Therapeutic Action of Ghrelin in a Mouse Model of Colitis Elena Gonzalez–Rey, Alejo Chorny, Mario Delgado  Gastroenterology  Volume 130, Issue 6, Pages 1707-1720 (May 2006) DOI: 10.1053/j.gastro.2006.01.041 Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 1 Treatment with ghrelin protects against TNBS-induced colitis. Colitis was induced by intracolonic administration of TNBS (3 mg/mouse) in 50% ethanol. Mice were treated IP with ghrelin (2 nmol/mouse, or different doses in B) 12 hours after TNBS injection. Mice treated with 50% ethanol were used as controls. Clinical evolution and severity was monitored by body weight changes (A and B), colitis score (C), and survival (D). N = 12–18 mice/group. *P < .001 vs TNBS-treated mice. Gastroenterology 2006 130, 1707-1720DOI: (10.1053/j.gastro.2006.01.041) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 2 Ghrelin prevents TNBS-induced pathology. Colitis was induced by intracolonic administration of TNBS (3 mg/mouse) in 50% ethanol. Mice were treated IP with ghrelin (2 nmol/mouse) 12 hours after TNBS injection. Mice treated with 50% ethanol were used as controls. (A) Macroscopic-damage score was determined at 3 days after TNBS administration. (B) Histopathologic analysis was determined in H&E-stained sections of colons obtained at day 3 of disease (original magnification, ×200). (C) Inflammatory infiltrates in the colons (day 3) were phenotypically characterized by immunostaining against TLR4, TNF-α-producing cells, CD11b cells, or CD4 T cells. Isotype FITC-labeled IgG Ab was used as negative control (original magnification, ×200). (D) Colonic MPO activity was determined in the acute phase of the disease (day 3). N = 12–18 mice/group. *P < .001 vs TNBS-treated mice. Gastroenterology 2006 130, 1707-1720DOI: (10.1053/j.gastro.2006.01.041) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 3 Treatment with ghrelin abrogates established colitis and reduces disease recurrence. (A) Established colitis. Colitis was induced by intracolonic administration of TNBS (1.5 mg/mouse) at days 0 and 6. Mice were treated daily for 3 consecutive days with ghrelin (2 nmol/mouse/day) starting 6 days after TNBS administration (arrow). Disease progression was assessed by body weight loss and macroscopic score at day 12. N = 8 mice/group. (B) Disease recurrence. Colitis was induced by intracolonic administration of TNBS (3 mg/mouse) at days 0 and 9 (arrows). Mice were treated IP with ghrelin (2 nmol/mouse) 12 hours after TNBS injection. Controls were given a second injection of ethanol at day 9. Disease progression was assessed by body weight loss, survival percentage, and macroscopic score at day 11. Numbers in parentheses represent daily mortality percentage after the second TNBS infusion. N = 8–10 mice/group. Gastroenterology 2006 130, 1707-1720DOI: (10.1053/j.gastro.2006.01.041) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 4 Ghrelin protects against established DSS-induced colitis. Colitis was induced by DSS intake. Ghrelin (2 nmol/mouse) was injected IP on 2 alternate days starting at day 4. Body weight loss, macroscopic appearance of the colon, colonic MPO activity, and disease activity index were determined 8 days after the induction of DSS colitis. N = 8 mice/group. *P < .001 vs DSS-treated mice. Gastroenterology 2006 130, 1707-1720DOI: (10.1053/j.gastro.2006.01.041) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 5 Ghrelin decreases systemic and colonic inflammatory responses in the TNBS model of colitis. Colitis was induced by intracolonic administration of TNBS. Mice were treated IP with ghrelin (2 nmol/mouse) 12 hours after TNBS injection. Mice treated with ethanol alone were used as controls. Serum was collected, and protein extracts and total RNA were obtained from colons at the acute phase of the disease (day 3). (A) Cytokine/chemokine contents in protein extracts were determined by ELISA. N = 5 or 6 mice/group. (B) Gene expression of several inflammatory/autoimmune mediators was determined by using a microarray. Results are representative of 2 separate