In vitro modeling of inflammatory bowel diseases using a newly developed immunocompetent colon epithelial monolayer co-culture model.

Clinical treatment of inflammatory bowel diseases (IBD) remains challenging due to the complex interplay between the epithelial barrier, immune system, and gut microbiota. While in vitro models are pivotal for studying barrier dysfunction, developing a standardized and functionally relevant system for IBD remains challenging. To overcome this, we established an immunocompetent murine colon epithelium monolayer to model IBD-like conditions. Colons from wild-type mice were digested into single cells and seeded onto Matrigel-coated transwells. Within seven days, monolayers showed strong barrier properties and displayed epithelial cell lineage, including goblet, stem, and enteroendocrine cells. However, exposure to pro-inflammatory cytokines as well as infection with pathogenic bacteria, including Clostridium rodentium and Salmonella Typhimurium, disrupted epithelial integrity. To better reflect the in vivo state, polarized T cells and macrophages were co-cultured with the epithelium. Pro-inflammatory Th1 and Th17 cells impaired barrier function, while M0 and M2 macrophages maintained it, representing both homeostatic and disrupted conditions of the gut. Upon Salmonella Typhimurium infection, M1 macrophages produced IFN-γ, and M2 macrophages secreted IL-10 and enhanced ZO-1 expression. Overall, our model presents a promising platform to study epithelial barrier dysfunction, immune-epithelial cross-talk, and host-pathogen interactions, offering valuable insights into IBD mechanisms and potential therapeutic approaches.