Using Multi-Omics to Define Regulators and Drivers of Granulomatous Inflammation and Chronic Beryllium Disease

使用多组学来定义肉芽肿性炎症和慢性铍病的调节因素和驱动因素

• 批准号: 10569103
• 负责人: LISA A MAIER
• 金额: $ 64.77万
• 依托单位: NATIONAL JEWISH HEALTH
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-09 至 2026-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10569103
• 关键词: ATAC-seq; Alleles; Amino Acids; Antigen Presentation; Antigen-Presenting Cells; Antigens; Berylliosis; Beryllium; Biological Markers; Bronchoalveolar Lavage; CD4 Positive T Lymphocytes; COVID-19; Cell Culture Techniques; Cell Differentiation process; Cell Line; Cells; Cellular Indexing of Transcriptomes and Epitopes by Sequencing; Chromatin; Chronic berylliosis; DNA Methylation; DNA analysis; Data; Development; Disease; Drug Targeting; Environment; Environmental Exposure; Epigenetic Process; Epitopes; Etiology; Exposure to; Flow Cytometry; Future; Gene Expression; Genes; Genetic; Genetic Polymorphism; Genetic Predisposition to Disease; Genomic approach; Glutamic Acid; Goals; Granuloma; Granulomatous; HLA-DPB1 gene; Health; Heritability; Hour; Immune; Immune Response Genes; Immune response; Inflammation; Inhalation; Inherited; Innate Immune Response; Investigation; Life; Lung; Macrophage; Measures; Mediating; Methods; Methylation; Modification; Molecular; Organ; Pathogenesis; Pathogenicity; Pathway interactions; Persons; Population; Positioning Attribute; Predisposition; Prevalence; Proliferating; Proteins; Pulmonary Fibrosis; Regulation; Regulatory Pathway; Risk; Risk Factors; Sampling; Site; Structure of parenchyma of lung; System; T-Lymphocyte; Technology; Testing; Time; Transcriptional Regulation; Validation; Variant; Work; XCL1 gene; airway epithelium; asthmatic airway; biomarker identification; chemokine; cytokine; disorder risk; epigenome; fibrotic lung; granulomatous lung disease; histone modification; member; multiple omics; neoantigens; novel; peripheral blood; recruit; response; single cell sequencing; targeted treatment; transcriptome; transcriptome sequencing

PROJECT SUMMARY/ABSTRACT: The goal of this study is to define cell-specific regulatory networks and key drivers of cellular response to beryllium (Be) that result in the granulomatous lung disease, chronic beryllium disease (CBD), at the site of organ involvement. Relying on the complementary, unique expertise of investigative team we will define pathogenic pathways and risk factors for CBD, its precursor (Be sensitization; BeS) and similar environmentally induced diseases. Exposure to an inhaled Be antigen(s), in the setting of a genetically susceptible host, initiates a Th1 immune response, with antigen presentation occurring via HLA Class II on antigen presenting cell (APC) in the context of CD4+ T cells. Subsequently, CD4+ T cells and APCs are recruited to the lung, proliferate, produce cytokines and chemokines, and eventually form granulomas. An increased prevalence of HLA-DPB1 alleles with a glutamic acid at amino acid position 69 (E69) is found in CBD and BeS, although this variant is found in up to 40% of Be exposed workers without BeS or CBD, suggesting that other factors or forms of genetic regulation are important in disease pathogenesis. Growing data in other immune-mediated diseases suggests that epigenetic mechanisms in combination with genetic susceptibility and environment may help explain disease risk. Epigenetic modifications determine T cell and APC/macrophage differentiation and the ensuing immune response through DNA methylation and histone modifications of key genes and thus impact health and disease. Our previous work demonstrated DNA methylation changes associated with alterations in gene expression and disease state in bronchoalveolar lavage (BAL) cells of CBD subjects compared to BeS and controls, including pivotal immune response genes and networks. Single cell sequencing technologies have emerged as a key method to characterize novel cell population and cell-specific changes in gene expression. Based on this information, our hypothesis is that exposure to Be alters epigenetic marks, impacting gene expression and immune cell differentiation in cell-specific manner, and ultimately risk of granulomatous lung disease. We will test this hypothesis through three specific aims, focusing mainly on macrophages and CD4+ T cells but acknowledging that other cell populations are important and considering them in alternative approaches and future directions. In Aim 1 we will characterize the lung immune cell response to Be exposure using single cell ATAC-seq and CITE- seq to measure chromatin accessibility, gene expression levels and protein epitopes at four timepoints to define key drivers in specific cell populations. In Aim 2 we will validate the regulatory networks in uncultured lung samples, focusing on macrophages and CD4+ Tcells, using bulk ATAC- Me and RNA-sequencing. We will functionally validate the results from our multi-omic analysis from Aims 1 and 2 using CRSIPR-dCas9 to change methylation, chromatin accessibility, and gene expression at loci identified as key drivers of response to Be in cultured macrophage and T cell lines in Aim 3. Ultimately, this proposal will enhance our understanding of the novel genes, regulatory pathways and networks, and molecular mechanisms involved in CBD etiology.

