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：
10303063
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 2023-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10303063
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 Intoxication 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 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.

项目摘要/摘要在拥挤的生活条件的地区，弧菌霍乱感染仍然是威胁生命的关注点和卫生不良。 El Tor变体的出现，这种菌株表现出增强的霍乱毒素（CT）生产和先天免疫激活，需要检查导致增加的因素疾病的严重程度最近暴发。 CT引起前列腺素E2（PGE2）的生产和分泌，5-羟色胺（5-HT）和许多可能调节肠道液体传输的细胞因子，而且可能有助于先天免疫反应。在我们的新颖观察结果中，发现CT B亚基诱导细胞周期停滞在主动分裂肠干/祖细胞时，我们假设这种现象可能来自与产生PGE2、5-HT，CAMP或细胞因子释放的相同信号传导。促进CT-的机理研究诱导的生物分子分泌和细胞周期停滞，我们将采用原发性未转换的人肠to 源自肠的成年干细胞。我们已经开发了一种将人类肠动物作为2-种植的方法维度上皮单层，克服了防止直接的3维培养物的局限性进入极化顶端细胞表面。该K01提案将建立对CT介导的理解使用肠环单层模型在以下目的：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，药理学抑制剂研究以及分泌的PGE2和细胞因子ELISA。使用最近报道的一种用肠球菌细胞谱系富集肠培养的方法，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。这会包括评估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）确定起源使用EDU掺入CT诱导的细胞周期停滞的持续时间，FACS分析细胞周期阶段，和RNA-seq比较CT，CTB，PGE2、5-HT，CAMP和特定的转录变化细胞因子。这些目标不仅会提高对CT诱导的病理生理学的理解临床意义，但它们还将在细胞因子检测/定量中促进技术技能的发展， CRISPR/CAS9驱动的基因编辑和计算生物信息学分析RNA-Seq数据。我的建议高素质的科学和专业榜样委员会以及资源丰富的环境约翰·霍普金斯大学医学院的胃肠病学系将指导我的发展从指导的研究人员到腹泻疾病和肠干领域的独立科学家由肠道微生物病原体引起的细胞作用。