Abstract Diarrhea-predominant Irritable Bowel Syndrome (IBS-D) is the most common cause of chronic diarrhea in the United States. A diet high in fermentable oligosaccharides, disaccharides, monosaccharides, and polyols (FODMAPs) (HFM) induces symptoms in IBS-D patients and a diet low in FODMAPs (LFM) provides some symptomatic relief in ~50-60% of these patients. However, we do not understand how FODMAPs affect IBS-D symptoms. In this proposal, I plan to delineate the mechanisms of FODMAP-mediated IBS-D pathophysiology, which will help identify a subgroup of IBS-D patients with a distinct pathophysiologic pathway ultimately leading to targeted therapy with improved efficacy. My strong preliminary data suggest LFM reduces the fecal abundance of Akkermansiaceae and Enterobacteriaceae and LPS levels in IBS-D patients. Moreover, I found that LFM improves colonic barrier function and reduces mast cell activation in LFM-responsive IBS-D patients. Finally, my preliminary work from in vivo rodent models suggests that luminal LPS and mast cells play a critical role in the FODMAP-mediated colonic barrier loss. Based on these preliminary data, my overall hypothesis is that HFM increases fecal LPS levels by increasing the abundance of pathogenic gram-negative bacteria. This luminal LPS initiates colonic epithelial barrier loss and activates mast cells causing the release of tryptase which further potentiates colonic barrier loss. I will test these hypotheses in two specific aims: In specific aim 1, I will elucidate the contributions of FODMAPs in IBS-D pathophysiology by determining the effect of a strictly-controlled 4-week LFM on clinical features, relative and absolute abundance of fecal gram-negative bacteria (including those detailed above) and LPS, colonic barrier function, and mucosal mast cell activation in IBS-D patients and comparing the changes in these pathophysiologic parameters among LFM-responders and non-responders. In specific aim 2, I will define the mechanisms by which FODMAPs induce epithelial barrier loss and mast-cell activation in IBS-D. I will do so by applying IBS-D fecal supernatants (pre- and post-LFM) to human colonoids with/without TLR4 antagonists. In parallel, I will also apply fecal supernatants to mast cells from wild type and tlr4-/- mice to assess tryptase release. Finally, I will determine the contributions of PAR2 receptor and myosin light chain kinase signaling in tryptase-mediated barrier loss in human colonoid model. These aims will address current gaps in our understanding of IBS-D pathophysiology and provide mentored training in the assessment of in vivo and ex vivo epithelial barrier structure and function, microbiome analysis, and in vitro human colonoid model to study epithelial barrier regulation. My mentorship committee comprises of internationally renowned NIH-funded investigators with an exceptional track record of mentoring- Drs. Owyang (expert in IBS pathophysiology), Drs. Nusrat and Turner (experts in barrie...