Epithelial relay of microbial signals coordinates intestinal macrophage supported barrier repair

The gastrointestinal tract is colonized by trillions of microorganisms collectively known as the gut microbiota. These microbes provide essential signals to support healthy gut function. The microbiota is separated from internal tissue by a single layer of intestinal epithelial cells that not only provides a physical barrier but also relays luminal signals to underlying gut immune cells. Altered microbiota composition including loss of anti-inflammatory microbes or outgrowth of mucosa-associated bacteria such as adherent-invasive E. coli (AIEC) are hallmarks of inflammatory disease including inflammatory bowel disease (IBD). In contrast to their hypothesized role in pathology, we recently identified select AIEC isolates that improve outcomes in mouse colitis models. These AIEC induce macrophage production of the anti-inflammatory cytokine IL-10 which limits gut inflammation and supports barrier repair. These benefits were lost if the AIEC was unable to attach to epithelial cells. However, the epithelial signaling underlying this protection remained unclear. To understand if intestinal epithelial cells signaled to immune cells after microbial attachment, we utilized human colonic organoid monolayers and found co-culture with a subset of AIEC isolates upregulated immune regulatory genes including CCL2, a macrophage recruiting chemokine. This effect was only observed in undifferenced epithelial cells, indicating epithelial stem cell recognition of microbes leads to macrophage recruitment. In vivo, antibody blockade of CCR2 abrogated the protective effect of AIEC colonization. Using bacterial transcriptome analysis, we identified high flagellin expression in AIEC isolates that activated epithelial signaling, with lost signaling in organoids deficient for TLR5, the receptor for flagellin. Together our findings suggest intestinal epithelial cells recognize microbial signals to coordinate macrophage recruitment that support intestinal repair, protecting from colitis. Overall design: To understand epithelial responses to adherent microbes, we utilized human colonoid-derived monolayers as a model to study epithelial responses to different E. coli strains. Three human colonoid lines were used in this study: ASC103, MSK161N and MSK162N. Human colonoid monolayers cultured in transwell inserts were either maintained as undifferentiated (UD) monolayers or differentiated for 5 days as differentiated (DF) monolayers. Both UD and DF monolayers were left untreated (NT) or colonized with AIEC 541-15 or non-AIEC T75. Cells were harvested at 4 hours post colonization using Qiagen RNeasy kits. Each treatment (NT, T75, 541-15) were technically duplicated or triplicated.