Volume 131, Issue 2, Pages 579-588 (August 2006) Deletion of Mtgr1 Sensitizes the Colonic Epithelium to Dextran Sodium Sulfate–Induced Colitis  J. Andres Martinez, Christopher S. Williams, Joseph M. Amann, Tiffany C. Ellis, Isabel Moreno–Miralles, M. Kay Washington, Paul Gregoli, Scott W. Hiebert  Gastroenterology  Volume 131, Issue 2, Pages 579-588 (August 2006) DOI: 10.1053/j.gastro.2006.06.009 Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 1 Expression of Mtg family members in the colon. (A) Reverse-transcription polymerase chain reaction (RT-PCR) of isolated small intestinal and colon crypt epithelium. Agarose gel electrophoresis of the crypt epithelial samples taken from the quantitative RT-PCR (30 cycles) experiment shown in B. (−), samples lacking reverse transcriptase as controls for DNA contamination; SI, small intestine. (B) QRT-PCR shows that Mtgr1 and Mtg16 are expressed in the colon. Bar graphs of the cycle threshold values (CT) from real-time RT-PCR. (−), samples lacking reverse transcriptase as controls for DNA contamination. RNA from isolated colon or small intestinal crypt epithelium was compared with RNA from portions of the whole organ. Please note that in the experiment shown, the Mtg8 sample did not reach the CT and is therefore not graphed. Gastroenterology 2006 131, 579-588DOI: (10.1053/j.gastro.2006.06.009) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 2 Effects of targeted deletion of Mtgr1 in the colonic epithelium. (A) Histologic sections of nontreated wild-type (WT) littermate controls and Mtgr1-null mice (Null) were stained with H&E (original magnification, 200×). (B) Proliferating cells were detected using BrdU incorporation and anti-BrdU immunostaining. Positive cells at the bottom of the crypts are reddish brown. (C) Sections were probed with antibodies that recognize the activated form of caspase-3. Apoptotic cells at the top of the crypts are reddish brown (arrows). Gastroenterology 2006 131, 579-588DOI: (10.1053/j.gastro.2006.06.009) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 3 Mtgr1-null mice display a pronounced weight loss and colon shortening in response to DSS. (A) DSS-treated Mtgr1-null mice lose a larger percentage of their initial body weight compared with wild-type (WT) mice. Wild-type and Mtgr1-null mice were fed drinking water supplemented with 3% DSS for days 1–4 and changed to normal drinking water for days 5–7. Their relative weights were graphed in relation to the initial weight set at 1.0 (WT and Null H2O, n = 10; WT and Null DSS, n = 32). (B) The colon lengths of treated Mtgr1-null mice were significantly decreased compared with wild-type mice. Colons were measured for length after water or DSS treatment as indicated (*P = .013, n = 4). Gastroenterology 2006 131, 579-588DOI: (10.1053/j.gastro.2006.06.009) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 4 DSS treatment causes a more severe colitis in Mtgr1-null mice. (A) DSS-treated Mtgr1-null mice display denuding of the colonic epithelium. H&E-stained sections of nontreated (H2O control) wild-type (WT) and Mtgr1-null mice, mice treated with DSS for 4 days (DSS day 4), or mice treated with DSS for 4 days and allowed to recover for 3 days (DSS day 7) (original magnification, 200×). (B) DSS-treated Mtgr1-null mice display a more extensive degree of colonic injury. Low-power image (original magnification, 40×) with high-power insets (original magnification, 200×) of an H&E section of the colon 7 days after initiation of DSS treatment that was rolled before embedding in paraffin. The center of the roll represents the distal colon. Note the inflammatory changes of the mid- and distal colon by day 7. Wild-type mice developed full-thickness inflammation but in a limited and patchy distribution (arrow). The colitis in Mtgr1-null mice is more severe and extensive, involving most of the mid- and distal colon. (C) Quantification of DSS-induced injury. Grades were assigned, in blinded fashion, according to the percentage of colon involved with crypt damage: 0 (none), 1 (1%–25%), 2 (26%–50%), 3 (51%–75%), or 4 (>76%); *Statistical significance at day 7 (P = .011). Gastroenterology 2006 131, 579-588DOI: (10.1053/j.gastro.2006.06.009) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 5 Mice who underwent transplantation with Mtgr1-null marrow responded like wild-type mice to DSS treatment. Wild-type mice or wild-type mice that were lethally irradiated and underwent transplantation with bone marrow from either wild-type or Mtgr1-null