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： 这项研究的目标是确定细胞特有的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疾病--慢性铍病(CBD) 参与其中。依靠调查团队互补的、独特的专业知识，我们将定义病原体 CBD、其前体(BE敏化；BES)和类似环境诱导的途径和风险因素 疾病。暴露于吸入的BE抗原(S)，在遗传易感宿主的环境中，启动Th1 免疫应答，通过抗原提呈细胞(APC)上的HLAII类递呈发生 关于CD4+T细胞的背景。随后，CD4+T细胞和APC被招募到肺中，增殖，产生 细胞因子和趋化因子，最终形成肉芽肿。随着人类白细胞抗原-DPB1等位基因的增加， 在CBD和BES中发现了位于氨基酸第69位(E69)的谷氨酸，尽管该变体在 40%的暴露于BE的工人没有BES或CBD，这表明其他因素或形式的基因调控 在疾病的发病机制中很重要。其他免疫介导性疾病的不断增长的数据表明，表观遗传学 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 修饰通过以下途径决定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 证实DNA甲基化变化与基因表达和疾病状态的变化有关 CBD患者的BAL细胞与BES和对照组的比较，包括关键免疫 反应基因和网络。单细胞测序技术已经成为一种关键方法 描述新的细胞群体和细胞特有的基因表达变化。根据这一信息，我们的 假说是，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，最终有患肉芽肿性肺部疾病的风险。我们将对此进行测试 通过三个特定的目标提出假说，主要关注巨噬细胞和CD4+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上考虑它们。在……里面 目的1我们将用单细胞ATAC-SEQ和CITE-SEQ来表征暴露的肺免疫细胞反应。 SEQ在四个时间点测量染色质可及性、基因表达水平和蛋白质表位，以确定 特定细胞群体中的关键驱动因素。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CD4+T细胞，使用批量ATAC-Me和RNA测序。我们会 使用CRSIPR-dCas9更改从功能上验证来自AIMS 1和2的多体分析的结果 甲基化、染色质可及性和基因表达被确定为反应的关键驱动因素 在AIM 3中培养巨噬细胞和T细胞系。最终，这项建议将增强我们对 涉及CBD病因学的新基因、调控途径和网络以及分子机制。