A Human Enteroid Model of Cholera Toxin Pathophysiology

霍乱毒素病理生理学的人肠模型

基本信息

批准号：
10062959
负责人：
Jennifer Foulke-Abel
金额：
$ 15.82万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-01-09 至 2022-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10062959
关键词：
3-Dimensional Acute Acute Diarrhea Address Adult Advisory Committees Affect Agonist Animal Model Apical Bioinformatics CRISPR/Cas technology Cell Cycle Cell Cycle Arrest Cell Cycle Regulation Cell Lineage Cell Separation Cell surface Cells Cholera Cholera Toxin Cholera Toxin Protomer B Crowding Cyclic AMP Data Detection Development Diarrhea Dinoprostone Disease Disease Outbreaks Drosophila genus EP4 receptor Enteral Enterochromaffin Cells Enterochromaffin-like Cells Enterocytes Enteroendocrine Cell Enterotoxins Environment Enzyme-Linked Immunosorbent Assay Epithelial Epithelial Cells Evaluation Exhibits Exposure to Fluids and Secretions Fostering Functional disorder Gastroenterology Genes Genetic Transcription Goals Goblet Cells Growth Factor Homeostasis Human IL8 gene Immune Inflammation Inflammatory Infrastructure Innate Immune Response Intention Intestinal Mucosa Intestines Knock-out Knowledge Life Link Liquid substance Mediating Mentors Methods Midgut Modeling NF-kappa B Patients Pharmacology Pharmacotherapy Phase Population Process Production Professional Role Public Health Recovery Reporting Research Research Personnel Resources Rest Role Salts Sanitation Scientist Serotonin Severities Severity of illness Signal Transduction Symptoms TLR4 gene TNF gene Technical Expertise Techniques Testing Universities Variant Vibrio cholerae Vibrio cholerae infection Villus adult stem cell cell dimension cell motility cell type clinically significant cytokine diarrheal disease experience extracellular immune activation inhibitor/antagonist insight intestinal epithelium medical schools monolayer novel pathogenic microbe prevent prototype receptor recruit response role model serotonin receptor serotonin transporter skill acquisition stem stem cell population stem cells symptom management transcriptome sequencing transmission process two-dimensional

项目摘要

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.

项目概要/摘要在生活条件拥挤的地区，霍乱弧菌感染仍然是威胁生命的问题和卫生条件差。 El Tor 变种的出现，一种表现出增强霍乱毒素 (CT) 的菌株产生和先天免疫激活，有必要检查导致增加的因素最近爆发的疾病严重程度。 CT 引发前列腺素 E2 (PGE2)、血清素的产生和分泌 (5-HT) 和许多细胞因子不仅可以调节肠道液体运输，还可能有助于先天免疫反应。在我们的新观察中，发现 CT B 亚基可诱导细胞周期停滞参与活跃分裂的肠干/祖细胞，我们假设这种现象可能是由于产生 PGE2、5-HT、cAMP 或细胞因子释放的相同信号传导。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，我们将采用原代未转化的人肠类源自肠道成体干细胞。我们开发了一种将人类肠类培养为 2- 三维上皮单层，克服了三维培养的限制，防止直接进入极化的顶端细胞表面。该 K01 提案将建立对 CT 介导的理解使用肠样单层模型的先天免疫反应和细胞周期失调如下目标：我们将 1) 确定 CT 如何通过 CRISPR/Cas9 诱导 PGE2 合成和 IL-8 分泌- 指导受体敲除、药理学抑制剂研究以及分泌型 PGE2 和细胞因子 ELISA。使用最近报道了用肠嗜铬细胞谱系丰富肠类培养物的方法，我们将 2) 确定 CT 如何增加细胞外 5-HT 以及这如何促进细胞因子释放。这将包括评估直接或间接刺激 5-HT 分泌、血清素转运蛋白的功能 (SERT) 和肠细胞 5-HT 受体在细胞因子产生中的作用。最后，我们将3）确定原点和使用 EdU 掺入的 CT 诱导细胞周期停滞的持续时间、细胞周期阶段的 FACS 分析、和 RNA-Seq 来比较 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 诱发的病理生理学的理解，并具有潜在的潜力临床意义，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来分析 RNA-Seq 数据。我的咨询高素质的科学和专业榜样委员会以及丰富的资源环境约翰·霍普金斯大学医学院胃肠病学部将指导我的发展从腹泻疾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。