Causes and consequences of neutrophil dysfunction in early onset Crohn's disease

项目摘要/摘要 克罗恩病(CD)是一种复发和缓解性胃肠道炎性疾病，可 对受影响儿童的健康和生活质量造成破坏性影响。尽管增加了抗肿瘤坏死因子的使用 在过去的二十年里，狭窄并发症和手术的发生率在人群水平上有所下降 还没有被观察到。我们多中心研究计划的长期目标是定义细胞和 驱动CD儿童疾病并发症的分子过程，并利用这一新知识 新的治疗方法。在本奖项颁发期间，我们取得了重大进展 建立儿童CD反应性(ROS)产生变异的遗传基础，而我们反过来也有 与儿童的狭窄并发症有关。在治疗前多变量模型中，较高的回肠表达 编码线粒体蛋白的基因与较低的狭窄率相关。动物模型的研究 表明益生素2‘-岩藻糖基乳糖可诱导保护性线粒体基因信号。基座 根据这些令人兴奋的数据，我们假设NADPH氧化酶ROS产生的基因组变异调节 细胞线粒体转录组和2‘-岩藻糖基乳糖启动的微生物代谢产物提供了一种新的 加强这一保护机制的方法。我们提出了以下补充目标，以机械地 测试我们的假设： ·在目标1中，我们将定义CD患者的回肠上皮细胞和中性粒细胞甲基组和转录组 有无NADPH氧化酶复合体基因突变。这些研究将定义甲基组和 携带基因变异的CD患者回肠上皮细胞和中性粒细胞的转录组 与ROS的产生相关，并将测试这些信号在患者来源的肠样体中的稳定性。 ·在目标2中，我们将测试错义变体和2‘-岩藻糖基乳糖启动的微生物代谢物的影响 诱导多能干细胞产生ROS及细胞代谢转录组的研究 派生模型系统。我们将把携带NADPH氧化酶错义变体的IPSC区分为 中性粒细胞和小肠上皮类物质，并将决定脂质过氧化的影响 产物和2‘-岩藻糖基乳糖启动微生物代谢产物对ROS产生和线粒体的影响 这些模型系统中的生物发生、甲基组和转录组。 创新和影响力。我们将利用原代回肠细胞和新的IPSC衍生模型系统来测试基因 以及环境对IEC和PMN ROS产生和线粒体功能的调节，以及2‘- FL启动微生物代谢物以改善细胞功能。这些研究将为 调节儿童CD狭窄并发症的基本细胞过程，并将为新的 根据分子图谱分类的患者的个性化治疗。

PROJECT SUMMARY/ABSTRACT Crohn's Disease (CD) is a relapsing and remitting gastrointestinal inflammatory disorder which can have devastating effects upon the health and quality of life of affected children. Despite the increased use of anti-TNF therapy over the past two decades, a population-level decline in rates of stricturing complications and surgeries has not been observed. The long-term goal of our multi-center research program is to define the cellular and molecular processes driving disease complications in children with CD, and to use this new knowledge to inform novel therapeutic approaches. We have made significant progress during the period of the current award in establishing a genetic basis for variation in reactive (ROS) production in pediatric CD, which we have in turn linked to stricturing complications in children. In a pre-treatment multivariable model, higher ileal expression of genes encoding mitochondrial proteins was associated with lower rates of stricturing. Studies in an animal model showed that the prebiotic 2'-Fucosyllactose would induce the protective mitochondrial gene signature. Based upon these exciting data, we hypothesize that genomic variation in NADPH oxidase ROS production regulates the cellular mitochondrial transcriptome, and 2'-Fucosyllactose primed microbial metabolites afford a novel approach to boost this protective mechanism. We propose the following complementary Aims to mechanistically test our hypotheses: • In Aim 1, we will define the ileal epithelial cell and neutrophil methylome and transcriptome in CD patients with and without NADPH oxidase complex gene mutations. These studies will define the methylome and transcriptome of ileal epithelial cells and neutrophils from CD patients carrying genetic variants associated with ROS production, and will test for stability of these signatures in patient-derived enteroids. • In Aim 2, we will test the effects of missense variants and 2'-Fucosyllactose primed microbial metabolites on ROS production and the cellular metabolic transcriptome using induced pluripotent stem cell (iPSC) derived model systems. We will differentiate iPSC carrying NADPH oxidase missense variants into neutrophils and small bowel epithelial organoids and will determine the effects of lipid peroxidation products and 2'-Fucosyllactose primed microbial metabolites upon ROS production and the mitochondrial biogenesis methylome and transcriptome in these model systems. Innovation and Impact. We will utilize primary ileal cells and novel iPSC-derived model systems to test genetic and environmental regulation of IEC and PMN ROS production and mitochondrial function, and the ability of 2'- FL primed microbial metabolites to improve cell function. These studies will provide critical insights into fundamental cellular processes regulating stricturing complications in pediatric CD, and will inform novel personalized therapies in patients classified using molecular profiles.