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

我们最近在小鼠TB模型中使用荧光适应性报告基因Mtb菌株方面的进展， 我们对巨噬细胞谱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，通过成功开发Dual RNA-seq方案， 直接从鼠肺分离的感染细胞的特征提供了进一步的理解 宿主营养免疫如何成为控制细菌生长的核心，至少在感染早期是如此。 我们建议在马拉维与亨利姆万杜姆巴博士合作的基础上， 通过使用人支气管肺泡灌洗（BAL）细胞的离体Mtb攻击模型的观察， 功能表型的巨噬细胞谱系存在于人肺气道。此外，我们最近 证明了转录活性HIV-1基因组在ART-1的肺泡巨噬细胞中的持久性。 我们相信，我们可以利用这种独特的人类 受试者队列，以表征已知发生在HIV-1感染者中的肺免疫功能受损， 使他们对结核病和其他下呼吸道感染过敏。 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控制模型 这将为未来的疫苗开发计划提供信息。 具体目的1：评估BAL离体激发模型中的抗Mtb免疫功能。这一目标将 由马拉维的Mwandumba博士监督。我们将使用荧光读数的细菌健身量化 并优化来自HIV-1未感染和感染供体的人BAL细胞培养物中的抗微生物活性 用Mtb离体攻击。 具体目标2。利用SILAC标记和单细胞RNA-seq来鉴定细胞凋亡的可溶性调节剂。 Mtb宿主巨噬细胞在HIV-1阴性和HIV-1阳性供体中的功能。这一目标将由 博士拉塞尔在康奈尔大学对来自马拉维的人类巨噬细胞和BAL样本进行了研究。我们将执行（一） 通过代谢标记研究（SILAC）对来自大肠杆菌的分泌蛋白进行释放的效应蛋白的蛋白质组学分析。 马拉维人BAL细胞离体脉冲。(ii)我们将进行单细胞（scRNA-seq）RNA-seq分析， 来自HIV-1未感染和感染供体的Mtb攻击的BAL细胞群，以鉴定巨噬细胞 Mtb生长的免疫控制依赖途径和HIV-1对其的损害。 具体目标3。使用功能丧失和功能获得方法评估候选基因 以及成功控制细胞内Mtb感染的途径。我们将使用siRNA和合成mRNA 操纵宿主HMDM和BAL巨噬细胞，以验证参与限制性内切酶的候选基因/途径 细菌生长，以及它是如何在HIV-1肺部环境中受损的。

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.