Volume 132, Issue 4, Pages 1331-1343 (April 2007) R-spondin1, A Novel Intestinotrophic Mitogen, Ameliorates Experimental Colitis in Mice  Jingsong Zhao, Josephine de Vera, Seiko Narushima, Eric X. Beck, Servando Palencia, Pauline Shinkawa, Kyung–Ah Kim, Yi Liu, Michael D. Levy, Daniel J. Berg, Arie Abo, Walter D. Funk  Gastroenterology  Volume 132, Issue 4, Pages 1331-1343 (April 2007) DOI: 10.1053/j.gastro.2007.02.001 Copyright © 2007 AGA Institute Terms and Conditions

Figure 1 Therapeutic effect of Rspo1 in treating DSS-induced colitis in mice. (A) IBD disease activity index in mice from day 7 to 14 (n = 4 mice/group). Therapeutic treatment of Rspo1 reduces established DSS-induced colitis activity in mice. *P < .05, DSS/saline vs DSS/Rspo1. (B) H&E-stained tissue sections of mouse small intestine (a–d) and colon (e–h; a and e, water/saline; b and f, water/Rspo1; c and g, DSS/saline; d and h, DSS/Rspo1). DSS-induced lesions in mucosa of both small intestine and colon are partially restored by therapeutic treatment of Rspo1 in mice. Bar = 100 μm. (C) Histometric analysis of H&E-stained sections for villus height and crypt depth in mucosa of mouse small intestine. Treatment of Rspo1 reverses DSS-induced reduction of both villus height and crypt depth in small intestinal mucosa in mice. *P < .05, water/saline vs water/Rspo1; #P < .05, water/saline vs DSS/saline; **P < .05, DSS/saline vs DSS/Rspo1. (D) Histometric analysis of H&E-stained section for crypt depth in mouse colonic mucosa. Rspo1 reverts DSS-induced shortening of crypt depth in mucosa in mouse colon. *P < .05, water/saline vs water/Rspo1; #P < .05, water/saline vs DSS/saline; **P < .05, DSS/saline vs DSS/Rspo1. Gastroenterology 2007 132, 1331-1343DOI: (10.1053/j.gastro.2007.02.001) Copyright © 2007 AGA Institute Terms and Conditions

Figure 2 Effect of Rspo1 in modulating intestinal crypt growth and goblet cell number in DSS-treated mice. (A) BrdU immunohistochemistry in mouse small intestine (a–c) and colon (d–f; a and d, water/saline; b and e, DSS/saline; c and f, DSS/Rspo1). Rspo1 reverses DSS-induced suppression of crypt cell proliferation, shown by BrdU incorporation, in both small intestine and colon in mice. Bar = 100 μm. (B) The resultant crypt proliferative index (percentage of BrdU-positive cells) in small intestinal crypt compartment (n = 3 mice/group). Rspo1 demonstrates a stimulatory effect in crypt cell mitosis in DSS-treated crypt epithelial cells in mouse small intestine. *P < .05, DSS/saline vs DSS/Rspo1. (C) Alcian blue staining of mucins in mouse colon (a, water/saline; b, water/Rspo1; c, DSS/saline; d, DSS/Rspo1). Addition of Rspo1 increases the number of Alcian blue–positive cells in normal and DSS-treated mouse colon. Bar = 100 μm. Gastroenterology 2007 132, 1331-1343DOI: (10.1053/j.gastro.2007.02.001) Copyright © 2007 AGA Institute Terms and Conditions

Figure 3 Effect of Rspo1 on intestinal MPO and cytokine levels in DSS-treated mice. (A) MPO activity in mouse colonic tissue (n = 3 mice/group). Rspo1 reduces DSS-induced overproduction of MPO in mouse colon. *P < .05, water/saline vs DSS/saline; #P < .05, DSS/saline vs DSS/Rspo1. (B) Cytokine levels of TNF-α, IL-1α, IL-6, IFN-γ, and GM-CSF in mouse small intestinal tissue. Rspo1 suppresses DSS-induced overproduction of TNF-α, IL-1α, IL-6, IFN-γ, and GM-CSF in mouse small intestine. *P < .05, water/saline vs DSS/saline; #P < .05, DSS/saline vs DSS/Rspo1. Gastroenterology 2007 132, 1331-1343DOI: (10.1053/j.gastro.2007.02.001) Copyright © 2007 AGA Institute Terms and Conditions

