specimens vs (C and D) uninflamed and (E and F) inflamed paired regions of CD specimens stained for the detection of (A, C, and E) αSMA, (B, D, and F) Tn-C, or both. In control and uninflamed CD mucosa, staining for both (A and C) αSMA and (B and D) Tn-C showed a thin and relatively continuous labeling in the (A–D) subepithelial myofibroblastic layer all along the crypt-villus axis (arrows). (E and F) In inflamed CD mucosa, the subepithelial layer remained positive for Tn-C in the villus region (arrows) but was found to be weak or negative for both αSMA and Tn-C in the majority of the crypts (asterisks). αSMA- and Tn-C–positive muscle cells were frequent in the superficial lamina propria (lp) under all conditions. Magnifications: A–F, 209×. e, epithelium; mm, muscularis mucosa. Gastroenterology 2009 136, 268-277.e3DOI: (10.1053/j.gastro.2008.10.014) Copyright © 2009 AGA Institute Terms and Conditions

Figure 3 Expression of αSMA and Tn-C in the crypts of control and CD small intestine. Proportion of crypts expressing αSMA and/or Tn-C in the pericryptal myofibroblastic layer was determined by indirect immunofluorescent staining in controls as well as in paired U-CD and I-CD small intestinal sections by counting a minimum of 100 crypts on each section. Partially stained crypts were scored as positive. Six distinct specimens were used as controls, whereas 11 paired specimens were analyzed in the CD group. Results are expressed as means ± SEM. Statistics were calculated on raw data. *P < .001. Gastroenterology 2009 136, 268-277.e3DOI: (10.1053/j.gastro.2008.10.014) Copyright © 2009 AGA Institute Terms and Conditions

Figure 4 HIM cell growth and survival under proinflammatory conditions. HIM cells were maintained under basal serum-free (sf) conditions in the presence of various cytokines added alone or in combination for 72 hours. (A) The number of cells treated with individual cytokines remained comparable with the control condition. Only the combination TNFα/IFNγ led to a decrease in cell number whereas 4% fetal bovine serum induced proliferation. Bars represent mean ± SEM of 3 to 7 different experiments. *P < .05. (B) As observed by phase-contrast microscopy, the morphology of the cells treated with individual cytokines or TNFα/IL-1β was comparable with SF conditions whereas a significant increase in the number of dead cells was observed with the TNFα/IFNγ combination. Gastroenterology 2009 136, 268-277.e3DOI: (10.1053/j.gastro.2008.10.014) Copyright © 2009 AGA Institute Terms and Conditions

Figure 5 Effect of TNFα and IFNγ alone and in combination on HIM apoptosis. TNFα and IFNγ used separately did not induce HIM cell apoptosis whereas the combination of the 2 cytokines had a significant effect (*P < .0001). The caspase inhibitors zVAD and zDEVD completely prevented apoptosis in the TNFα/IFNγ-treated HIM cells. Bars represent the mean ± SEM of a minimum of 3 separate experiments. Gastroenterology 2009 136, 268-277.e3DOI: (10.1053/j.gastro.2008.10.014) Copyright © 2009 AGA Institute Terms and Conditions

Figure 6 Effect of proinflammatory cytokines on CMF apoptosis. Five primary adult myofibroblast cell cultures were treated with the TNFα/IFNγ combination, with or without zVAD, and compared with HIM. (A) Adult myofibroblasts were not significantly affected by this treatment. (B) The experiment was repeated using a more complete cocktail of proinflammatory cytokines, TNFα/IFNγ/IL-1β/IL-6. This cytokine combination caused a significant increase in the number of apoptotic cells in the HIM (*P < .01) and in the normal adult myofibroblasts (**P < .001), and was reversed by the caspase inhibitor, zVAD. Three to 5 fields for each condition from 2 to 3 separate experiments were compiled and analyzed using the chi-squared test. ncmf, normal colonic myofibroblast cultures isolated from the resection margin of uninflamed mucosa; icmf, inflamed colonic myofibroblast cultures isolated from the inflamed mucosa of IBD patients. Gastroenterology 2009 136, 268-277.e3DOI: (10.1053/j.gastro.2008.10.014) Copyright © 2009 AGA Institute Terms and Conditions

Figure 7 Effect of cytokines on HIM cell differentiation. (A) Relative amounts of αSMA in HIM cells were determined by Western blot relative to vimentin. Bars represent the mean ± SEM of at least 3 different experiments. *P < .05. Reduction of αSMA expression by the TNFα/IFNγ combination and its induction by TGFβ were investigated further at the transcript level by (B) semiquantitative and (C) quantitative reverse-transcription polymerase chain reaction relative to S14. Results are representative of 6 separate experiments and bars represent the mean ± SEM. *P < .02. sf, serum-free; fbs, fetal bovine serum. Gastroenterology 2009 136, 268-277.e3DOI: (10.1053/j.gastro.2008.10.014) Copyright © 2009 AGA Institute Terms and Conditions

