Altered chromatin accessibility in lamina propria macrophages in Crohn’s disease

摘要 克罗恩病(CD)是一种慢性、异质性炎症性疾病，对 胃肠道。CD是无法治愈的。进一步加深我们对CD理解的研究可能会引起 更有效的诊断标记物和疗法。巨噬细胞是免疫系统不可或缺的一部分。 他们的工作是吞噬有害微生物，消化它们，并为T细胞和B细胞的适应性反应做好准备。 健康人固有层巨噬细胞(LP MΦ)功能与正常人相比有显著差异 CD.CD患者LP MΦS具有分泌炎性细胞因子并促进Th1、Th17的能力 免疫应答，而LP MΦS在健康中促进更多的保护性T细胞应答。分子 CD患者LP MΦS异常的发病机制尚不清楚。我假设这一点改变了 染色质可及性驱动与结肠炎表型/CD相关的LP MΦ表型改变。 这一假设将通过两个目标进行检验。目标1将确定LP MΦ的操作是否可访问 IL10-/-巨噬细胞中的染色质足以恢复生理性LP MΦ表型。使用高- 通过小分子筛选确定溴结构域和末端外结构域抑制剂 蛋白质，如()-JQ1，足以改变可获得的染色质并防止IL10-/-的炎症 巨噬细胞。我将评估()-JQ1在IL-10降低全基因组染色质可及性的能力 可变区，并防止炎性细胞因子的体外分泌。我还会决定是否 用()-JQ1治疗无菌IL10-/-小鼠足以预防结肠炎的发生 无特定病原体的细菌。在目标2中，我将确定LP MΦ调节性的功能特征 与人类CD关联的程序。对于目标2的第一部分，我将从健康和CD患者中分离LP MΦS 并通过atac-seq和rna-seq测定全基因组染色质可及性的变化和基因表达。 在匹配的未刺激和细菌刺激的样本中。对于目标2的第二部分，我将量化 基于其巨噬细胞特异性的可访问染色质区域的强度，已知参与 炎症信号通路以及它们是否已知含有与CD相关的变体。我会评估一下 用电泳迁移率改变分析和Will检测这些选定区域结合转录因子的能力 通过荧光素酶报告实验评估它们对转录水平的影响。综合起来，这些目标将突出 Lp MΦ染色质可及性的改变对Lp MΦ功能的改变有显著作用 CD的发病机制，并满足我作为遗传学和医学博士研究生课程的技术要求 分子生物学。

Abstract Crohn’s disease (CD) is a chronic, heterogeneous inflammatory disease that causes damage to the gastrointestinal tract. There is no cure for CD. Research that furthers our understanding of CD may give rise to more effective diagnostic markers and therapeutics. Macrophages are an integral part of the immune system. Their job is to engulf harmful microbes, digest them and prime T cells and B cells for an adaptive response. There are marked differences in lamina propria macrophage (LP MΦ) function in health when compared with CD. LP MΦs from CD patients have the ability to secrete inflammatory cytokines and promote Th1 and Th17 immune responses, while LP MΦs in health promote more protective T cell responses. The molecular mechanism giving rise to abnormal LP MΦs in CD patients remains poorly understood. I hypothesize that altered chromatin accessibility drives the LP MΦ phenotypic changes that are associated with colitis phenotypes/CD. This hypothesis will be tested through two aims. Aim 1 will determine if manipulation of LP MΦ accessible chromatin in Il10-/- macrophages is sufficient to restore the physiological LP MΦ phenotype. Using a high- throughput small molecule screen I determined that inhibitors of bromodomain and extra-terminal domain proteins, such as (+)-JQ1, are sufficient to alter accessible chromatin and prevent inflammation in Il10-/- macrophages. I will evaluate the ability of (+)-JQ1 to decrease genome-wide chromatin accessibility at IL-10 variable accessible regions and to prevent inflammatory cytokine secretion in vitro. I will also determine if treatment of germ free Il10-/- mice with (+)-JQ1 is sufficient to prevent onset of colitis after colonization with specific-pathogen free bacteria. In Aim 2 I will identify and functionally characterize the LP MΦ regulatory program associated with human CD. For the first part of Aim 2, I will isolate LP MΦs from healthy and CD patients and determine genome-wide changes in chromatin accessibility by ATAC-seq and gene expression by RNA-seq in matched unstimulated and bacterially-stimulated samples. For the second part of Aim 2, I will quantify the strength of select accessible chromatin regions based on their macrophage-specificity, known involvement in inflammatory signaling pathways and if they are known to contain CD associated variants. I will evaluate the ability of these selected regions to bind transcription factors using electrophoretic mobility shift assays and will evaluate their impact on levels of transcription by luciferase reporter assays. Together, these aims will highlight that changes in LP MΦ chromatin accessibility contributes significantly to altered LP MΦ function in the pathogenesis of CD and fulfil my technical requirements as a Ph.D. candidate in the curriculum in Genetics and Molecular Biology.