Figure 4 Effect of Rspo1 on TNBS-treated mice. (A) Animal body weight change on day 7 (n = 6 mice/group). Rspo1 treatment reverses TNBS-induced body weight loss in mice at a dose of either 100 or 50 μg. *P < .05, vehicle/saline vs TNBS/saline; #P < .05, TNBS/saline vs TNBS/Rspo1. (B) Macroscopic images of mouse colon harvested on day 7 (a, vehicle/saline; b, TNBS/saline; c, TNBS/Rspo1 [100-μg dose]; d, TNBS/Rspo1 [50-μg dose]). Addition of Rspo1 reduces TNBS-induced atrophy and hyperemia in the colon. Bar = 1 cm. (C) Colitis score of morphologic grading of mouse colon on day 7. Treatment of Rspo1, at a daily dose of either 100 μg or 50 μg, reduces colitis activity in mouse colon receiving TNBS. *P < .05, vehicle/saline vs TNBS/saline; #P < .05, TNBS/saline vs TNBS/Rspo1. Gastroenterology 2007 132, 1331-1343DOI: (10.1053/j.gastro.2007.02.001) Copyright © 2007 AGA Institute Terms and Conditions

Figure 5 Effect of Rspo1 in treating TNBS-induced colitis in mice. (A) H&E sections of mouse distal colon on day 7 (a, vehicle/saline; b, TNBS/saline; c, TNBS/Rspo1 [100-μg dose]; d, TNBS/Rspo1 [50-μg dose]). Rspo1 (100-μg or 50-μg dose) partially abrogates colonic damage in TNBS-treated mice. Bar = 100 μm. (B) Histologic grading of colonic colitis score in mice (n = 6 mice/group). Rspo1 treatment down-regulates colitis activity in TNBS-treated mouse colon. *P < .05, vehicle/saline vs TNBS/saline; #P < .05, TNBS/saline vs TNBS/Rspo1. (C) MPO activity in mouse colon on day 7. Rspo1 therapy reduces TNBS-induced MPO activity in mouse colon. *P < .05, vehicle/saline vs TNBS/saline; #P < .05, TNBS/saline vs TNBS/Rspo1. Gastroenterology 2007 132, 1331-1343DOI: (10.1053/j.gastro.2007.02.001) Copyright © 2007 AGA Institute Terms and Conditions

Figure 6 Effect of Rspo1 in modulating piroxicam-induced colitis in IL-10−/− mice. (A) H&E sections of proximal colon (a and b), middle colon (c and d), and distal colon (e and f) in IL-10−/− mice exposed to piroxicam (a, c, and e, IL-10 knockout/saline; b, d, and f, IL-10 knockout/Rspo1; n ≥ 5 mice/group). Rspo1 treatment reduces the histologic severity of colitis in IL-10 knockout mice treated with piroxicam. Bar = 100 μm. (B) Histologic scoring of colitis severity in IL-10 null mutant mice. Administration of Rspo1 decreases colitis disease activity in whole as well as proximal, middle, and distal segments of mouse colon deficient of IL-10. *P < .05, saline vs Rspo1. (C) SAA level in mouse peripheral blood upon harvest. Elevated SAA level in saline-treated IL-10–deficient mice was moderately suppressed by Rspo1 treatment. *P < .05, wild-type vs IL-10 knockout/saline; #P < .05, IL-10 knockout/saline vs IL-10 knockout/Rspo1. Gastroenterology 2007 132, 1331-1343DOI: (10.1053/j.gastro.2007.02.001) Copyright © 2007 AGA Institute Terms and Conditions

Figure 7 Rspo1 immunohistochemistry in mouse small intestine. Using a polyclonal Rspo1 antibody, endogenous Rspo1 immunoreactivity in (B) small intestine is detected in the epithelium lining of the villus compartment (inset C for higher magnification) and in Paneth cells in the crypt compartment (inset D for higher magnification). Only background staining is seen in small intestine when normal rabbit immunoglobulin G was used (A). (A and B) Bar = 100 μm. (C and D) Bar = 50 μm. Gastroenterology 2007 132, 1331-1343DOI: (10.1053/j.gastro.2007.02.001) Copyright © 2007 AGA Institute Terms and Conditions