Enteroendocrine regulation of intestinal barrier function - PROJECT SUMMARY Enteroendocrine cells (EECs) are rare sensory cells scattered throughout the gastrointestinal epithelium that interface between environmental stimuli like nutrients and microbes and the body's response, secreting over 20 distinct hormones that act locally and systemically. Our lab uses EEC-deficient mice and human intestinal organoids to uncover the intestinal functions regulated by EECs and uses these models as a blank slate to test the roles of individual EEC-derived products on intestinal physiology. EECs are often dysregulated in metabolic and gastrointestinal diseases, such as inflammatory bowel disease (IBD), although their roles in disease pathogenesis remain unknown. Many of these diseases are associated with impaired function of the intestinal epithelial barrier, allowing undigested food, microbes, metabolites, and toxins to cross the epithelium, triggering local and systemic inflammation. Tight junctions are essential for barrier integrity and are composed of several proteins which are supported by sphingolipids called ceramides, best characterized in the skin. In the intestine, barrier proteins are often reduced in IBD, along with several species of ceramides. Similarly, loss of ceramides within the intestinal epithelium exacerbates chemically-induced colitis and supplementation of ceramides improves colitis in mouse models. We discovered that several species of ceramides are reduced or absent in EEC-deficient mouse small intestine. Moreover, EEC-deficient human intestinal organoids display increased barrier permeability and upregulate an inflammatory gene signature. In preliminary experiments, we found that EEC-deficient mice also display impaired barrier function with increased permeability and markers of inflammation. When challenged with oral ingestion of dextran sulfate sodium (DSS), which classically induces colitis, EEC-deficient mice displayed significantly greater weight loss compared to controls. These data led us to hypothesize that EECs promote a strong barrier by regulating ceramide abundance and may be effective therapeutics for inflammation-mediated barrier dysfunction. Our first aim is to determine the role of EECs in the structure-function relationship between tight junctions and ceramides using in vitro human intestinal organoid cultures. Our second aim is to define the role of EECs in DSS-induced disease progression and recovery, and to test the role of the EEC hormone PYY in protecting the gut against DSS-induced damage. Our third aim is to perform targeted analysis of sphingolipid metabolic pathways of human intestinal organoids and murine intestine to define the mechanism by which EECs participate in ceramide biosynthesis in homeostasis and in disease. We expect that restoration of exogenous PYY to EEC-deficient models will improve barrier function and mitigate the severity of DSS-induced disease by increasing the abundance of ceramides within the epithelium. These experiments will define a new role for EECs in maintaining gastrointestinal homeostasis, uncover a novel mechanism regulating barrier integrity in health and disease, and provide a basis for future therapies aimed at repairing a leaky gut.
