Volume 139, Issue 6, Pages 2083-2092 (December 2010) Corticotropin-Releasing Factor Regulates TLR4 Expression in the Colon and Protects Mice From Colitis  Zoi Chaniotou, Panagiotis Giannogonas, Stamatis Theoharis, Thalia Teli, Jerome Gay, Tor Savidge, Yassemi Koutmani, James Brugni, Efi Kokkotou, Charalabos Pothoulakis, Katia P. Karalis  Gastroenterology  Volume 139, Issue 6, Pages 2083-2092 (December 2010) DOI: 10.1053/j.gastro.2010.08.024 Copyright © 2010 AGA Institute Terms and Conditions

Figure 1 Intestinal indices in Crh+/+ and Crh−/− mice. (A) H&E staining of colonic tissue from control Crh+/+ and Crh−/− mice. (B) Intestinal permeability assessment by fluorescein isothiocyanate/dextran and (C) water consumption over a 7-day period of DSS treatment revealed no differences between the 2 groups. Gastroenterology 2010 139, 2083-2092DOI: (10.1053/j.gastro.2010.08.024) Copyright © 2010 AGA Institute Terms and Conditions

Figure 2 DSS-induced colitis in Crh+/+ and Crh−/− mice following 7 days of DSS treatment. (A) H&E staining of colonic tissue from Crh+/+ and Crh−/− mice. The lower part of the micrograph shows an enlargement of the boxed portion in the upper part of the micrograph. The extended destruction of the epithelial barrier, the associated loss of crypts, and the massive leukocytic infiltration in the injured tissue are depicted. (B) Number of preserved crypts in the affected colon. Results express mean values from 5 slides per mouse, including the entire length of the excised colon, from a representative experiment (n = 3–4 mice per group). **P < .01. (C) Cumulative inflammatory index. Quantitative representation of inflammatory changes, epithelial destruction, number of crypts, and hyperplastic/dysplastic changes in the colonic tissue of Crh+/+ and Crh−/− mice (n = 4–5 per genotype per treatment). *P < .05. (D) Tissue levels of IL-12 and PGE2 in Crh−/− and Crh+/+ mice. *P < .05. (Figure 3D) Body weight loss expressed in grams lost over the initial body weight (Figure 2B) over the course of DSS treatment. Gastroenterology 2010 139, 2083-2092DOI: (10.1053/j.gastro.2010.08.024) Copyright © 2010 AGA Institute Terms and Conditions

Figure 3 Effects of corticosterone on DSS-induced colitis in Crh−/− mice. (A) H&E staining of colonic tissue from Crh−/− mice treated with DSS and corticosterone together. Enlargement of the boxed portion in the upper part of the micrograph is shown in the lower part of the micrograph, depicting the severe inflammatory changes in the injured tissue. (B) Cumulative inflammatory index. Quantitative representation of inflammatory changes, epithelial destruction, number of crypts, and hyperplastic/dysplastic changes in the colonic tissue of Crh−/− mice following 7 days of DSS and corticosterone, or vehicle, cotreatment (n = 3 per treatment). (C) H&E staining of colonic tissue and (D) viability of DSS-treated Crh−/− mice administered corticosterone for 7 days before and through the DSS treatment or not. Gastroenterology 2010 139, 2083-2092DOI: (10.1053/j.gastro.2010.08.024) Copyright © 2010 AGA Institute Terms and Conditions

Figure 4 Effects of CRH deficiency on TLR4 expression in the mouse colon. (A) TLR4 messenger RNA expression normalized by expression of glyceraldehyde-3-phosphate dehydrogenase. (B) MyD88 protein expression normalized by expression of actin. Values represent mean densitometry values ± SEM (n = 4). *P < .05. (C) Real-time polymerase chain reaction analysis of TNFα expression in Crh+/+ and Crh−/− mouse intestinal isografts 4 hours after intraperitoneal administration of LPS (100 μg/mouse) or saline (P < .05). Gastroenterology 2010 139, 2083-2092DOI: (10.1053/j.gastro.2010.08.024) Copyright © 2010 AGA Institute Terms and Conditions

Figure 5 Effects of glucocorticoid on colon Tlr4 expression in Crh−/− mice treated or not with DSS. (A) Glucocorticoid-mediated regulation of TLR4 in the Crh−/− fetal mouse colon. Western blot for TLR4 abundance in embryonic day 18.5 Crh+/+ and Crh−/− intestine, treated with corticosterone or vehicle, and quantitative representation of densitometric analysis of Tlr4 messenger RNA expression (n = 4 per group). *P < .05. (B) Tlr4 expression in Crh−/− colonic tissue from control or DSS-treated, cotreated or not with corticosterone, mice. Tlr4 expression is induced in Crh−/− mice treated with DSS alone. *P < .05. (C) Schematic representation of the regulation of Tlr4 by CRH and glucocorticoid. Solid and dashed lines indicate stimulatory and inhibitory interactions, respectively. Gastroenterology 2010 139, 2083-2092DOI: (10.1053/j.gastro.2010.08.024) Copyright © 2010 AGA Institute Terms and Conditions

Figure 6 Survival and tissue repair in DSS-treated mice. (A) Kaplan–Meier cumulative survival index of Crh+/+ (n = 20) and Crh−/− (n = 17) mice treated for 7 days with DSS (3%), followed by regular drinking water. (B) Body weight loss in Crh+/+ and Crh−/− mice over the course of DSS treatment and in the recovery phase. (C) H&E staining of colonic tissue from Crh+/+ and Crh−/− mice 4 days after completion of DSS treatment. (D) IL-12 and (E) expression of phospho-p38 expression in the colon of Crh+/+ and Crh−/− mice 2 days after completion of DSS treatment. *P < .05. Gastroenterology 2010 139, 2083-2092DOI: (10.1053/j.gastro.2010.08.024) Copyright © 2010 AGA Institute Terms and Conditions