Volume 140, Issue 4, Pages 1272-1282 (April 2011) Modifying the Protease, Antiprotease Pattern by Elafin Overexpression Protects Mice From Colitis  Jean–Paul Motta, Laurent Magne, Delphyne Descamps, Corinne Rolland, Camila Squarzoni–Dale, Perrine Rousset, Laurence Martin, Nicolas Cenac, Viviane Balloy, Michel Huerre, Leopold F. Fröhlich, Dieter Jenne, Julien Wartelle, Azzaq Belaaouaj, Emmanuel Mas, Jean–Pierre Vinel, Laurent Alric, Michel Chignard, Nathalie Vergnolle, Jean–Michel Sallenave  Gastroenterology  Volume 140, Issue 4, Pages 1272-1282 (April 2011) DOI: 10.1053/j.gastro.2010.12.050 Copyright © 2011 AGA Institute Terms and Conditions

Figure 1 Modulation of colonic protease activity by elafin after DSS- or TNBS-induced colitis. Male C57/Bl6 WT, MCMV-EL, and Endo-EL mice drank either water (n = 9, 6, and 5, respectively) or DSS (n = 11, 8, and 8, respectively), or received TNBS intracolonically (n = 7, 5, and 4, respectively), and were killed 7 days later. Activity of (A and B) elastase/PR-3 and (C and D) trypsin were assessed in colonic extracts. Significant differences compared with WT inflamed: *P < .05 and ***P < .001. Gastroenterology 2011 140, 1272-1282DOI: (10.1053/j.gastro.2010.12.050) Copyright © 2011 AGA Institute Terms and Conditions

Figure 2 Modulation of colonic macroscopic damage (A and B) and MPO activity (C and D) in elafin transgenic mice after DSS- or TNBS-induced colitis. Male C57/Bl6 WT, MCMV-EL, and Endo-EL mice (after H2O, DSS, or TNBS treatment) were assessed for colonic damage and MPO activity at day 7. Significant differences compared with the WT inflamed group: *P < .05, **P < .01, and ***P < .001. Gastroenterology 2011 140, 1272-1282DOI: (10.1053/j.gastro.2010.12.050) Copyright © 2011 AGA Institute Terms and Conditions

Figure 3 Modulation of tissue architecture and proinflammatory cytokines in elafin transgenic mice after DSS- or TNBS-induced colitis. (A–C) Histology of colons and (D) cytokine protein content from C57/Bl6 WT, MCMV-EL, and Endo-EL mice 7 days after H2O, DSS, or TNBS treatment. Scale bar, 20 μm. Significant differences compared with WT corresponding treatment: *P < .05. Gastroenterology 2011 140, 1272-1282DOI: (10.1053/j.gastro.2010.12.050) Copyright © 2011 AGA Institute Terms and Conditions

Figure 4 Modulation of in vivo NF-κB activity in elafin transgenic mice after DSS-mediated colitis. (A) Study design, (B and C) luciferase bioluminescence measurements (B, pictures, C, quantification), and (D) macroscopic damage score in male C57/Bl6 WT, MCMV-EL, and Endo-EL mice that drank H2O or DSS and received 2.5 × 109 pfu Ad–NF-κB–luciferase intracolonically, 5 days after starting water or DSS treatments. Numbers represent mean ± standard deviation. *P < .05 compared with WT water. **P < .05 compared with WT DSS. ***P < .001 compared with WT Adeno–NF-κB + DSS–treated mice. Gastroenterology 2011 140, 1272-1282DOI: (10.1053/j.gastro.2010.12.050) Copyright © 2011 AGA Institute Terms and Conditions

Figure 5 Inhibition of TNF-α–stimulated permeability by Ad-elafin in intestinal epithelial cells. Permeability (apical-to-basal transport of a dextran probe conjugated to fluorescein isothiocyanate [FITC]) was measured in Caco-2 cells grown as a monolayer and exposed to apical and basolateral cytokines (TNFα, 10 ng/mL), and Ad-null or Ad-elafin (all at multiplicity of infection of 5) for 48 hours. Data are expressed as a percentage of paracellular flux compared with vehicle-treated cells and are representative of 3 experiments of n = 12 for the TNF group and n = 6 for the TNF + Ad-EL group. *Significant difference compared with TNF group was noted at P < .05. Gastroenterology 2011 140, 1272-1282DOI: (10.1053/j.gastro.2010.12.050) Copyright © 2011 AGA Institute Terms and Conditions

Figure 6 Colonic Ad-elafin expression in WT mice recapitulates the elafin protection observed in transgenic mice. Three days after the induction of colitis by DSS (water was given to controls, n = 5), male C57/Bl6 WT mice were injected intracolonically with either PBS (n = 8) or 2 × 108 pfu of either Ad-null (n = 6) or Ad-elafin (n = 7). (A) Elastase/PR-3, (B) trypsin activity, (C) colonic inflammation, (D) histology, and (E) cytokine content were assessed in tissues of those mice 7 days after the induction of colitis. (A and B) Significant difference compared with PBS: *P < .05 and **P < .01. ΨSignificant difference compared with the Ad-null group: P < .05. (C) Significant difference compared with PBS: ***P < .001. ΨΨΨSignificant difference compared with the Ad-null group: P < .001. (D) Scale bar, 20 μm. (E) Significant differences compared with DSS alone: *P < .05. ΨSignificant difference compared with the DSS + Ad-null–treated group: P < .05. Gastroenterology 2011 140, 1272-1282DOI: (10.1053/j.gastro.2010.12.050) Copyright © 2011 AGA Institute Terms and Conditions