Genetic Regulation of Tissue Fibrosis in Human Intestinal Organoids

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 克罗恩病的并发症(CD)。持续的狭窄并发症需要手术的比率，尽管 增加抗肿瘤坏死因子α疗法的使用增加了这项工作的紧迫性。肠上皮细胞间的相互作用 CD中的细胞、髓样细胞和肌成纤维细胞调节伤口愈合和纤维化之间的平衡。这个 该项目的总体目标将是开发一种新型的人类肠道器官(HIO)模型系统来测试 调节组织炎症反应和胶原产生的遗传机制。我们的初步数据 提示肌成纤维细胞在肠道产生外源性活性氧(ROS)中起着关键作用 上皮性DUOX2和中性粒细胞NOX2-NADPH氧化酶调节上皮样细胞和髓样细胞的产生 包括转化生长因子β在内的促进肌成纤维细胞活化和细胞外基质的炎性细胞因子 制作。我们发现，DUOX2中功能丧失的遗传变异与低三倍相关 Cd狭窄的比率，同时Cyba、NCF1、NCF2和NCF4基因的功能丧失遗传变异 由NOX2复合体组成的血管狭窄发生率高出三倍。CD患者的临床分析 回肠活检确定了编码上皮和髓系炎症信号驱动的基因表达程序 在那些后来发展为狭窄的人中，肌成纤维细胞的激活和胶原的产生。一种新型的微扰剂 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。 我们已经建立了可诱导多能干细胞(IPSC)来源的人类肠道器官和中性粒细胞- 伴有和不伴有DUOX2和NCF1突变的CD患者的类细胞，并证实ROS的变异 制作。这将为检测组织纤维化的基因调控提供一种新的模型系统。我们假设 NADPH氧化酶功能的遗传变异调节ROS的产生以及上皮和髓系 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 以下目标中的假设：在目标1中，我们将区分带有和不带有DUOX2的等基因IPSC 突变成人类肠道器官。我们将测试微生物产品对有机物ROS和 炎性细胞因子的产生、胶原含量和组织硬度。我们将使用基因验证研究结果 CD患者回肠活检和手术切除标本的表达和胶原含量数据。在……里面 目的2我们将具有和不具有NCF1突变的等基因IPSC分化为中性粒样细胞和人类 肠道器官。我们将确定中性粒细胞产物和小分子对有机物的影响 ROS和炎性细胞因子的产生、胶原含量和组织硬度。我们将使用以下工具验证调查结果 CD患者中性粒细胞和回肠标本。这些研究将解决该计划的目标 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。

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.