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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要体内非免疫细胞的数量大大超过专业免疫细胞，提供细胞内病原体有很多庇护和避难的机会，免疫系统如何保护这片广阔的土地？我之前的工作描述了免疫细胞因子干扰素-g (IFN-g) 的能力，通常被认为是巨噬细胞激活蛋白，广泛激活非免疫细胞并赋予其杀菌能力通过干扰素刺激基因 (ISG) 编码的效应子产生的细胞内在反应，统称为然而，我们对 ISG 如何执行组织局部反应的理解还处于起步阶段，这些效应器中很少有被充分表征的，并且几乎不知道如何多重 ISG 在功能上相互作用以实现宿主防御，理解 ISG 的主要障碍是它们很少。孤立地工作；相反，它们是复杂遗传网络的一部分，不受表型效应的影响。我提出了一种全新的策略，通过映射函数来利用这种遗传复杂性。使用组合正向遗传筛选来研究 ISG 之间的关系我的中心假设是系统地绘制遗传相互作用图谱将揭示局部 IFN-g 信号传导的效应器，并使得将 ISG 产品分层组织成执行特定功能的复合体和路径（网络）在我的博士后工作中，在小范围内使用单个查询基因，这就是保护性和病理性反应。该方法揭示了一对协同的 ISG，它们对细菌执行出乎意料的有效防御细胞溶胶的进一步开发将提供框架，以便在我自己的实验室中更大规模地使用。为了将干扰素完全分解为不同的宿主抗性途径，我将开发一个实验管道。该项目采用了一个基于 CRISPR 的混合筛选系统，用于多位点基因扰动，并与一种新颖的这一创新的单细胞成像和表达分析平台专注于人类气道上皮。该方法将用于剖析 ISG 编码的效应器，这些效应器介导三种不同的细胞内在控制肺部病原体（肺炎衣原体、百日咳博德氏菌和 RSV）以及介导肺组织损伤的病原体新发现的 ISG 及其连接的保护作用将得到验证。或使用来自捐赠的人肺和人肺模型的组织外植体系统进行病理活动随后的遗传相互作用网络将为效应子提供前所未有的见解。这些发现将揭示 1 型免疫反应期间人类肺部的感染结果。局部治疗目标并建立了解全方位免疫的范例。这里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器其综合研究将定义对人类健康至关重要的新生物景观。