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: 10339740
• 负责人: LISA A MAIER
• 金额: $ 65.96万
• 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/10339740
• 关键词: ATAC-seq; Alleles; Amino Acids; Antigen Presentation; Antigen-Presenting Cells; Antigens; 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; Genetic Transcription; 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; 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; base; chemokine; cytokine; disorder risk; epigenome; fibrotic lung; gene network; genomic locus; granulomatous lung disease; histone modification; macrophage; 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： 这项研究的目的是定义细胞特异性调节网络和细胞对铍反应的关键驱动因素 （BE）在器官部位导致肉芽肿性肺部疾病，慢性铍病（CBD） 参与。依靠调查团队的互补，独特的专业知识，我们将定义病原 CBD的途径和风险因素，其前体（敏化； BES）和类似的环境诱导 疾病。在遗传易感宿主的情况下，接触吸入的是抗原，启动了Th1 免疫反应，抗原呈递通过HLA II类在抗原呈递细胞（APC）中发生 CD4+ T细胞的上下文。随后，CD4+ T细胞和APC被募集到肺部，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了 在CBD和BES中发现了氨基酸位置69（E69）处的谷氨酸 40％的无BES或CBD的暴露工人表明，其他因素或遗传调节的形式是 在疾病发病机理中很重要。其他免疫介导的疾病中的数据增长表明表观遗传 结合遗传易感性和环境的机制可能有助于解释疾病的风险。表观遗传学 修改确定T细胞和APC/巨噬细胞分化以及随之而来的免疫反应 关键基因的DNA甲基化和组蛋白修饰，从而影响健康和疾病。我们以前的工作 证明了与基因表达和疾病状态改变有关的DNA甲基化变化 与BES和对照组相比，CBD受试者的支气管肺泡灌洗（BAL）细胞，包括关键免疫 响应基因和网络。单细胞测序技术已作为关键方法出现 表征新的细胞群体和基因表达细胞特异性变化。基于此信息，我们 假设是暴露是改变表观遗传标记，影响基因表达和免疫细胞 细胞特异性方式的分化，并最终有肉芽肿性肺部疾病的风险。我们将测试这个 通过三个特定目标的假设，主要集中于巨噬细胞和CD4+ T细胞，但承认 其他细胞群体很重要，并以其他方法和未来的方向考虑它们。在 AIM 1我们将使用单细胞ATAC-SEQ和CITE-表征肺免疫细胞反应是暴露的 在四个时间点上测量染色质访问性，基因表达水平和蛋白质表位以定义的SEQ 特定细胞种群中的关键驱动因素。在AIM 2中，我们将验证未培养的肺部的监管网络 使用大量的Atacme和RNA测序，以巨噬细胞和CD4+ TCELLS为重点。我们将 使用CRSIPR-DCAS9从AIMS 1和2验证我们的多摩尼克分析的结果 甲基化，染色质的可及性和基因座的基因表达，被确定为反应的关键驱动因素 AIM 3中培养的巨噬细胞和T细胞系。最终，该建议将增强我们对 涉及CBD病因的新型基因，调节途径和网络以及分子机制。