experiments (n = 4 or 5 mice/group). (C) Cytokine/chemokine and SAA contents in the serum were determined by ELISA. N = 5 or 6 mice/group. *P < .001 vs TNBS-treated mice. Gastroenterology 2006 130, 1707-1720DOI: (10.1053/j.gastro.2006.01.041) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 6 Ghrelin decreased chronic inflammatory response in established colitis. Colitis was induced by intracolonic administration of TNBS (1.5 mg/mouse) at days 0 and 6. Mice were treated daily for 3 consecutive days with ghrelin (2 nmol/mouse/day) starting 6 days after TNBS administration (arrow). Protein extracts were obtained from colons on different phases of the disease, and the cytokine contents were determined by ELISA. N = 5 or 6 mice/group. *P < .001 vs TNBS-treated mice. Gastroenterology 2006 130, 1707-1720DOI: (10.1053/j.gastro.2006.01.041) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 7 Ghrelin down-regulates Th1 cytokine response and stimulates IL-10 production in TNBS-induced colitis. Colitis was induced by intracolonic administration of TNBS, and mice were treated IP with ghrelin (2 nmol/mouse) 12 hours after TNBS injection. Mice treated with ethanol alone were used as controls. Mesenteric lymph node cells (MLN) and lamina propria mononuclear cells (LPMN) were isolated from the different experimental groups at the peak of the disease (day 3) and cultured with medium alone (unstimulated) or with PMA plus ConA (stimulated). (A) Proliferative response was determined after 4 days, culture as described in the Materials and Methods section. Cytokine contents in the supernatants were determined after 48-hour culture by ELISA. (B) Stimulated lymphocytes were analyzed for CD4 and intracellular cytokine expression by flow cytometry. Double staining for IFN-γ/IL-2 or IL-4/IL-10 expression was performed in gated CD4 T cells. Flow cytometry plots are representative of LPMC. The number of IFN-γ- and IL-10-expressing T cells relative to 104 CD4 T cells was determined (right panels). Data shown represent pooled values from 2 independent experiments (n = 5 or 6 mice/group/experiment). *P < .001 vs TNBS-treated mice. Gastroenterology 2006 130, 1707-1720DOI: (10.1053/j.gastro.2006.01.041) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 8 Ghrelin induces the appearance of CD4+CD25+Foxp3+ T cells in TNBS-induced colitis. Colitis was induced by intracolonic administration of TNBS. Mice were treated IP with ghrelin (2 nmol/mouse) 12 hours after TNBS injection. MLN cells were isolated from the different experimental groups at different times after TNBS infusion, and the percentage of CD4+CD25+Foxp3+ cells was determined by flow cytometry. Alternatively, a second TNBS dose (3 mg/mouse) was given at day 8 (arrow) to both untreated (solid circle) and ghrelin-treated (open circle) mice, and percentage of CD4+CD25+Foxp3+ cells was determined 2 days later. N = 5 mice/group. Gastroenterology 2006 130, 1707-1720DOI: (10.1053/j.gastro.2006.01.041) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 9 GH and IGF-I are not involved in the therapeutic effect of ghrelin. Colitis was induced by intracolonic administration of TNBS. Mice were treated IP with ghrelin (2 nmol/mouse) 12 hours after TNBS injection. (A) GH and IGF-I levels were determined at different times in serum. N = 4 mice/group. (B) Mice treated with TNBS and ghrelin were injected with neutralizing anti-GH and/or anti-IGF-I Abs (200 μg/mouse/dose) 2 hours and 24 hours after ghrelin administration. Disease severity was determined by body weight changes at day 3. N = 4 mice/group. (C) MLN cells (5 × 105 cells/mL) isolated from TNBS-treated mice were stimulated with PMA (10 ng/mL) plus ConA (2.5 μg/mL) in the absence (none) or presence of ghrelin (10-7 mol/L). Neutralizing anti-GH and/or anti-IGF-I Abs (20 μg/mL) were added to the cultures. After 48-hour culture, cytokines were determined in supernatants by ELISA. N = 3 experiments performed in duplicate. Gastroenterology 2006 130, 1707-1720DOI: (10.1053/j.gastro.2006.01.041) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions