Atherogenic diet causes lethal ileo-ceco-colitis in cyclooxygenase-2 deficient mice

Abstract

Cyclooxygenases (COX) regulate a variety of inflammatory diseases, including inflammatory bowel disease (IBD). While the pathological effects of COX-1 inhibition by NSAIDs on intestinal ulceration are well established, the role of COX-2 on intestinal inflammation remains under investigation. In this paper, we report a protective role for COX-2 against diet-mediated intestinal inflammation in mice. COX-2(-/-) mice fed an atherogenic diet or diet containing cholate, but not chow or fat alone, had a high mortality whereas COX-1(-/-) mice and wild-type mice were unaffected by the dietary changes. Histological analysis identified the cause of death in COX-2(-/-) mice due to severe intestinal inflammation that was surprisingly limited to the ileo-ceco-colic junction. COX-2 expression is induced in the cecum of wild-type mice fed an atherogenic diet. Our findings show that COX-2 plays an anti-inflammatory role at the ileo-ceco-colic junction in mice, and the pathology of diet-mediated intestinal inflammation in COX-2(-/-) mice offers an excellent model system to elucidate the molecular mechanisms of intestinal inflammation.

Figure 1. Percent difference in body weight after 3 weeks of experimental diet

Body weights of individual mice on a specific diet (n=8–12 per group) were measured after fasting overnight. Weight difference is calculated as the difference in weight between the groups on experimental diet versus their chow counterparts. Mice that died were included in this analysis. * p < 0.05 vs. wild-type, ** p < 0.05 vs. chow, Student’s t-test. WT = COX-2^+/+ (wild-type), KO = COX-2^−/− (knockout), Athero = atherogenic diet

Figure 2. Gross pathology of ceca from COX-2^−/− mice

Ceca from COX-2^−/− mice on chow, atherogenic, fat or cholate diet for 3 weeks were resected and contents evacuated. A perforation at the ileo-cecal junction is noted in the specimen from the mouse on atherogenic diet (arrow). PC = proximal colon, TI = terminal ileum, C = cecum

Figure 3. COX-2 expression in cecum

Total protein (15 μg) extracted from cecum of wild-type or COX-2^−/− mice fed chow or atherogenic diet for 3 weeks were loaded on SDS-PAGE and immunoblotted for COX-2 or β-actin (upper panel). Cell lysate from NIH3T3 cells treated with phorbol 12-myristate 13-acetate (PMA: 1 μM) for 4 hours was used as a positive control for COX-2. The density of COX-2 protein band was quantified by ImageJ and normalized to β-actin (lower panel). Each bar represents the average with one standard deviation. NIH = NIH3T3, WT = COX-2^+/+ (wild-type), KO = COX-2^−/− (knockout), P = PMA, C = Chow, A = atherogenic diet

Figure 4. Inflammation at the ileo-ceco-colonic junction in COX-2^−/− mice is mediated by cholate in the diet

Ceca from male and female COX-2^+/+ or COX-2^−/− mice fed specific diets for 3 weeks were fixed and stained with H & E. All are shown at 20X magnification. Athero = atherogenic, WT = COX-2^+/+ (wild-type), KO = COX-2^−/− (knockout)

Figure 5. Histological inflammation scores

Inflammatory scores of ceca from COX-2^−/− mice fed the indicated diet for 3 weeks. Scores were equal to 0 in COX-2^−/− mice fed chow or fat and in wild-type (COX-2^+/+) littermate controls fed chow, atherogenic, cholate, or high fat diets (n=8 to 12 per group). Two-way factorial ANOVA followed by Mann Whitney tests were performed for statistical analysis. p < 0.0001 for COX-2^−/− mice fed atherogenic or cholate diet vs any other group. p = 0.304 for atherogenic vs cholate diet in COX-2^−/− mice. For detailed criteria of the inflammation score, see Materials and Methods.

Figure 6. Cecal wall of a COX-2^−/− mouse fed atherogenic diet for 4 weeks

(A) Cecum was resected, fixed with formalin and stained with H&E (4X). um = mucosal ulceration, mm = muscularis mucosae, sm = submucosa, mp = muscularis propria, and s = peritoneum (serosa). (B) Cecal wall of a COX-2^−/− mouse fed an atherogenic diet is shown at 20X. Lower panel, shown at 40X: The submucosa and muscularis propria shows a predominance of plasma cells (green arrows) and macrophages (black arrow) with some eosinophils (red arrows). m = mucosa, lp = lamina propria, sm = submucosa, mm = muscularis mucosa, and mp = the two layers of the muscularis propria.