Reactive oxygen species limit intestinal mucosa-bacteria homeostasis in vitro

Interactions between epithelial and immune cells with the gut microbiota have wide-ranging effects on many aspects of human health. Therefore, there is value in developing in vitro models capable of performing highly controlled studies of such interactions. However, several critical factors that enable long term homeostasis between bacterial and mammalian cultures have yet to be established. In this study, we explored a model consisting of epithelial and immune cells, as well as four different bacterial species (Bacteroides fragilis KLE1958, Escherichia coli MG1655, Lactobacillus rhamnosus KLE2101, or Ruminococcus gnavus KLE1940), over a 50 hour culture period. Interestingly, both obligate and facultative anaerobes grew to similar extents in aerobic culture environments during the co-culture period, likely due to measured microaerobic oxygen levels near the apical surface of the epithelia. It was demonstrated that bacteria elicited reactive oxygen species (ROS) production, and that the resulting oxidative damage heavily contributed to observed epithelial barrier damage in these static cultures. Introduction of a ROS scavenger significantly mitigated oxidative damage, improving cell monolayer integrity and reducing lipid peroxidation, although not to control (bacteria-free culture) levels. These results indicate that monitoring and mitigating ROS accumulation and oxidative damage can enable longer term bacteria-intestinal epithelial cultures, while also highlighting the significance of additional factors that impact homeostasis in mammalian cell-bacteria systems.