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

人类结核感染中的肺免疫功能及其受 HIV-1 的干扰。

基本信息

批准号：
10085075
负责人：
Henry Charles Mwandumba
金额：
$ 67.7万
依托单位：
CORNELL UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-06-04 至 2025-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10085075
关键词：
Acquired Immunodeficiency Syndrome Address Africa South of the Sahara Alveolar Macrophages Bacillus Bronchoalveolar Lavage Candidate Disease Gene Cause of Death Cell Culture Techniques Cell Lineage Cells Clinical Research Collaborations Data Development Disease Progression Environment Equilibrium Fluorescent in Situ Hybridization Foundations Future Genetic Transcription Glycolysis Granuloma Growth HIV HIV Genome HIV-1 Human Immune Immunity Immunize Immunological Models Immunomodulators Impairment In Vitro Individual Infection Knowledge Label Link Lower Respiratory Tract Infection Lung Lymphocyte Lymphoid Cell Maintenance Malawi Mass Spectrum Analysis Mediating Mediator of activation protein Messenger RNA Metabolic Metabolism Modality Modeling Molecular Mus Mycobacterium tuberculosis Myeloid Cells Nature Nutritional Immunity Participant Pathway interactions Phenotype Physiologic pulse Population Program Development Proteins Proteomics Protocols documentation Reporter Role Sampling Small Interfering RNA Tissues Transcript Treatment Efficacy Tuberculosis Universities Vaccines Viral active control antimicrobial bacterial fitness cohort comorbidity design disorder control experimental study fatty acid oxidation fetal stem cell fitness gain of function global health human subject immune function in vivo inhibitor/antagonist interstitial loss of function macrophage novel pathogen permissiveness programs recruit single-cell RNA sequencing small molecule inhibitor therapy design transcriptome transcriptome sequencing vaccine development volunteer

项目摘要

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.

我们在小鼠结核病模型中使用荧光适应度报告 Mtb 菌株的最新进展提供了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，通过成功开发双 RNA-seq 协议扩展了这些观察结果对直接从小鼠肺部分离的感染细胞进行表征提供了进一步的了解宿主营养免疫如何成为控制细菌生长的核心，至少在早期感染中如此。我们就与马拉维 Henry Mwandumba 博士的合作制定提案并扩展这些通过使用人支气管肺泡灌洗 (BAL) 细胞的离体 Mtb 攻击模型观察到此外，我们最近对人类肺气道中存在的巨噬细胞谱系进行了功能表型分析。在 ART 的肺泡巨噬细胞中证明了具有转录活性的 HIV-1 基因组的持续存在- 马拉维天真的和受 ART 抑制的捐赠者相信我们可以利用这种独特的人类受试者队列来表征已知 HIV-1 感染者中发生的肺部免疫损伤，使他们对结核病和其他下呼吸道感染高度敏感。我们的假设是 Mtb 感染的人肺巨噬细胞的功能和表型分型来自健康和 HIV-1 感染志愿者的子集将生成用于免疫介导控制的可测试模型结核分枝杆菌的增长将为未来的疫苗开发计划提供信息。具体目标 1：评估 BAL 离体攻击模型中的抗 Mtb 免疫功能。由马拉维的 Mwandumba 博士监督，我们将使用细菌适应性的荧光读数来量化。并优化未感染和感染 HIV-1 供体的人类 BAL 细胞培养物的抗菌活性离体 Mtb 挑战。具体目标 2. 利用 SILAC 标记和单细胞 RNA-seq 鉴定可溶性调节剂 HIV-1 阴性和 HIV-1 阳性捐赠者中 Mtb 宿主巨噬细胞的功能将由该目标监督。康奈尔大学的 Russell 博士研究来自马拉维的人类巨噬细胞和 BAL 样本，我们将执行 (i)。通过蛋白质分泌蛋白的代谢标记研究 (SILAC) 对释放的效应蛋白进行蛋白质组学分析在马拉维进行离体脉冲的人类 BAL 细胞 (ii) 我们将对以下细胞进行单细胞 (scRNA-seq) RNA-seq 分析。来自未感染和感染 HIV-1 的供体的 Mtb 攻击的 BAL 细胞群可识别巨噬细胞 Mtb 生长的免疫控制及其受 HIV-1 损害的依赖途径。具体目标 3. 使用功能丧失和功能获得方法来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 巨噬细胞来验证参与限制的候选基因/途径细菌生长，以及它如何在 HIV-1 肺部环境中受到损害。