Lung immune function in human TB infection and its perturbation by HIV-1.

我们在小鼠结核病模型中使用荧光适合性报告Mtb菌株的最新进展 我们对巨噬细胞系在控制和促进结核分枝杆菌生长中的作用有独特的认识。 此外，通过成功地开发双重RNA-SEQ方案，这些观察结果得到了推广 为鉴定直接从小鼠肺中分离的感染细胞提供了进一步的了解 了解宿主营养免疫是如何控制细菌生长的核心，至少在早期感染时是这样。 我们建议在马拉维与Henry Mwandumba博士合作的基础上，扩大这些 人支气管肺泡灌洗(BAL)细胞体外Mtb攻击模型的观察 功能表型人肺内存在的巨噬细胞系。此外，我们最近 证实在ART的肺泡巨噬细胞中持续存在转录活性的HIV-1基因组- 马拉维天真和抑制艺术的捐赠者，并相信我们可以利用这个独特的人类 受试者队列描述已知的HIV-1携带者肺免疫功能受损的特征 使它们对结核病和其他下呼吸道感染都具有超敏性。 我们的假设是结核分枝杆菌感染的人肺巨噬细胞的功能和表型 来自健康和HIV-1感染志愿者的子集将产生可测试的免疫介导性控制模型 结核分枝杆菌的增长情况将为未来的疫苗开发计划提供信息。 具体目标1：在BAL体外激发模型中评估抗结核分枝杆菌免疫功能。这一目标将 由马拉维的Mwandumba博士监督。我们将使用细菌适合性的荧光读数来量化 并优化来自HIV-1未感染和感染捐赠者的人BAL细胞培养的抗微生物活性 在体外用结核分枝杆菌挑战。 具体目的2.利用SILAC标记和单细胞RNA-SEQ鉴定可溶性调节子 HIV-1阴性和HIV-1阳性献血者结核杆菌宿主巨噬细胞功能。这一目标将由 康奈尔大学的拉塞尔博士对来自马拉维的人类巨噬细胞和BAL样本进行了研究。我们将演出(一) 应用新陈代谢标记研究(SILAC)对释放的效应蛋白进行蛋白质组学分析 马拉维的人类BAL细胞在体外产生脉冲。(Ii)我们将进行单细胞(scRNA-seq)RNA-seq分析 MTB挑战来自HIV-1未感染和感染捐赠者的BAL细胞群以鉴定巨噬细胞- 结核分枝杆菌生长的免疫控制依赖途径及其HIV-1的损害。 具体目标3.使用功能丧失和功能获得的方法来评估候选基因 以及成功控制细胞内结核分枝杆菌感染的途径。我们将使用siRNA和合成的mrna。 操纵宿主HMDM和BAL巨噬细胞以验证参与限制的候选基因/途径 细菌生长，以及它在HIV-1肺部环境中是如何受到损害的。

Our recent advances in the use of fluorescent fitness reporter Mtb strains in the murine TB model has afforded us a unique appreciation of the role of macrophage lineages in both the control and promotion of Mtb growth. Moreover, the extension of these observations through the successful development of Dual RNA-seq protocols for characterization of infected cells isolated directly from the murine lung has provided a further understanding of how host nutritional immunity is central to the control of bacterial growth, at least in early infection. We propose building on our collaboration with Dr. Henry Mwandumba in Malawi and extending these observations through an ex vivo Mtb challenge model with human bronchoalveolar lavage (BAL) cells to functionally phenotype the macrophage lineages present in the human lung airways. Furthermore, we recently demonstrated the persistence of transcriptionally-active HIV-1 genomes in the alveolar macrophages of ART- naïve and ART-suppressed donors in Malawi and believe that we can take advantage of this unique human subjects cohort to characterize the impairment of lung immunity known to occur in people living with HIV-1 that renders them hypersusceptible to both TB and other lower respiratory tract infections. Our hypothesis is that the functional and phenotypic typing of Mtb-infected human lung macrophage subsets from healthy and HIV-1-infected volunteers will generate testable models for immune-mediated control of Mtb growth that will inform future vaccine development programs. Specific Aim 1: Assessment of anti-Mtb immune function in a BAL ex vivo challenge model. This aim will be overseen by Dr. Mwandumba in Malawi. We will use our fluorescent readouts of bacterial fitness to quantify and optimize anti-microbial activities in human BAL cell cultures from HIV-1 uninfected and infected donors challenged with Mtb ex vivo. Specific Aim 2. Utilization of SILAC labeling and single cell RNA-seq to identify soluble modulators of Mtb host macrophage function in HIV-1 negative and HIV-1 positive donors. This aim will be overseen by Dr. Russell at Cornell University on human macrophages and BAL samples from Malawi. We will perform (i) Proteomic analysis of released effector proteins by metabolic labeling studies (SILAC) of secreted proteins from human BAL cells pulsed ex vivo in Malawi. (ii) We will perform single cell (scRNA-seq) RNA-seq analysis of Mtb-challenged BAL cell populations from HIV-1 uninfected and infected donors to identify macrophage- dependent pathways of immune control of Mtb growth and their impairment by HIV-1. Specific Aim 3. The use of Loss of function and Gain of function approaches to assess candidate genes and pathways in successful control of intracellular Mtb infection. We will use siRNA and synthetic mRNA to manipulate host HMDMs and BAL macrophages to validate candidate genes/pathways involved in restriction of bacterial growth, and how it is compromised in the HIV-1 lung environment.