ObjectiveThe epithelial layer of the GI tract is equipped with innate immune receptors to sense invading pathogens. Dysregulation in innate immune signalling pathways is associated with severe inflammatory diseases, but the responsiveness of GI epithelial cells to bacterial stimulation remains unclear.DesignWe generated 42 lines of human and murine organoids from gastric and intestinal segments of both adult and fetal tissues. Genome-wide RNA-seq of the organoids provides an expression atlas of the GI epithelium. The innate immune response in epithelial cells was assessed using several functional assays in organoids and two-dimensional monolayers of cells from organoids.ResultsResults demonstrate extensive spatial organisation of innate immune signalling components along the cephalocaudal axis. A large part of this organisation is determined before birth and independent of exposure to commensal gut microbiota. Spatially restricted expression of Toll-like receptor 4 (Tlr4) in stomach and colon, but not in small intestine, is matched by nuclear factor kappa B (NF-κB) responses to lipopolysaccharide (LPS) exposure. Gastric epithelial organoids can sense LPS from the basal as well as from the apical side.ConclusionWe conclude that the epithelial innate immune barrier follows a specific pattern per GI segment. The majority of the expression patterns and the function of TLR4 is encoded in the tissue-resident stem cells and determined primarily during development.

The proliferative and functional heterogeneity among seemingly uniform cells is a universal phenomenon. Identifying the underlying factors requires single-cell analysis of function and proliferation. Here we show that the pancreatic beta-cells in zebrafish exhibit different growth-promoting and functional properties, which in part reflect differences in the time elapsed since birth of the cells. Calcium imaging shows that the beta-cells in the embryonic islet become functional during early zebrafish development. At later stages, younger beta-cells join the islet following differentiation from post-embryonic progenitors. Notably, the older and younger beta-cells occupy different regions within the islet, which generates topological asymmetries in glucose responsiveness and proliferation. Specifically, the older beta-cells exhibit robust glucose responsiveness, whereas younger beta-cells are more proliferative but less functional. As the islet approaches its mature state, heterogeneity diminishes and beta-cells synchronize function and proliferation. Our work illustrates a dynamic model of heterogeneity based on evolving proliferative and functional beta-cell states.

Epstein-Barr virus (EBV) is best known for infection of B cells, in which it usually establishes an asymptomatic lifelong infection, but is also associated with the development of multiple B cell lymphomas. EBV also infects epithelial cells and is associated with all cases of undifferentiated nasopharyngeal carcinoma (NPC). EBV is etiologically linked with at least 8% of gastric cancer (EBVaGC) that comprises a genetically and epigenetically distinct subset of GC. Although we have a very good understanding of B cell entry and lymphomagenesis, the sequence of events leading to EBVaGC remains poorly understood. Recently, ephrin receptor A2 (EPHA2) was proposed as the epithelial cell receptor on human cancer cell lines. Although we confirm some of these results, we demonstrate that EBV does not infect healthy adult stem cell-derived gastric organoids. In matched pairs of normal and cancer-derived organoids from the same patient, EBV only reproducibly infected the cancer organoids. While there was no clear pattern of differential expression between normal and cancer organoids for EPHA2 at the RNA and protein level, the subcellular location of the protein differed markedly. Confocal microscopy showed EPHA2 localization at the cell-cell junctions in primary cells, but not in cancer cell lines. Furthermore, histologic analysis of patient tissue revealed the absence of EBV in healthy epithelium and presence of EBV in epithelial cells from inflamed tissue. These data suggest that the EPHA2 receptor is not accessible to EBV on healthy gastric epithelial cells with intact cell-cell contacts, but either this or another, yet to be identified receptor may become accessible following cellular changes induced by inflammation or transformation, rendering changes in the cellular architecture an essential prerequisite to EBV infection.

The human gastric epithelium forms highly organized gland structures with different subtypes of cells. The carcinogenic bacterium Helicobacter pylori can attach to gastric cells and subsequently translocate its virulence factor CagA, but the possible host cell tropism of H. pylori is currently unknown. Here, we report that H. pylori preferentially attaches to differentiated cells in the pit region of gastric units. Single-cell RNA-seq shows that organoid-derived monolayers recapitulate the pit region, while organoids capture the gland region of the gastric units. Using these models, we show that H. pylori preferentially attaches to highly differentiated pit cells, marked by high levels of GKN1, GKN2 and PSCA. Directed differentiation of host cells enable enrichment of the target cell population and confirm H. pylori preferential attachment and CagA translocation into these cells. Attachment is independent of MUC5AC or PSCA expression, and instead relies on bacterial TlpB-dependent chemotaxis towards host cell-released urea, which scales with host cell size.

The human gastrointestinal tract is in constant contact with microbial stimuli. Its barriers have to ensure co-existence with the commensal bacteria, while enabling surveillance of intruding pathogens. At the centre of the interaction lies the epithelial layer, which marks the boundaries of the body. It is equipped with a multitude of different innate immune sensors, such as Toll-like receptors, to mount inflammatory responses to microbes. Dysfunction of this intricate system results in inflammation-associated pathologies, such as inflammatory bowel disease. However, the complexity of the cellular interactions, their molecular basis and their development remains poorly understood. In recent years, stem cell–derived organoids have gained increasing attention as promising models for both development and a broad range of pathologies, including infectious diseases. In addition, organoids enable the study of epithelial innate immunity in vitro. In this review, we focus on the gastrointestinal epithelial barrier and its regional organization to discuss innate immune sensing and development.

No abstract