PROJECT SUMMARY Inflammatory bowel disease (IBD), comprised of Crohn’s disease and Ulcerative colitis, is a chronic relapsing disorder that affects 1.4 million Americans including an estimated 600 per 100,000 veterans causing malabsorption, bloody diarrhea, strictures, fistulas, and infection. In addition, the chronic inflammatory state increases the risk for colorectal cancer with poorer prognosis and reduced survival rates compared to non-IBD patients. Immunotherapy is increasingly effective; however, refractory disease and relapse are not well understood. My long-term goal is to apply my expertise in epithelial cell biology to understand how the intestinal epithelium forms an effective barrier against microbes. The goal of this proposal is to understand how the microscopic surface architecture of the gut is disrupted in and contributes to IBD pathogenesis and bacterial infection. The epithelial cells lining the intestine are positioned at the interface of the intestinal microbes and the gut-associated immune system. Enterocytes are the most abundant cell type and are coated with ~2000 small hair-like protrusions, microvilli (MV) that facilitate nutrient absorption and serve as a protective barrier from the millions of bacteria concentrated inside the gut. MV are the initial site of contact between intestinal epithelial cells and luminal bacteria. Perturbations in MV structure and density result in nutrient malabsorption and osmotic diarrhea as seen in IBD and infection with pathogenic bacteria. A recent study showed that even in uninflamed tissue, patients with Crohn’s disease have lower levels of MV structural proteins. These findings imply MV defects are not only present in IBD during active inflammation but persist despite therapy and may contribute to relapse. Interestingly, our preliminary data show that mice lacking the MV structural protein CDHR2 recapitulate several of the epithelial defects seen in Crohn’s patients, providing a unique in vivo model system. In this proposal, we will test the hypothesis that defects in normal enterocyte apical surface structure and function persist in the absence of active inflammation and provide a path for microbial entry driving inflammation, infection, and disease relapse. In Aim 1, we will use Crohn’s patient biopsy tissue and patient stem cell-derived intestinal enteroids to characterize the fundamental ultrastructural defects present in Crohn’s disease. In Aim 2, we will challenge CDHR2 intestinal knockout mice and Crohn’s patient stem cell-derived intestinal enteroids with Fusobacterium nucleatum and Enterococcus faecalis infection to assess the functional consequences of defective MV structure on defense against bacterial infection/colonization, susceptibility to colitis, and healing in response to inflammation. By the completion of these studies, we will have identified defects in enterocyte structure in Crohn’s patients and characterized how these defects contribute to persistent changes in the microbial content. These epithelial structural defects may provide a new target for therapeutic intervention. Finally, through these studies, a promising young physician scientist will gain new skills in basic and translational studies of intestinal inflammation and bacterial infection under the guidance of a highly accomplished mentorship committee of experts in epithelial biology, host- microbe interactions, and inflammation. These new mentored skills will form the foundation for this junior investigator to achieve a long-term goal to be an independently funded academic physician scientist.

PROJECT NARRATIVE Inflammatory bowel disease, comprised of Crohn’s disease and Ulcerative colitis, impacts an estimated 600 per 100,000 veterans causing significant morbidity (malabsorption, bloody diarrhea, strictures, fistulas, infection, and surgical resection) as well as mortality (increased rates of colorectal cancer with decreased survival). Medical therapy has improved but relapse remains common and is not well understood. The luminal surface of the intestinal epithelium is covered with a densely-packed array of microvilli that function in nutrient absorption and host defense. Crohn’s patients have decreased levels of proteins that maintain the structural integrity of the intestinal microvilli in areas of the gut without significant inflammation. In this proposal we will perform experiments using Crohn’s patient biopsy tissue, patient-derived enteroid culture, and in vivo mouse experiments to understand how defects in microvilli contribute to Crohn’s relapse and infection.