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

摘要 炎症性肠病(IBD)是一种复杂的肠道病理类型，其特征是 遗传易感宿主适应性免疫反应和先天免疫反应的失调。精准的 病理生理学和免疫失调的分子机制尚未完全阐明。 已澄清。更好地理解这种疾病的发病机制的一个重要障碍是 肠道组织。此外，即使在相同的表型细胞子集内也可能存在实质上的异质性， 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 然而，结合单细胞蛋白质鉴定代表了一项重大的技术进步， 识别先前无法检测到的新的小区子集和状态的能力。我们建议 利用这项技术生成肠道和外周血液免疫反应的单细胞图谱 IBD的两个亚型：溃疡性结肠炎(UC)和回肠克罗恩病。这项提议的总体目标是 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的差异 开发利用外周血诊断IBD的新方法。具体目标1建议识别异常 肠道组织和外周组织中丰富的分化状态和致病细胞亚型 UC和克罗恩氏回肠炎的血液与健康对照。在基因表达上的差异 来自IBD患者和健康对照的肠道细胞将提供对IBD分子决定因素的洞察 UC和克罗恩回肠炎所特有的疾病。具体目标2建议利用单个细胞中的差异 在更大的队列患者中检测外周血中的基因表达，以开发UC和 克罗恩回肠炎。单细胞基因表达的非侵入性检测不仅有可能预测疾病 不仅包括疾病活动的严重程度，还包括潜在的治疗目标。总而言之，本提案旨在使用 单细胞基因和蛋白表达有助于了解IBD的疾病病理生理机制并开发新的研究方向 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.