Genetic Regulation of Tissue Fibrosis in Human Intestinal Organoids

我们研究计划的长期目标是确定基因组和微生物驱动疾病的机制 克罗恩病（Crohn's Disease，CD）需要手术的狭窄并发症的持续发生率， 抗TNF α治疗的增加了这项工作的紧迫性。肠上皮细胞之间相互作用 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。的 本项目的总体目标是开发一种新的人类肠道类器官（HIO）模型系统，以测试 调节组织炎症反应和胶原蛋白产生的遗传机制。我们的初步数据 表明肠内肌成纤维细胞产生外源性活性氧（ROS）的关键作用， 上皮DUOX2和中性粒细胞NOX2 NADPH氧化酶在调节上皮和髓样产生中的作用 炎性细胞因子，包括促进肌成纤维细胞活化和细胞外基质的TGFβ 生产我们发现DUOX2中的功能缺失遗传变异与三倍降低的 CD狭窄率，而CYBA、NCF 1、NCF 2和NCF 4基因的功能丧失遗传变异 包含NOX2复合物的蛋白质与三倍高的狭窄率相关。CD患者分析 回肠活组织检查确定了编码上皮和骨髓炎性信号的基因表达程序， 肌成纤维细胞活化和胶原蛋白的产生。一种新型微扰剂， 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性多能干细胞（iPSC）衍生的人类肠道类器官和中性粒细胞， 来自具有和不具有DUOX2和NCF 1突变的CD患者的Like细胞，并证实了ROS的变化 生产这将提供一种新的模型系统来测试组织纤维化的遗传调控。我们假设 NADPH氧化酶功能的遗传变异调节ROS的产生， 驱动肌成纤维细胞活化和细胞外基质产生的炎性信号。我们将测试这个 在目标1中，我们将区分具有和不具有DU0X 2的同基因iPSC 突变成人类肠道类器官。我们将测试微生物产品对类器官ROS的影响， 炎性细胞因子产生、胶原蛋白含量和组织硬度。我们将使用基因验证发现， 来自CD患者回肠活检和手术切除样品的表达和胶原含量数据。在 目的2我们将有和没有NCF 1突变的同基因iPSC分化为嗜中性粒细胞样细胞和人iPSC。 肠类器官我们将确定中性粒细胞产物和小分子对类器官的影响， ROS和炎性细胞因子的产生、胶原蛋白含量和组织硬度。我们将使用 CD患者中性粒细胞和回肠样本。这些研究将解决本方案的目标 宣布利用可识别的人类标本来描绘疾病状态下的候选基因功能， 并更精确地定义导致终末器官损伤的病理生理机制。

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.