Figure 8 Effects of the TNFα/IFNγ combination on Tn-C expression by HIM cells. By Western blot, Tn-C was found to be expressed under 2 major isoforms, 210 and 300 kilodaltons, in HIM cells under basal serum-free (sf) conditions as well as in TNFα/IFNγ-treated HIM cells. Although comparable in cells, a decrease of Tn-C was noted in the medium. (A) Equal amounts of cell lysates (50 g/well) and equal volumes of culture medium (50 L/well) were used for the representative experiment shown. (B) However, densitometric analysis of the data normalized relative to vimentin for the cells and to 105 cells for the media showed constant Tn-C expression under both conditions. Bars represent the mean ± SEM of 3 separate experiments. Gastroenterology 2009 136, 268-277.e3DOI: (10.1053/j.gastro.2008.10.014) Copyright © 2009 AGA Institute Terms and Conditions

Supplementary Figure 1 Characterization of the pericryptal myofibroblastic sheath in the colon from controls and patients with IBD. Immunohistochemistry micrographs from representative fields of the colonic mucosa from various pathologic situations including (A and B) noninfectious colitis, (C) acute infectious colitis, (D and E) ulcerative colitis, and (F) CD stained for the detection of (A–F) αSMA or (A''F') desmin on serial sections. As seen in controls and noninfectious and acute infectious colitis, αSMA was detected in the (A–C, arrows) pericryptal myofibroblastic layer, (A, arrowhead) a few isolated smooth muscle cells in the lamina propria, and the (A and C) muscularis mucosa (mm), whereas desmin was detected only in isolated smooth muscle cells of the (A', arrowhead) lamina propria and of the (A' and C') mm. In inflamed mucosa from IBDs, the staining for (D–F) αSMA showed a (E and F, arrows) regular thin staining at the base of the epithelium in a few regions, whereas (E and F, arrowheads) an irregular and thicker pattern was observed around other crypts and many other crypt regions were negative for αSMA (asterisks). (H) Staining for desmin showed that most pericryptal structures stained for αSMA were also (E' and F', arrowheads) desmin positive. Magnifications: A, 146×; B, C, E, and F, 191×; and D, 58×. Gastroenterology 2009 136, 268-277.e3DOI: (10.1053/j.gastro.2008.10.014) Copyright © 2009 AGA Institute Terms and Conditions

Supplementary Figure 2 Expression of the Tn-C transcript in the small intestine of patients with CD. (A) Representative reverse-transcription polymerase chain reaction analysis of Tn-C mRNA in uninflamed (u) and corresponding inflamed (i) CD specimens. –RT, no RT. The S14 transcript was used as the reference gene. (B) Amounts of Tn-C in uninflamed (u) and inflamed (i) specimens were determined on 6 series of paired samples relative to S14 by quantitative reverse-transcription polymerase chain reaction. Results are expressed as means ± SEM and show similar expression of Tn-C in I-CD when compared with U-CD. Gastroenterology 2009 136, 268-277.e3DOI: (10.1053/j.gastro.2008.10.014) Copyright © 2009 AGA Institute Terms and Conditions

Supplementary Figure 3 Characterization of the HIM myofibroblastic cell line. (A) Western blot analysis for the cell markers vimentin, αSMA, and desmin on proteins extracted from subconfluent HIM cells and intestinal smooth muscle (sm). SM was dissected from the muscularis propria of the fetal small intestine. Both HIM cells and SM were positive for vimentin and αSMA whereas only the SM extract was positive for the specific smooth muscle marker desmin, confirming the myofibroblastic phenotype of the HIM cells. For HIM cell staining experiments, 8-well LabTek chambers (Nalge–Nunc, Rochester, NY) were seeded with HIM cells at a density of 5 × 104 cells/well. Cells were incubated for 24 hours in the presence of 4% fetal bovine serum. Cells were stained for αSMA and desmin as described in the Materials and Methods section. Vimentin was detected using the monoclonal V9 antibody (Chemicon). Indirect immunofluorescent staining of subconfluent HIM cells confirmed expression of (B) vimentin and (C) αSMA and the (D) lack of desmin. Gastroenterology 2009 136, 268-277.e3DOI: (10.1053/j.gastro.2008.10.014) Copyright © 2009 AGA Institute Terms and Conditions

Supplementary Figure 4 Effect of TGFβ on desmin expression in HIM cells. Cells were treated with or without TGFβ in the absence of serum for 72 hours. (A) Desmin was not detected in subconfluent HIM cells (Figure 5), although basal levels can be detected in HIM cells at postconfluence. (B) Immunofluorescent staining showed that only a few HIM cells (approximately 10%) were stained under basal serum-free (sf) conditions. Treatment of postconfluent HIM cells with TGFβ (A) increased levels of desmin expression, (B) although the number of desmin-positive HIM cells remained around 10%. Gastroenterology 2009 136, 268-277.e3DOI: (10.1053/j.gastro.2008.10.014) Copyright © 2009 AGA Institute Terms and Conditions