Dietary Indole‐3‐Carbinol Supplementation Restores Epithelial Abnormalities in SAMP1/YitFc Mouse Model of Ileitis

Abstract

SAMP1/YitFC (SAMP) mice exhibit spontaneous ileitis as they age with morphological and functional epithelial abnormalities mimicking human Crohn’s disease, a chronic disease characterized by ileal inflammation. Indole‐3‐carbinole (I3C), found in broccoli and Brussel sprouts, plays a crucial role in the anti‐inflammatory processes by activating the aryl hydrocarbon receptor (AhR), a transcription factor vital for epithelial and immune cell functions. The impact of I3C diet on disease progression and epithelial integrity in SAMP model of ileitis is not known.Methods6‐week‐old male and female (n=8 per group) SAMP and control AKR/J mice (Jackson) were fed for 14 weeks either a standard chow diet or a purified diet supplemented with 200 ppm with indole‐3‐carbinole (+I3C diet), or purified diet depleted of AhR ligands (‐I3C diet). Ileum samples were collected for qPCR and histological analyses. Statistical analysis was performed by Tukey’s multiple comparison test for all three diets. To detect changes between +I3C vs. ‐I3C, delta values (Δ +/‐I3C = SAMP‐AKR) were analyzed by unpaired T‐test.ResultsAs expected, H&E staining for the ileum of SAMP mice fed chow diet, showed thickening of the muscle layer, neutrophil infiltration, and epithelial tufting vs. AKR mice. Severity of inflammation in SAMP mice, as assessed by fecal calprotectin ELISA, mRNA expression of cytokines, and histological analysis was not significantly impacted by dietary depletion or supplementation of I3C. With respect to epithelial abnormalities, Periodic Acid Schiff staining showed increased number of goblet cells per villi (~47‐75%) in SAMP mice on all three diets. While mRNA levels of the major goblet cell secretory proteins, mucin2 (Muc2) and intestinal trefoil factor (ITF), were dramatically decreased in SAMP mice vs. AKR, supplementation of I3C attenuated this decrease in Muc2 and ITF by ~35% and 51% (p<.001), respectively, indicating defective synthesis or secretion. Similarly, I3C diet significantly (p<.01) attenuated the decrease in Paneth cell markers lysozyme C‐1 (LYZ1) alpha defensin 5 (Def5α) and regenerating islet‐derived protein 3 alpha (Reg3α) in SAMP mice by ~65%, 60%, and 24%, respectively. A decrease in fibroblast growth factor 15 (FGF15) and the epithelial ion transporters serotonin transporter (SERT) and organic solute transporter (OST) were observed in ileal mucosa of SAMP mice vs. AKR, and the supplementation of I3C mitigated these decreases by 30‐58% (p<.01). There were no significant changes in the expression of tryptophan hydroxylase 1 (tph‐1), C‐X‐C motif chemokine ligand 2 (CXCL2), or AhR by I3C diet in SAMP mice. In summary, I3C supplementation significantly attenuated the goblet cell, Paneth cell, and epithelial‐specific abnormalities seen in SAMP mice. These data imply that dietary supplementation of AhR ligands may be a viable therapeutic strategy for Crohn’s patients to restore epithelial integrity.