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

项目概要/摘要 克罗恩病（CD）是一种复发和缓解的胃肠道炎症性疾病， 对受影响儿童的健康和生活质量造成毁灭性影响。尽管抗肿瘤坏死因子的使用增加， 在过去的二十年里，人口水平的狭窄并发症和手术率下降， 没有被观察到。我们的多中心研究计划的长期目标是定义细胞和 分子过程驱动CD儿童的疾病并发症，并利用这一新知识， 新的治疗方法。我们在本合同期内取得了重大进展， 建立儿童CD中活性氧（ROS）产生变异的遗传基础， 与儿童狭窄并发症有关。在治疗前的多变量模型中，回肠表达较高的 编码线粒体蛋白质的基因与狭窄率较低相关。动物模型研究 显示益生元2 '-岩藻糖基乳糖将诱导保护性线粒体基因签名。基于 基于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 '-岩藻糖基乳糖引发的微生物代谢物提供了一种新的 加强这种保护机制。我们提出以下补充目标， 测试我们的假设： ·在目标1中，我们将定义CD患者中的回肠上皮细胞和中性粒细胞甲基化组和转录组 有和没有NADPH氧化酶复合物基因突变。这些研究将定义甲基化组， 携带遗传变异的CD患者的回肠上皮细胞和中性粒细胞的转录组 与ROS产生相关，并将测试这些特征在患者来源的类肠中的稳定性。 ·在目标2中，我们将测试错义变体和2 '-岩藻糖基乳糖引发的微生物代谢产物的影响 使用诱导多能干细胞（iPSC）对ROS产生和细胞代谢转录组的影响 衍生模型系统。我们将区分携带NADPH氧化酶错义变体的iPSC， 中性粒细胞和小肠上皮类器官，并将决定脂质过氧化的影响 产物和2 '-岩藻糖基乳糖引发的微生物代谢产物后，ROS的生产和线粒体 在这些模型系统中的生物发生甲基化组和转录组。 创新与影响。我们将利用原代回肠细胞和新的iPSC衍生模型系统来测试遗传学。 IEC和PMN ROS产生和线粒体功能的环境调节，以及2 '- FL引发微生物代谢产物以改善细胞功能。这些研究将提供重要的见解， 基本的细胞过程调节狭窄并发症在儿科CD，并将告知新的 使用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.