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.

项目摘要/摘要： 本研究的目标是定义细胞特异性调控网络和细胞对铍反应的关键驱动因素 (Be) 导致器官部位的肉芽肿性肺病、慢性铍病 (CBD) 参与。依靠调查团队互补且独特的专业知识，我们将定义致病性 CBD、其前体（Be 致敏；BeS）和类似环境诱导的途径和风险因素 疾病。在遗传易感宿主的环境中，暴露于吸入的 Be 抗原会启动 Th1 免疫反应，通过 HLA II 类抗原呈递细胞 (APC) 上的抗原呈递发生 CD4+ T 细胞的背景。随后，CD4+ T 细胞和 APC 被募集至肺部、增殖、产生 细胞因子和趋化因子，最终形成肉芽肿。 HLA-DPB1 等位基因的患病率增加 在 CBD 和 BeS 中发现了第 69 位氨基酸 (E69) 的谷氨酸，尽管这种变体最多见于 40% 的暴露工人没有 BeS 或 CBD，这表明其他因素或基因调控形式 在疾病发病机制中具有重要意义。其他免疫介导疾病的越来越多的数据表明，表观遗传 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。表观遗传 修饰决定 T 细胞和 APC/巨噬细胞的分化以及随后的免疫反应 关键基因的 DNA 甲基化和组蛋白修饰，从而影响健康和疾病。我们之前的工作 证明 DNA 甲基化变化与基因表达和疾病状态的改变相关 CBD 受试者的支气管肺泡灌洗 (BAL) 细胞与 BeS 和对照组相比，包括关键免疫 反应基因和网络。单细胞测序技术已成为一种关键方法 表征新的细胞群和基因表达的细胞特异性变化。根据这些信息，我们 假设接触 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，最终导致肉芽肿性肺病的风险。我们将测试这个 通过三个具体目标提出假设，主要关注巨噬细胞和 CD4+ T 细胞，但承认 其他细胞群很重要，并以替代方法和未来方向考虑它们。在 目标 1 我们将使用单细胞 ATAC-seq 和 CITE 来表征肺免疫细胞对 Be 暴露的反应 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来定义 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监管网络 使用批量 ATAC-Me 和 RNA 测序对样本进行分析，重点关注巨噬细胞和 CD4+ T 细胞。我们将 使用 CRSIPR-dCas9 对目标 1 和 2 的多组学分析结果进行功能验证以进行更改 甲基化、染色质可及性和基因表达被确定为 Be in 反应的关键驱动因素 目标 3 中培养的巨噬细胞和 T 细胞系。最终，该提案将增强我们对 CBD 病因学涉及的新基因、调控途径和网络以及分子机制。