Diversity and function of mononuclear phagocytes in the lung driven by mycobacterium tuberculosis infection

项目摘要/摘要 结核分枝杆菌(Mtb)在单核巨噬细胞(MPS)中存活和复制的能力是 它的致病性和感染世界四分之一人口的能力至关重要。乔尔博士的实验室 Ernst(共同导师)和其他人已经证明了结核分枝杆菌在不同种群之间的不同增长和复制 国会议员。然而，MP亚集的复杂性和多样性直到最近才在转录分辨率上被定义 在结核分枝杆菌感染期间没有得到很好的描述。因此，不清楚哪些子集对结核分枝杆菌是最允许的。 生长和如果结核分枝杆菌指导单核细胞分化。应用流式细胞术和单细胞RNA测序 (scRNA-seq)，初步数据显示不同的感染率和对结核分枝杆菌的反应。 在两个招募的巨噬细胞亚群中。这引导了核心假设，即结核分枝杆菌的毒力 在感染过程中，因素驱动肺内新招募的MPS的分化状态，为Mtb提供肥沃的生态位。 增长和复制。这项工作的长期目标是勾勒出结核分枝杆菌和MPS之间的关系 在理解肺免疫细胞异质性的最新革命的背景下，使更有效的 宿主导向疗法。利用荧光表达Mtb、流式细胞术和单细胞-RNA测序 在感染了结核分枝杆菌实验室菌株(H37Rv)和缺乏ESX-1分泌系统的菌株的小鼠中， 特定目标1将检验以下假设：结核分枝杆菌毒力系统驱动单核细胞分化为特定的 巨噬细胞亚群。通过使用流式细胞术、免疫组织化学和体外感染模型，特异性 目标2将验证招募的巨噬细胞亚群具有不同的限制结核分枝杆菌的能力的假设 复制。 培训计划的重点是发展对单核分枝杆菌的免疫反应方面的专业知识。 吞噬细胞和肺环境生物学、分枝杆菌的遗传操作、单链RNA序列和生物信息学， 领导力和负责任的研究实践。培训将包括授课课程、讲习班、研讨会、 本地和国际会议。主要导师和联合导师是奥伦·罗森博格博士和乔尔博士 恩斯特既是成功的内科科学家，又是免疫学、结核分枝杆菌生物学和宿主方面的综合专业知识- 病原体相互作用。一个主要的导师团队还包括一名生物信息学的科学顾问， 下一代测序的合作者，成就卓著的医生-科学家职业导师。查医生已经 加州大学旧金山分校任务湾的实验室和办公空间，与BSL2和BSL3实验室共用空间， 获得这项研究所需的设备和鼠标模型。本年度职业发展奖 申请有创新的研究建议，严谨的培训计划，分枝杆菌领导者的指导 免疫学和生物学，医生-科学家顾问团队，有将科学家过渡到 独立性，以及一位被驱使在分枝杆菌宿主领域走向独立职业生涯的杰出候选人 相互作用与慢性肺病。

Project Summary/Abstract Mycobacterium tuberculosis’s (Mtb) ability to survive and replicate in mononuclear phagocytes (MPs) is central to its pathogenicity and capability to infect a quarter of the world’s population. The laboratory of Dr. Joel Ernst (co-mentor) and others have demonstrated differential Mtb growth and replication between populations of MPs. Yet, the complexity and diversity of MP subsets is only recently being defined at transcriptional resolution and not well described during Mtb infection. It is therefore unclear which subsets are most permissive to Mtb growth and if Mtb directs monocyte differentiation. Using flow cytometry and single cell RNA-sequencing (scRNA-seq) from infected mice, preliminary data demonstrates differential infection rates and responses to Mtb within two recruited macrophage subpopulations. This has directed the central hypothesis that Mtb virulence factors drive differentiation states of recruited MPs in the lung during infection, providing a fertile niche for Mtb growth and replication. The long-term goal of this work is to delineate the relationship between Mtb and MPs in the context of a recent revolution in understanding lung immune cell heterogeneity, enabling more effective host-directed therapies. Utilizing fluorescently expressing Mtb, flow cytometry, and single cell-RNA sequencing in mice infected with a laboratory strain of Mtb (H37Rv), and a strain lacking a functional ESX-1 secretion system, specific Aim 1 will test the hypothesis that an Mtb virulence system drives differentiation of monocytes to specific macrophage subsets. With use of flow cytometry, immunohistochemistry, and ex vivo infection models, specific Aim 2 will test the hypothesis that recruited macrophage subsets have differential ability to restrict Mtb replication. The training plan is focused to develop expertise in immunologic responses to mycobacterium, mononuclear phagocyte and lung milieu biology, genetic manipulation of mycobacterium, scRNA-seq and bioinformatics, leadership, and responsible research practices. Training will include didactic courses, workshops, seminars, local and international conferences. The primary mentor and co-mentors are Dr. Oren Rosenberg and Dr. Joel Ernst, both successful physician-scientists with combined expertise in immunology, Mtb biology, and host- pathogen interactions. An overarching team of mentors also include a scientific advisor in bioinformatics, collaborator in next generation sequencing, and accomplished physician-scientist career mentors. Dr. Zha has laboratory and office space at UCSF Mission Bay, with shared space in a BSL2 and BSL3 laboratories, with access to equipment and mouse models necessary for this research. This Career Development Award application has an innovative research proposal, rigorous training plan, mentorship of leaders in mycobacterial immunology and biology, team of physician-scientist advisors with a track record of transitioning scientists to independence, and an exceptional candidate driven toward an independent career in mycobacterial-host interaction and chronic lung disease.