USING SINGLE-CELL RNA AND PROTEIN APPROACHES TO ELUCIDATE MOLECULAR DETERMINANTSAND PREDICTORS OF INFLAMMATORY BOWEL DISEASES

摘要 炎症性肠病（IBD）代表一系列复杂的肠道病理学，其特征在于 遗传易感宿主的适应性和先天性免疫反应失调。的精确 病理生理学和免疫失调的分子机制尚未完全阐明， 阐明。更好地理解疾病发病机制的一个重要障碍是疾病的异质性。 肠组织此外，即使在相同的表型细胞亚群内也可能存在实质性异质性， 限制了使用批量RNA测序或流式细胞术检测差异的能力。单细胞rna测序 和单细胞蛋白鉴定相结合，然而，代表了一个重大的技术进步， 识别先前无法检测到的新细胞子集和状态的能力。我们提出 利用该技术生成肠和外周血免疫应答的单细胞图谱 在IBD的两个子集中：溃疡性结肠炎（UC）和回肠克罗恩病。本建议的总体目标是 确定组织和血液中IBD的分子决定因素，然后利用这些基因表达差异， 利用外周血开发IBD的新型诊断测试。具体目标1建议识别异常 在肠组织和外周血中富集的分化状态和致病细胞亚型 与健康对照相比，来自UC和克罗恩氏回肠炎的血液。基因表达的差异， 来自IBD患者和健康对照的肠细胞将提供对IBD的分子决定因素的深入了解。 UC和克罗恩氏回肠炎的特异性疾病。具体目标2建议利用单个细胞中的差异 在一个更大的患者队列中从外周血中检测基因表达，以开发UC的诊断检测， 克罗恩氏回肠炎。单细胞基因表达的非侵入性检测不仅可以预测疾病， 状态以及疾病活动的严重程度与潜在的治疗靶点。总而言之，这项建议旨在利用 单细胞基因和蛋白质表达，以了解IBD疾病病理生理学并开发新的 诊断生物标志物。

Abstract Inflammatory bowel diseases (IBD) represent a spectrum of complicated intestinal pathology characterized by dysregulation of the adaptive and innate immune responses in genetically susceptible hosts. The precise molecular mechanisms underlying the pathophysiology and immune dysregulation have yet to be fully elucidated. A significant barrier to better understanding of the disease pathogenesis is the heterogeneity of intestinal tissue. Furthermore, substantial heterogeneity may exist even within the same phenotypic cell subset, limiting the ability to detect differences using bulk RNA sequencing or flow cytometry. Single cell RNA sequencing and single cell protein identification in combination, however, represent a significant technologic advance with the capability to identify novel cell subsets and states that could not be previously detected. We propose exploiting this technology to generate a single cell atlas of the intestinal and peripheral blood immune response in two subsets of IBD: ulcerative colitis (UC) and ileal Crohn’s disease. The overall objective of this proposal is to define molecular determinants of IBD in tissue and blood, then to harness these differences in gene expression to develop novel diagnostic testing for IBD using peripheral blood. Specific Aim 1 proposes to identify abnormal states of differentiation and pathogenic cell subtypes that are enriched in the intestinal tissue and peripheral blood from UC and Crohn’s ileitis as compared to healthy controls. The differences in gene expression between the intestinal cells from IBD patients and healthy controls will provide insight into molecular determinants of disease that are specific to UC and Crohn’s ileitis. Specific Aim 2 proposes to utilize the differences in single cell gene expression from the peripheral blood in a larger cohort of patients to develop diagnostic testing for UC and Crohn’s ileitis. Non-invasive testing with single cell gene expression has the potential to predict not only disease status but also severity of disease activity with potential therapeutic targets. In sum, this proposal aims to use single cell gene and protein expression to understand disease pathophysiology of IBD and develop novel diagnostic biomarkers.