donors were treated with 3% DSS for 4 days and then allowed to recover for 3 days. Segments of the colon were splayed and rolled and were stained with H&E. Lower-power photographs (original magnification, 40×) and higher-power insets (original magnification, 200×) show a similar degree of inflammation and crypt damage at day 7. WT Control, a section from a wild-type mouse treated with DSS for 7 days; Null marrow, a wild-type recipient mouse that underwent transplantation with Mtgr1-null bone marrow. (B) Weight loss in mice that underwent transplantation with Mtgr1-null marrow. Relative weights of mice that underwent transplantation with Mtgr1-null bone marrow were graphed in relation to the initial weight set at 1.0 and compared with wild-type controls. H2O Control, water control mice. (C) Quantification of DSS-induced injury in the transplanted mice. Grades were assigned, in blinded fashion, according to the percentage of colon involved with crypt damage: 0 (none), 1 (1%–25%), 2 (26%–50%), 3 (51%–75%), and 4 (>76%) for the indicated groups. Gastroenterology 2006 131, 579-588DOI: (10.1053/j.gastro.2006.06.009) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 6 Loss of Mtgr1 sensitizes the colonic epithelium to apoptosis. (A) Identification of cells undergoing apoptosis. Colon sections taken from nontreated (H2O control) littermate control mice, mice treated for 4 days with DSS in their drinking water (day 4), and mice treated with DSS and allowed to recover for 3 days (day 7) were stained with anti-activated caspase-3 (original magnification, 200×). Positive cells are stained reddish brown (arrow). (B) DSS increases the number of TUNEL-positive cells in the colons of Mtgr1-null mice. Wild-type and Mtgr1-null mice were fed water or 3% DSS in water for 4 days and apoptotic cells identified using TUNEL assays. Sections from day 7 are not shown due to the extensive amount of apoptosis. Positive cells are stained reddish brown. (C) The apoptosis index was calculated as the number of caspase-3–positive cells per 100 consecutive crypts (*statistical significance at day 4, P = .043, n = 8). Quantification of the apoptotic index in the Mtgr1-null mice at day 7 was not performed due to the low number of surviving crypts in the Mtgr1-null mice (N/A). Gastroenterology 2006 131, 579-588DOI: (10.1053/j.gastro.2006.06.009) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 7 Surviving Mtgr1-null stem cells and progenitor cells retain the capacity to proliferate. (A) BrdU staining showed increased proliferation in Mtgr1-null mice compared with wild-type mice (H2O control). Proliferation decreased acutely after DSS treatment in both wild-type and null mice (day 4) but was increased from baseline during the recovery phase (day 7). Surviving crypts of Mtgr1-null mice showed a similar increase in proliferation (original magnification, 200×). (B) The proliferation index was calculated as the number of BrdU-positive staining cells per crypt (*P = .04, n = 9). N/A, not applicable because there were too few surviving crypts to enumerate. Gastroenterology 2006 131, 579-588DOI: (10.1053/j.gastro.2006.06.009) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions

Figure 8 DSS induces long-term histologic changes in the colons of Mtgr1-null mice. (A) Mtgr1-null mice show slow weight recovery after DSS treatment. Groups of 4–5 wild-type and 4–5 Mtgr1-null mice were allowed to recover on normal water for a longer period of time. The graph depicts daily weights for the first 11 days posttreatment of one of the replicates. One Mtgr1-null mouse died at day 7. (B) DSS-treated Mtgr1-null mice display a long-term colonic injury. At 6 and 10 weeks after the initiation of DSS treatment, the colon was sectioned and stained with H&E. Wild-type (WT) colons had mostly healed by week 6 and completely recovered by week 10. However, Mtgr1-null (Null) colons showed changes at weeks 6 and 10 that were consistent with chronic colitis, including acute and chronic inflammatory infiltration (white arrow) and colonic architectural distortion, including gland dropout and bifid glands (black arrowheads). Shown in the upper panels are low-magnification photographs (original magnification, 50×) with higher-power insets (original magnification, 200×). Lower panels show high-power photographs so that the inflammatory cell infiltrates can be observed. Gastroenterology 2006 131, 579-588DOI: (10.1053/j.gastro.2006.06.009) Copyright © 2006 American Gastroenterological Association Institute Terms and Conditions