Impact of Mycobacterium tuberculosis on monocyte differentiation in vivo

项目摘要 从2007年到2019年，结核病是全球死亡的主要传染病，直到2020年才被取代 新冠肺炎大流行与COVID-19不同，目前还没有有效的结核病疫苗，因此它仍将是一种 是世界范围内最主要的传染性死亡原因随着多重耐药菌株的出现，迫切需要 开发新的疗法，增强对感染的免疫反应。结核分枝杆菌用来逃避 消除不完全理解。恩斯特实验室观察到，在感染M.肺结核， 某些来源于骨髓单核细胞的吞噬细胞（单核细胞亚群1; MNC 1）尤其是 "放任"：它们比其他吞噬细胞更频繁地被感染， 分枝杆菌然而，单核细胞衍生细胞在炎症组织中的积累与病原体相关。 清除其他感染。最近的研究还表明，骨髓单核细胞群不是 相反，单核细胞的亚群具有不同的转录谱，并且具有不同的 对不同炎症刺激的反应。这些数据提示了M.结核病重编程 骨髓产生单核细胞，单核细胞分化成肺细胞，肺细胞高度允许 分枝杆菌的生长和存活。为了检验这一假设，将使用光谱流式细胞术来确定 在MNC 1细胞中观察到的表型的获得是否开始于来自Mtb的骨髓单核细胞， 感染的老鼠来自未感染和感染的小鼠的骨髓单核细胞也将共转移到 感染的受体以确定单核细胞来源是否影响分化成MNC1细胞。的 还将通过单细胞RNA测序来评估骨髓单核细胞的异质性，以测试多个细胞的异质性。 关于允许肺细胞分化的潜在机制的假说。来自结核分枝杆菌的单核细胞 将感染的小鼠与来自未感染小鼠和来自施用脂多糖的小鼠的那些进行比较。 在另一项实验中，将比较结核分枝杆菌感染小鼠骨髓单核细胞中的基因表达 感染减毒分枝杆菌M.牛卡介苗 Guérin Pasteur，将Mtb特异性基因与改变的单核细胞发育联系起来。拟议的研究将改善 了解M。结核病影响从骨髓招募到肺部的单核细胞，并且可能 导致宿主导向疗法以增强其杀分枝杆菌活性，这是一个创新的靶点， 提高目前抗分枝杆菌药物的有效性。所有工作将在加州大学旧金山分校进行，提供 为像申请人这样的医生-科学家提供支持性的、资源充足的培训环境。拟议 研究将为他提供单核细胞开发的专业知识，在高封闭设施中进行实验， 和生物信息学分析，促进他转变为研究免疫反应的独立研究者 肺部感染

PROJECT SUMMARY Tuberculosis was the leading infectious cause of mortality worldwide from 2007 to 2019, only dethroned in 2020 by the Covid-19 pandemic. Unlike Covid-19, there is no effective vaccine for tuberculosis, so it will remain a leading infectious cause of death worldwide. With multidrug-resistant strains emerging, there is a critical need to develop new therapies that enhance the immune response to infection. The mechanisms used by Mtb to evade elimination are incompletely understood. The Ernst lab has observed that in mice infected with M. tuberculosis, certain phagocytes (mononuclear cell subset 1; MNC1) descended from bone marrow monocytes are especially “permissive”: they are more frequently infected than are other phagocytes and poorly eliminate engulfed mycobacteria. However, accumulation of monocyte-derived cells in inflamed tissues is associated with pathogen clearance in other infections. Recent work has also shown that the bone marrow monocyte population is not homogeneous; rather, subpopulations of monocytes have differing transcriptional profiles and have varying responses to distinct inflammatory stimuli. These data prompt the hypothesis that M. tuberculosis reprograms the bone marrow to produce monocytes that differentiate into lung cells that are highly permissive of mycobacterial growth and survival. To test this hypothesis, spectral flow cytometry will be used to determine whether acquisition of the phenotype observed in MNC1 cells begins in bone marrow monocytes from Mtb- infected mice. Bone marrow monocytes from uninfected and infected mice will also be co-transferred into infected recipients to determine whether monocyte origin influences differentiation into MNC1 cells. The heterogeneity of bone marrow monocytes will also be assessed by single-cell RNA sequencing to test multiple hypotheses about mechanisms underlying the differentiation of permissive lung cells. Monocytes from Mtb- infected mice will be compared to those from uninfected mice and from mice administered lipopolysaccharide. In a separate experiment, gene expression in bone marrow monocytes from Mtb-infected mice will be compared to that in monocytes from mice infected with the attenuated mycobacterial strain M. bovis bacillus Calmette- Guérin Pasteur, to link Mtb-specific genes to altered monocyte development. The proposed studies will improve understanding of how M. tuberculosis affects monocytes recruited to the lung from the bone marrow and could lead to host-directed therapies to augment their mycobactericidal activity, an innovative target that would increase the effectiveness of current anti-mycobacterial drugs. All work will be performed at UCSF, which offers a supportive, well-resourced training environment for physician-scientists like the applicant. The proposed studies will provide him with expertise in monocyte development, experimentation in a high-containment facility, and bioinformatic analysis, facilitating his transition to an independent investigator studying immune responses to lung infections.