Abstracts Category : Other

Evaluation of potential antioxidant probiotics in in vitro models of the gut epithelium

by Sahitya Raja

Inflammatory bowel disease (IBD) is a heterogeneous group of inflammatory disorders of the gut, which includes Crohns disease (CD) and ulcerative colitis (UC). IBD is characterized by chronic intestinal inflammation and increased permeability of the gut epithelial barrier. Reactive oxygen species (ROS), generated by the immune system and epithelium as part of the inflammatory response, increase epithelial barrier permeability through lipid peroxidation, apoptosis, DNA damage, and tight junction damage. Dysbiosis of the gut microbiota plays a critical role in exacerbating inflammation; therefore, there is significant interest in the use of beneficial microbes (probiotics) in the treatment of IBD to restore a healthy microbial community. Many Lactobacillus species can reduce oxygen in culture, and are potential candidates for probiotic-based interventions to counter oxidative stress.Herein, primary epithelial cell monolayers derived from murine colonic crypts were grown in a culture system allowing for physiological polarization with apical and basolateral access. In a pilot experiment to model the intestinal barrier damage caused by ROS in IBD, monolayers were apically treated with hydrogen peroxide. Lactobacillus strains exhibiting oxygen reduction capabilities were co-cultured with epithelial monolayers. Effects to barrier permeability were assessed by measuring changes in trans-epithelial electrical resistance (TEER) and expression patterns of the tight junction marker ZO-1. Monolayers of Caco-2 BbE cells (human epithelial colorectal adenocarcinoma), often used in in vitro studies of the epithelial barrier, were similarly treated. Further, candidate strains were evaluated for potential antioxidant activity using RT-qPCR analysis of target genes in respiratory pathways.Damage to tight junctions and the cytoskeleton of Caco-2 BbE cells were observed after hydrogen peroxide treatment, with corresponding reductions in TEER. A similar treatment resulted in broad cell death of primary colonic epithelial cells, demonstrating a response distinct from typically used cell culture methods. The primary epithelial monolayer system used here to model inflammation-induced barrier damage will allow for further in vitro study of epithelial permeability; furthermore, the identified strains may be promising candidates for probiotic therapies for IBD.Advisors/Committee Members: Garrett, Wendy (committee member), Fenn, Kathrin (committee member).