Genetic Regulation of Tissue Fibrosis in Human Intestinal Organoids

人类肠道类器官组织纤维化的基因调控

基本信息

批准号：
10191137
负责人：
LEE ARMISTEAD DENSON
金额：
$ 23.85万
依托单位：
CINCINNATI CHILDRENS HOSP MED CTR
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-06-15 至 2023-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10191137
关键词：
Address Atomic Force Microscopy Automobile Driving Bioinformatics Biological Models Biopsy CYBA gene Candidate Disease Gene Cells Clinical Collagen Complex Crohn&apos s disease Data Disease Epithelial Epithelial Cells Equilibrium Excision Extracellular Matrix Fibrosis Flow Cytometry Gene Expression Gene Expression Profile Genes Genetic Genetic Variation Genomics Genotype Goals Human Image Inflammasome Inflammatory Inflammatory Response Intestines Lead Measures Mesenchymal Microbe Mus Mutation Myelogenous Myeloid Cells Myofibroblast NADPH Oxidase NIH Program Announcements Operative Surgical Procedures Organoids Patients Pluripotent Stem Cells Production Proteomics Reactive Oxygen Species Regulation Research Role Sampling Severities Signal Transduction Specimen TNF gene Testing Tissues Transforming Growth Factor beta Variant Work cytokine gene function genetic testing genetic variant innovation intestinal epithelium loss of function microbial neutrophil novel novel strategies organ injury precision medicine prevent programs response second harmonic small molecule wound healing

项目摘要

The long-term goal of our research program is to define genomic and microbial mechanisms driving disease complications in Crohn's Disease (CD). Persistent rates of stricturing complications requiring surgery despite increased use of anti-TNFα therapy increase the urgency of this work. Interactions between intestinal epithelial cells, myeloid cells, and myofibroblasts regulate the balance between wound healing and fibrosis in CD. The overall objective of this project will be to develop a novel human intestinal organoid (HIO) model system to test genetic mechanisms regulating tissue inflammatory responses and collagen production. Our preliminary data suggests a critical role for myofibroblast extrinsic reactive oxygen species (ROS) production by the intestinal epithelial DUOX2 and neutrophil NOX2 NADPH oxidases in regulating production of epithelial and myeloid inflammatory cytokines including TGFβ which promote myofibroblast activation and extra-cellular matrix production. We found that loss-of-function genetic variants in DUOX2 were associated with three-fold lower rates of stricturing in CD, while loss-of-function genetic variants in the CYBA, NCF1, NCF2, and NCF4 genes comprising the NOX2 complex were associated with three-fold higher rates of stricturing. Analysis of CD patient ileal biopsies defined a gene expression program encoding epithelial and myeloid inflammatory signals driving myofibroblast activation and collagen production in those who later developed strictures. A novel perturbagen bioinformatics approach prioritized small molecules likely to reverse the pro-fibrotic gene expression signature. We have established inducible pluripotent stem cell (iPSC)-derived human intestinal organoids and neutrophil- like cells from CD patients with and without DUOX2 and NCF1 mutations, and confirmed variation in ROS production. This will provide a novel model system to test genetic regulation of tissue fibrosis. We hypothesize that genetic variation in NADPH oxidase function regulates ROS production and epithelial and myeloid inflammatory signals driving myofibroblast activation and extra-cellular matrix production. We will test this hypothesis in the following Aims: In Aim 1 we will differentiate isogenic iPSC with and without DUOX2 mutations into human intestinal organoids. We will test the effects of microbial products upon organoid ROS and inflammatory cytokine production, collagen content, and tissue stiffness. We will validate findings using gene expression and collagen content data derived from CD patient ileal biopsy and surgical resection samples. In Aim 2 we will differentiate isogenic iPSC with and without NCF1 mutations into neutrophil-like cells and human intestinal organoids. We will determine the effects of neutrophil products and small molecules upon organoid ROS and inflammatory cytokine production, collagen content, and tissue stiffness. We will validate findings using CD patient neutrophils and ileal samples. These studies will address the goals of this Program Announcement to utilize identifiable human specimens to delineate candidate gene functions in disease states, and to more precisely define the pathophysiologic mechanisms that lead to end-organ injury.

我们研究计划的长期目标是确定驱动疾病的基因组和微生物机制克罗恩病 (CD) 的并发症尽管需要手术，但狭窄并发症的持续发生率。抗 TNFα 治疗的增加增加了这项工作的紧迫性。细胞、骨髓细胞和肌成纤维细胞调节 CD 中伤口愈合和纤维化之间的平衡。该项目的总体目标是开发一种新型人类肠道类器官（HIO）模型系统来测试我们的初步数据是调节组织炎症反应和胶原蛋白产生的遗传机制。表明肠道对肌成纤维细胞外源性活性氧 (ROS) 产生的关键作用上皮细胞 DUOX2 和中性粒细胞 NOX2 NADPH 氧化酶在调节上皮细胞和骨髓细胞生成中的作用炎症细胞因子，包括促进肌成纤维细胞活化和细胞外基质的 TGFβ 我们发现 DUOX2 中功能丧失的基因变异与产量降低三倍相关。 CD 狭窄率，而 CYBA、NCF1、NCF2 和 NCF4 基因功能丧失遗传变异包含 NOX2 复合物的 CD 患者的狭窄率高出三倍。回肠活检定义了编码上皮和骨髓炎症信号驱动的基因表达程序肌成纤维细胞的激活和胶原蛋白的产生在后来出现狭窄的人中是一种新的扰动因素。生物信息学方法优先考虑可能逆转促纤维化基因表达特征的小分子。我们已经建立了诱导多能干细胞（iPSC）衍生的人类肠道类器官和中性粒细胞类似来自有或没有 DUOX2 和 NCF1 突变的 CD 患者的细胞，并证实了 ROS 的变化这将为测试组织纤维化的遗传调控提供一个新的模型系统。 NADPH 氧化酶功能的遗传变异调节 ROS 的产生以及上皮细胞和骨髓细胞驱动肌成纤维细胞激活和细胞外基质产生的炎症信号我们将对此进行测试。假设在以下目标中：在目标 1 中，我们将区分有和没有 DUOX2 的同基因 iPSC 我们将测试微生物产品对类器官 ROS 和 ROS 的影响。我们将使用基因验证炎症细胞因子的产生、胶原蛋白含量和组织硬度。来自 CD 患者回肠活检和手术切除样本的表达和胶原蛋白含量数据。目标 2 我们将具有和不具有 NCF1 突变的同基因 iPSC 分化为中性粒细胞样细胞和人类我们将确定中性粒细胞产物和小分子对类器官的影响。我们将使用 ROS 和炎症细胞因子的产生、胶原蛋白含量和组织硬度来验证研究结果。 CD 患者中性粒细胞和回肠样本这些研究将实现该计划的目标。宣布利用可识别的人类样本来描绘疾病状态下的候选基因功能，并更准确地定义导致终末器官损伤的病理生理机制。