Decoding the Interferome by Mapping Genetic Interactions in Human Tissue

通过绘制人体组织中的遗传相互作用来解码干扰素

• 批准号: 10725446
• 负责人: Ryan Gilbert Gaudet
• 金额: $ 48.31万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-10 至 2028-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10725446
• 关键词: 3-Dimensional; Anatomy; Antibacterial Response; Bacterial Infections; Biological; Biological Assay; Bordetella pertussis; Buffers; CRISPR screen; Cell Communication; Cell Lineage; Cells; Chlamydophila pneumoniae; Chromosome Mapping; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Complex; Cytokine Signaling; Cytosol; Endothelium; Epithelium; Fibroblasts; Frustration; Future; Gene Combinations; Genes; Genetic; Genetic Screening; Genetic Transcription; Genomics; Goals; Health; Host Defense; Host resistance; Human; Human Genome; Image; Immune; Immune response; Immune signaling; Immune system; Immunity; Immunology; Individual; Infection; Interferon Type II; Interferons; Intestines; Link; Lung; Lymphocyte; Macrophage; Maps; Mediating; Mediator; Methods; Microbe; Modeling; Molecular; Network-based; Organ; Organ Donor; Organoids; Outcome; Pathologic; Pathology; Pathway interactions; Pattern; Phenotype; Postdoctoral Fellow; Predisposition; Proteins; Protozoan Infections; Role; Shapes; Shelter facility; Signal Transduction; Site; Structure; Structure of parenchyma of lung; System; Techniques; Tissues; Validation; Virus Diseases; Vision; Work; airway epithelium; arm; bactericide; cell type; cellular imaging; combat; combinatorial; cytokine; cytokine release syndrome; empowerment; functional outcomes; gene function; gene network; gene product; genomic locus; human model; human tissue; immunopathology; infancy; innovation; insight; intestinal epithelium; lung pathogen; mutant; novel; novel strategies; pathogen; programs; response; single-cell RNA sequencing; synergism; technology development; therapeutic target; virtual

PROJECT SUMMARY Non-immune cells greatly outnumber professional immune cells in the body, providing intracellular pathogens with many opportunities to shelter and take refuge. How does the immune system protect this huge landscape? My previous work described the ability of the immune cytokine interferon-g (IFN-g), classically considered a macrophage activating protein, to broadly activate non-immune cells and confer the ability to mount sterilizing cell-intrinsic responses through effectors encoded by Interferon-Stimulated Genes (ISGs), collectively termed the ‘interferome’. However, our understanding of how ISGs execute tissue localized responses is in its infancy, with remarkably few of these effectors being well characterized and virtually nothing known about how multiple ISGs functionally interact to achieve host defense. The principal barrier to understanding ISGs is that they rarely work in isolation; rather they are part of complex genetic networks that are buffered from the phenotypic effects of perturbation. I propose a radical new strategy that exploits this genetic complexity by mapping functional relationships between ISGs using combinatorial forward genetic screens. My central hypothesis is that systematically mapping genetic interactions will uncover the effectors of localized IFN-g signalling and enable hierarchical organization of ISG products into functional complexes and pathways (network) that execute specific protective and pathological responses. On a small scale with a single query gene in my postdoctoral work, this approach unveiled a pair of synergistic ISGs that execute unexpectedly potent bactericidal defense of the cytosol. Further development of this technology for use at a larger scale in my own lab will provide the framework to fully deconvolve the interferome into distinct host resistance pathways. I will develop an experimental pipeline that enlists a pooled CRISPR-based screening system for multi-locus gene perturbation paired with a novel single cell imaging and expression analysis platform. Focusing on the human airway epithelium, this innovative approach will be applied to dissect the ISG-encoded effectors that mediate cell-intrinsic control of three diverse pulmonary pathogens (C. pneumoniae, B. pertussis, and RSV), and those that mediate lung tissue damage downstream of cytokine storm. Newly identified ISGs and their connections will be validated for their protective or pathological activities using tissue explant systems from donated human lungs and models of human lung organoids. The ensuing genetic interaction network will provide unprecedented insight into the effectors that dictate infection outcome in the human lung during type 1 immune responses. These findings will reveal new local therapeutic targets and establish a paradigm for appreciating the full spectrum of immunity. The approaches pioneered here will also extend to the downstream effectors of other non-immune cells and tissues whose integrated study will define a new biological landscape of critical importance to human health.

项目摘要 非免疫细胞在体内大量的外部数字专业免疫细胞，提供细胞内病原体 有很多机会庇护和避难。免疫系统如何保护这一巨大的景观？ 我以前的工作描述了免疫细胞因子干扰素G（IFN-G）的能力，从经典看来 巨噬细胞激活蛋白质，以广泛激活非免疫细胞并赋予安装灭菌的能力 通过由干扰素刺激的基因（ISG）编码的效应来产生细胞中的响应，统称为 “干扰素”。但是，我们对ISGS如何执行组织局部响应的理解仍处于起步阶段， 这些效果中很少有很好的特征，并且几乎对多重效果一无所知 ISG在功能上进行互动以实现主机防御。理解ISGS的主要障碍是他们很少 孤立工作；相反，它们是从表型效应中缓冲的复杂遗传网络的一部分 扰动。我提出了一种根本的新策略，该策略通过绘制功能来利用这种遗传复杂性 ISG之间使用组合远期遗传筛选之间的关系。我的中心假设是 系统地绘制遗传相互作用将发现局部的IFN-G信号传导的影响，并启用 ISG产品的层次结构组织成功能复合物和途径（网络），以执行特定 保护性和病理反应。在我的博士后工作中，在小规模上带有一个查询基因，这 方法揭示 细胞质。在我自己的实验室中，该技术的进一步开发将在我自己的实验室中更大规模使用 完全将干扰素解析为不同的宿主电阻途径。我将开发一个实验管道 这招募了一个基于CRISPR的基于CRISPR的筛选系统，用于多基因基因扰动与新颖 单细胞成像和表达分析平台。专注于人类气道上皮，这种创新性 将应用方法来剖析ISG编码的效果，以介导三个潜水员的细胞内部控制 肺病原体（C.肺炎，B。百日咳和RSV），以及介导肺组织损伤的病原体 细胞因子风暴的下游。新识别的ISG及其连接将得到其受保护的验证 或使用捐赠的人肺和人肺模型的组织外植体系统的病理活动 器官。确保遗传相互作用网络将为对影响的影响提供前所未有的见解 在1型免疫复杂期间，在人肺中指示感染结果。这些发现将揭示新的 局部治疗靶标并建立一个范式来欣赏全部免疫力。这 此处开创的方法还将扩展到其他非免疫细胞和组织的下游效应 其综合研究将定义一种对人类健康至关重要的新生物学景观。