Background: Down Regulated in Adenoma (DRA) or SLC26A3 is the key anion transporter involved in chloride absorption in the intestine. Slc26a3 polymorphism has been linked to the pathogenesis of IBD, and DRA deficiency is a critical event underlying IBD-associated diarrhea. DRA knockout (DRAKO) mice exhibit some of the key features of IBD, including diarrhea, thinner mucus layer, compromised barrier integrity, and dysbiosis. Alterations in gut microbiota and their metabolic functions are increasingly implicated in IBD pathogenesis. In this regard, bile acids (BAs) have emerged as an important class of microbiota-associated metabolites that regulate intestinal homeostasis. The gut microbiome generates secondary BAs and is responsible for the deconjugation and desulfation of BAs, all impaired in dysbiosis. However, whether gut dysbiosis induced by DRA deficiency alters bile acid metabolism/homeostasis to promote inflammation and diarrhea remains unknown and was the focus of the current study. Methods: The analysis of BAs in feces (collected over a 24-hour period) of DRAKO and wildtype (WT) littermate mice was done using LC-MS/MS (McGuire Research Institute Mass Spectrometry Facility, Richmond, VA). RNA was extracted from the mucosa of the ileum of DRAKO and WT mice (Qiagen RNeasy mini kit). RT-PCR and western blotting were used to assess mRNA and protein expression changes. Results: Loss of DRA resulted in a significant increase in the total fecal bile acids (33.59 ± 12.04 nanomoles/24 hr WT 114.9 ± 28.55 nanomoles/24 hr DRA KO P< 0.03). DRAKO mice exhibited a significant decrease in the fecal secondary to the primary bile acid ratio (1.10 ± 0.36 WT vs. 0.22 ± 0.07 DRAKO, P< 0.04), similar to changes observed in IBD patients. The ratio of unconjugated to conjugated fecal bile acid in feces was significantly increased in DRA deficiency (79 ± 6.06 WT vs. 151 ± 15.44 DRAKO, P< 0.002). The relative abundance of Sulfated bile acids such as 7-sulfo-cholic acid (35.35 ± 7.3 WT vs. 69.53 ± 5.31 DRA KO, P< 0.005). In addition, the mRNA (1.21 ± 0.27 WT vs 2.46 ± 0.39 DRA KO, P< 0.03) and protein (0.82 ± 0.02 WT vs 1.24 ± 0.12 DRA KO, P< 0.04) expression of the ileal bile acid transporter ASBT were significantly increased in DRAKO mice. Also, there was a significant increase in the mRNA expression of hepatic CYP7A1(2.1 ± 0.46 WT vs 4.1 ± 0.70 DRA KO, P< 0.04), the enzyme responsible for the rate-limiting step of bile acid synthesis. Conclusion: DRAKO mice exhibit an altered fecal bile acid profile similar to that observed in IBD patients, which is likely due to associated dysbiosis. Our novel data suggests that the decrease in DRA expression in IBD leads to the pro-inflammatory profile of BA that may contribute to the exacerbation of intestinal inflammation and associated diarrhea. (Supported by VA/NIH).

Shubha Priyamvada

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