Defining Features of Bacterial Control in M. tuberculosis Granulomas Using Single-cell mRNA Sequencing

项目摘要 结核分枝杆菌是引起人类结核病的病原，其导致肺部和播散性 感染每年结核病感染导致130万人死亡，而据估计， 人群被潜伏感染。当结核分枝杆菌感染肺部时，它通常以复杂的聚集体形式被隔离， 免疫细胞和成纤维细胞称为肉芽肿。然而，单个肉芽肿内的免疫反应 往往导致不同的结果。在给定的个体内，许多病变可以同时存在于多个组织中。 状态-一些病变能够控制细菌复制，而另一些则支持细菌持续生长 最终导致疾病的传播。很可能这些细菌控制方面的差异 肉芽肿是由细胞类型组成、细胞内在活化状态和细胞- 每个肉芽肿内的细胞相互作用。直到最近，解开这一复杂程度似乎 无法克服的然而，我们最近开发并应用了单细胞mRNA的创新技术， 测序以分析已知细菌的限制性和允许性非人灵长类动物（NHP）肉芽肿 单细胞分辨率的负担。高维单细胞转录谱分析允许无与伦比的 多细胞社区的解决方案。到目前为止，我们已经恢复了超过 来自6只动物的40个MTB肉芽肿的200，000个单细胞。最重要的是，由于肉芽肿是从 在感染后10周，当限制性病变中的细菌负荷刚刚开始下降时，我们认为， 这些病变之间免疫生态系统的差异将与细菌控制有因果关系。在这里， 我建议将联合收割机计算和实验方法相结合来检验细胞差异 型丰度、表型同一性和细胞-细胞信号网络与结核分枝杆菌中的细菌控制相关 肉芽肿首先，我将构建一个肉芽肿细胞类型多样性的地图，并检查是否 细胞类型组成的差异预测肉芽肿水平的细菌控制。然后我将探讨表型 MTB肉芽肿内巨噬细胞的多样性影响细菌控制。最后，我将使用小说 计算分析来检查肉芽肿中细胞间相互作用的模式， 使用原位成像和体外扰动进行验证。如果成功，所提出的分析和实验将 提供了一个前所未有的了解免疫相关的结核病控制在个人的水平 疾病病变。我设想这项研究有可能识别以前未被重视的细胞类型， 结核分枝杆菌肉芽肿的多样性，并提出新的宿主导向治疗和预防策略， 结核分枝杆菌感染。

Project Summary Mycobacterium tuberculosis is the cause of human tuberculosis, which results in pulmonary and disseminated infection. Each year TB infection results in 1.3 million deaths, while it is estimated that 1/3 of the world’s population is latently infected. When MTB infects the lung, it is typically sequestered in complex aggregations of immune cells and fibroblasts known as granulomas. However, immune responses within individual granulomas often lead to divergent outcomes. Within a given individual, many lesions can simultaneously exist in multiple states – some lesions are able to control bacterial replication while others support persistent bacterial growth eventually leading to the spread of disease. It is likely that these differences in bacterial control across granulomas arises from combined differences in cell-type composition, cell-intrinsic activation states, and cell- cell interactions within each granuloma. Until recently, disentangling this level of complexity seemed insurmountable. However, we have recently developed and applied innovative technology for single-cell mRNA sequencing to profile restrictive and permissive non-human primate (NHP) granulomas of known bacterial burden at single-cell resolution. High-dimensional single-cell transcriptional profiling allows unparalleled resolution of multi-cellular communities. To date, we have recovered transcriptional transcriptomes of over 200,000 single cells from 40 MTB granulomas from 6 animals. Crucially, since the granulomas were harvested at 10 weeks post-infection when bacterial burden had just begun to decline in restrictive lesions, we believe that the differences in immune ecosystems between these lesions will be causally related to bacterial control. Here, I propose to combine computational and experimental approaches to test the hypothesis that differences in cell type abundance, phenotypic identity and cell-cell signaling networks correlate with bacterial control in MTB granulomas. Initially, I will construct a map of cell type diversity across granulomas and examine whether differences in cell-type composition predicts granuloma-level bacterial control. I will then explore how phenotypic diversity among macrophages within MTB granulomas influences bacterial control. Finally, I will use novel computational analyses to examine patterns of cell-cell interactions across granulomas that I will experimentally validate using in situ imaging and in vitro perturbation. If successful, the proposed analysis and experiments will provide an unprecedented understanding of the immune correlates of MTB control at the level of individual disease lesions. I envision that this study has potential to identify previously unappreciated cell types and diversity across MTB granulomas and nominate novel strategies for host-directed therapy and prophylaxis in MTB infection.