Premium
Reduced Expression of Down‐Regulated in Adenoma (DRA) in Murine Enteroid‐Derived Monolayers Following Infection with Salmonella
Author(s) -
Jayaratne Rashini Randika,
Lee BeomJae,
Lim Eileen,
Barrett Kim,
Das Soumita
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.1007.15
Subject(s) - salmonella , internalization , salmonella enterica , microbiology and biotechnology , enteroendocrine cell , salmonella infection , biology , crypt , paneth cell , cell , small intestine , bacteria , endocrinology , endocrine system , genetics , hormone
Non‐typhoidal Salmonella spp. are the second leading cause of foodborne illness worldwide. Despite its prevalence, the mechanism through which Salmonella infection causes diarrhea is not fully understood. The intestinal epithelium plays a major role in both interacting with bacteria and fluid accumulation. To understand the basic mechanism of Salmonella infection in intestinal epithelial cells, we developed a murine enteroid model that mimics the crypt‐villus axis of the intestines, and contains the four different epithelial subtypes found in the intestine (enterocytes, enteroendocrine, goblet, and Paneth cells). We have previously shown that infection with Salmonella causes decreased expression of the chloride/bicarbonate exchanger SLC26A3 (DRA; Downregulated in Adenoma) in the murine colon. We developed enteroid‐derived monolayers (EDM) from mouse ileum and colon to test whether DRA was similarly down‐regulated following infection with Salmonella . Monolayers were generated from enteroids on either permeable transepithelial supports or cell culture plates. Monolayers were also grown in 8‐well chamber slides to visualize DRA and bacterial internalization. After differentiation, EDM were infected with Salmonella enterica serovar Typhimurium. Expression of DRA and markers for different cell subtypes were studied using RT‐PCR, western blotting, and confocal microscopy. We also measured bacterial internalization using a gentamicin protection assay followed by colony counts. We found bacterial internalization after 1 h followed by decreased expression of DRA after 6 h of Salmonella infection. Interestingly, following infection the proliferation marker Ki67 was upregulated. The intestines contain both secretory (enteroendocrine, goblet, and paneth cells) and absorptive (enterocytes) cell types and their expression was evaluated following infection. The Wnt/NOTCH signaling pathways control cell fate decisions in the intestines. We found that Hes1, a downstream signaling molecule in the NOTCH signaling pathway, was up regulated following infection. Our work shows the potential for EDM to identify molecular mechanisms that may underlie intestinal dysfunction during infection. Ultimately, this model may identify signals that can be targeted for therapeutic development.