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， 胃肠道CD没有治愈方法。进一步了解裁谈会的研究可能会引起 更有效的诊断标志物和治疗方法。巨噬细胞是免疫系统的组成部分。 它们的工作是吞噬有害微生物，消化它们，并引发T细胞和B细胞的适应性反应。 健康人固有层巨噬细胞（LP M Φ）功能与正常人相比有显著差异。 CD. CD患者的LP M Φ具有分泌炎性细胞因子和促进Th1和Th17的能力 免疫反应，而健康的LP M Φ促进更多的保护性T细胞反应。分子 CD患者中引起LP M Φ异常的机制仍知之甚少。我假设 染色质可及性驱动与结肠炎表型/CD相关的LP M Φ表型变化。 这一假设将通过两个目标进行检验。目标1将确定LP M Φ的操作是否可行 IllO-/-巨噬细胞中的染色质足以恢复生理LP M Φ表型。使用高- 通过小分子筛选I确定溴结构域和末端外结构域的抑制剂 蛋白质，如（+）-JQ1，足以改变可接近的染色质并防止IL 10-/- 巨噬细胞我将评估（+）-JQ1降低IL-10水平下全基因组染色质可及性的能力 可变的可接近区域和防止体外炎性细胞因子分泌。我也会决定 用（+）-JQ1处理无菌Il10-/-小鼠足以预防结肠炎在用（+）-JQ1定殖后的发作。 无特定病原体的细菌。在目标2中，我将鉴定并功能性表征LP M Φ调节蛋白。 与人类CD相关的程序。对于目标2的第一部分，我将从健康和CD患者中分离LP M Φ 并通过ATAC-seq和RNA-seq确定染色质可及性的全基因组变化 无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选择可接近的染色质区域的强度，已知参与 炎症信号通路，并且如果已知它们含有CD相关变体。我将评估 使用电泳迁移率变动分析，这些选择的区域结合转录因子的能力， 通过荧光素酶报告基因测定评价它们对转录水平的影响。总之，这些目标将突出 LPM Φ染色质可及性的变化显著影响了LPM Φ在细胞内的功能改变， CD发病机制，并满足我作为博士的技术要求。候选人在遗传学课程和 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.