Abstract
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.
Highlights
Interactions between epithelial and immune cells with the gut microbiota have wide-ranging effects on many aspects of human health
The epithelial-immune cultures were exposed in the apical compartment to 6.67 × 1 04 CFU per mL of one of four bacterial species: L. rhamnosus KLE2101, B. fragilis KLE1958, E. coli MG1655, or R. gnavus KLE1940, which were selected based on their presence in the human commensal gut microbiota and the variety of oxygen requirements, motility, and Gram status that they represent (Table 1)
It is noted that L. rhamnosus is considered a probiotic species as it has been found to promote cell growth and wound healing, and is sold as a commercial supplement[16,33]
Summary
Interactions between epithelial and immune cells with the gut microbiota have wide-ranging effects on many aspects of human health. Reports of static cultures incorporating immune components, dendritic c ells[15,19] or macrophage simulating differentiated THP-1 cells[20], and bacteria result in high accumulation of dead epithelial cells and significant increases in paracellular permeability when compared to control cultures, even when cultured with bacteria for as little as 2 h This suggests that flow is an important factor in preserving epithelial health and barrier function. As the addition of an antioxidant can ameliorate damage to the epithelial monolayer induced by bacterial co-culture, oxidative damage likely plays a significant role in epithelial monolayer damage in the presence of microbes These findings highlight the significance of ROS and oxidation in the establishment of homeostasis in intestinal models incorporating microbes and demonstrate mechanisms underlying the importance of flow in microbe-mammalian co-cultures, and are of broad significance in development of in vitro systems for studying microbiome-host interactions and their role in health and disease
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