A Human Enteroid Model of Cholera Toxin Pathophysiology

项目摘要/摘要 霍乱弧菌感染在生活条件拥挤的地区仍然是一个威胁生命的问题。 以及糟糕的卫生条件。El Tor变种的出现，一种表现出增强霍乱毒素(CT)的菌株 生产和先天免疫激活，需要检查导致增加的因素 最近暴发的疾病严重程度。CT诱导前列腺素E2(PGE2)、5-羟色胺的产生和分泌 (5-羟色胺)，以及许多细胞因子，不仅可能调节肠道液体运输，还可能有助于 先天免疫反应。在我们新的观察中，发现CT B亚单位可以诱导细胞周期停滞 在积极分裂肠道干/祖细胞的过程中，我们假设这种现象可能是由 产生PGE2、5-羟色胺、cAMP或细胞因子释放的相同信号。为了促进CT的机理研究- 诱导生物分子分泌和细胞周期停滞，我们将使用原代未转化的人肠道样体 来源于肠道的成体干细胞。我们已经开发出一种方法，可以将人的肠样培养成2- 三维上皮单分子层，克服了三维培养阻止直接 接近极化的顶端细胞表面。此K01建议书将建立对CT中介的理解 使用肠样单层模型的先天性免疫反应和细胞周期失调 目的：我们将1)确定CT如何通过CRISPR/Cas9-诱导PGE2合成和IL-8分泌。 引导受体敲除，药物抑制剂研究，以及分泌PGE2和细胞因子的ELISA法。vbl.使用 最近报道的一种用肠嗜铬细胞系丰富肠样培养物的方法，我们将2) 确定CT如何增加细胞外5-羟色胺，以及这如何促进细胞因子的释放。这将是 包括直接或间接刺激5-羟色胺分泌、5-羟色胺转运体功能的评估 (SERT)和肠细胞5-羟色胺受体在细胞因子产生中的作用。最后，我们将确定原点 和使用EDU掺入的CT诱导的细胞周期停滞的持续时间，细胞周期时相的FACS分析， 和RNA-Seq比较CT、CTB、PGE2、5-HT、cAMP和特异性诱导的转录变化 细胞因子。这些目标不仅将促进对CT诱导的病理生理学的理解，而且具有潜在的 临床意义，但它们也将促进细胞因子检测/定量的技术技能发展， CRISPR/Cas9驱动的基因编辑，以及用于分析RNA-Seq数据的计算生物信息学。我的建议 高素质科学和专业模范委员会和资源丰富的环境 约翰霍普金斯大学医学院的胃肠病学部将指导我的发展 从腹泻病和肠干领域的指导研究员到独立科学家 肠道微生物病原体引起的细胞效应。

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.