A Human Enteroid Model of Cholera Toxin Pathophysiology

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PROJECT SUMMARY/ABSTRACT Vibrio cholerae infection continues to be a life-threatening concern in regions with crowded living conditions and poor sanitation. The emergence of El Tor variant, a strain that exhibits enhanced cholera toxin (CT) production and innate immune activation, necessitates examination of the factors that contribute to increased disease severity in recent outbreaks. CT elicits production and secretion of prostaglandin E2 (PGE2), serotonin (5-HT), and numerous cytokines that may not only regulate intestinal fluid transport, but may also contribute to the innate immune response. In our novel observation, the CT B subunit was found to induce cell cycle arrest in actively dividing intestinal stem/progenitor cells, and we hypothesize that this phenomenon may arise from the same signaling that yields PGE2, 5-HT, cAMP, or cytokine release. To facilitate mechanistic studies of CT- induced biomolecule secretion and cell cycle arrest, we will employ primary untransformed human enteroids derived from adult stem cells of the intestine. We have developed a method to grow human enteroids as 2- dimensional epithelial monolayers, overcoming the limitation of 3-dimensional cultures that prevents direct access to the polarized apical cell surface. This K01 proposal will establish understanding of CT-mediated innate immune response and cell cycle dysregulation using the enteroid monolayer model in the following aims: We will 1) identify how CT induces PGE2 synthesis and IL-8 secretion through CRISPR/Cas9- guided receptor knock-outs, pharmacological inhibitor studies, and secreted PGE2 and cytokine ELISA. Using a recently reported method to enrich enteroid cultures with the enterochromaffin cell lineage, we will 2) determine how CT increases extracellular 5-HT and how this contributes to cytokine release. This will include evaluation of direct or indirect stimulation of 5-HT secretion, function of the serotonin transporter (SERT), and enterocyte 5-HT receptor roles in cytokine production. Finally, we will 3) determine the origin and duration of CT-induced cell cycle arrest using EdU incorporation, FACS analysis of cell cycle phase, and RNA-Seq to compare transcriptional changes induced by CT, CTB, PGE2, 5-HT, cAMP, and specific cytokines. Not only will these aims advance understanding of CT-induced pathophysiology with potential clinical significance, but they will also foster technical skill development in cytokine detection/quantitation, CRISPR/Cas9-driven gene editing, and computational bioinformatics to analyze RNA-Seq data. My advisory committee of highly qualified scientific and professional role models and the resource-rich environment in the Division of Gastroenterology at the Johns Hopkins University School of Medicine will guide my development from a mentored researcher to an independent scientist in the field of diarrheal diseases and intestinal stem cell effects caused by enteric microbial pathogens.