A Human Enteroid Model of Cholera Toxin Pathophysiology

项目总结/摘要 在居住条件拥挤的地区，霍乱弧菌感染仍然是一个威胁生命的问题 卫生条件差。El Tor变种的出现，一种表现出增强的霍乱毒素（CT）的菌株 生产和先天免疫激活，有必要检查的因素，有助于增加 最近疫情的严重程度。CT促进前列腺素E2（PGE 2）、5-羟色胺（5-HT）的产生和分泌 （5-HT）和许多细胞因子，它们不仅可以调节肠道液体转运，还可以促进 先天免疫反应在我们的新观察中，发现CT B亚单位诱导细胞周期停滞 在活跃分裂的肠干/祖细胞中，我们假设这种现象可能源于 产生PGE 2、5-HT、cAMP或细胞因子释放的相同信号传导。为了促进CT的机制研究- 诱导的生物分子分泌和细胞周期阻滞，我们将采用原代未转化的人肠样细胞， 来源于肠的成体干细胞。我们已经开发出一种方法，以2- 三维上皮单层，克服了三维培养的限制， 进入极化的顶端细胞表面。本K 01提案将建立对CT介导的 先天性免疫应答和细胞周期失调使用肠单层模型， 目的：我们将1）确定CT如何通过CRISPR/Cas9- 1诱导PGE 2合成和IL-8分泌。 指导的受体敲除、药理学抑制剂研究以及分泌的PGE 2和细胞因子ELISA。使用 最近报道了一种用肠嗜铬细胞谱系富集肠样培养物的方法，我们将2） 确定CT如何增加细胞外5-HT以及这如何有助于细胞因子释放。这将 包括评价5-HT分泌的直接或间接刺激、5-羟色胺转运蛋白的功能 （SERT），和肠上皮细胞5-HT受体在细胞因子产生中的作用。最后，我们将确定 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、PGE 2、5-HT、cAMP和特异性 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， 临床意义，但它们也将促进细胞因子检测/定量方面的技术技能发展， CRISPR/Cas9驱动的基因编辑和计算生物信息学分析RNA-Seq数据。我的咨询 高素质科学和专业榜样委员会 约翰霍普金斯大学医学院胃肠病学系将指导我的发展 从一个指导研究员到一个独立的科学家在肠道疾病和肠道干细胞领域， 由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.