项目总结/摘要： 本研究的目的是确定细胞特异性调节网络和细胞对铍反应的关键驱动因素 (Be)导致肉芽肿性肺病，慢性铍病（CBD），在器官部位 参与。依靠调查团队的互补性和独特的专业知识，我们将确定致病性 CBD、其前体（Be敏化; BeS）和类似环境诱导的 疾病在遗传易感宿主的环境中，暴露于吸入的Be抗原，启动Th 1 免疫应答，其中抗原呈递通过HLA II类在抗原呈递细胞（APC）上发生， CD 4 + T细胞。随后，CD 4 + T细胞和APC被募集到肺中，增殖，产生 细胞因子和趋化因子，并最终形成肉芽肿。HLA-DPB 1等位基因的患病率增加， 在CBD和BeS中发现氨基酸位置69处的谷氨酸（E69），尽管该变体在多达100%的人中发现， 40%的Be暴露工人没有BeS或CBD，这表明其他因素或形式的遗传调节是 在疾病发病机制中重要。在其他免疫介导疾病中，越来越多的数据表明， 与遗传易感性和环境相结合的机制可能有助于解释疾病风险。后生 修饰决定T细胞和APC/巨噬细胞的分化以及随后的免疫应答， 关键基因的DNA甲基化和组蛋白修饰，从而影响健康和疾病。我们以前的工作 研究表明，DNA甲基化变化与基因表达和疾病状态的改变有关， 与BeS和对照相比，CBD受试者的支气管肺泡灌洗（BAL）细胞，包括关键免疫细胞， 反应基因和网络。单细胞测序技术已经成为一种关键方法， 表征新的细胞群体和基因表达的细胞特异性变化。根据这些信息，我们 一种假说认为，暴露于Be会改变表观遗传标记，影响基因表达和免疫细胞 以细胞特异性方式分化，并最终导致肉芽肿性肺病的风险。我们将测试这个 假设通过三个具体的目标，主要集中在巨噬细胞和CD 4 + T细胞，但承认 其他细胞群体也很重要，并在替代方法和未来方向中考虑它们。在 目的1：我们将使用单细胞ATAC-seq和CITE-1来表征肺免疫细胞对Be暴露的应答。 seq测量四个时间点的染色质可及性、基因表达水平和蛋白质表位， 特定细胞群的关键驱动因素。在目标2中，我们将验证未培养肺中的调控网络 样本，重点是巨噬细胞和CD 4 + T细胞，使用批量ATAC-Me和RNA测序。我们将 使用CRSIPR-dCas 9从功能上验证我们的目标1和2的多组学分析的结果， 甲基化、染色质可及性和基因表达，这些基因座被鉴定为对Be应答的关键驱动因素， 培养的巨噬细胞和T细胞系。最终，这一建议将加强我们对 新的基因，调控途径和网络，以及CBD病因学中涉及的分子机制。