Human genetics of TB resistance in HIV-infected persons

 描述（由申请人提供）： 很大一部分HIV相关的结核病（TB）是由新的结核分枝杆菌（Mtb）感染引起的，并迅速进展为活动性疾病。宿主对结核分枝杆菌感染的耐药性的存在得到了多方面证据的支持，了解耐药性的机制对于减少艾滋病毒感染者的结核病负担至关重要。Mtb感染导致潜伏性TB感染（LTBI），这是从T细胞免疫测定推断的。由于获得性抗分枝杆菌免疫的缺乏定义了耐药性，因此先天机制对于预防Mtb感染和LTBI的建立至关重要。我们的应用的假设是，肺泡巨噬细胞（AM），第一个肺细胞，遇到吸入的结核分枝杆菌，不同的先天性能力，以抵抗建立结核分枝杆菌感染，和达特个体间的差异，先天性AM阻力是由宿主遗传因素控制。因此，我们建议补充目前的LTBI检测方法（TST，IGRA）与AM感染抗性的细胞测定，将用于优化遗传研究中研究的抗性表型。在我们的研究目标1中，我们建议在南非（发现样本）和海地（复制样本）筛选大量HIV感染者，并确定TST/IGRA双阳性或双阴性的年龄分层人群。在目标2中，我们将通过支气管肺泡灌洗获得AM，并从健康和HIV感染受试者的血液中衍生单核细胞衍生的和诱导多能干细胞（iPSC）衍生的AM样细胞。我们将在存在和不存在HIV的情况下绘制基因组中MTB特异性表观遗传修饰。在目标3中，我们将从所有入选的南非受试者中获得诱导多能干细胞（iPSC），并使用这些iPSC AM样细胞进行测定，该测定通过测量Mtb摄取、细胞内生长、宿主细胞凋亡和细胞因子的产生以及转录和表观遗传变化来量化它们对Mtb感染的先天抗性。接下来，我们将进行数量性状基因座（QTL）分析，并绘制影响基因表达水平的遗传变异和功能性AM检测的定量读数。在目标4中，我们将对南非样本进行全基因组测序，并采用以患者为中心和以队列为基础的设计，寻找具有强烈个体效应的罕见变异，这些个体效应影响严格定义的感染抗性表型。遗传变异将通过多个标准进行优先排序，包括其在i）HIV感染受试者和初始或感染AM（目的2）的AM特异性表观遗传片段特征，和（ii）已知参与HIV阴性受试者感染抗性的区域中的位置。确定在海地样本中复制的遗传抗性因素将被 使用受试者PBMC和受试者iPSC AM样细胞进行功能验证。这些实验提供了一种强有力的综合方法来鉴定HIV感染者中Mtb感染抗性的分子基础，并指出了通过小分子筛选增强iPSCs细胞中抗性因子的自然途径。

 DESCRIPTION (provided by applicant): A large proportion of HIV related tuberculosis (TB) is caused by new Mycobacterium tuberculosis (Mtb) infection with rapid progression to active disease. Existence of host resistance to Mtb infection is supported by multiple lines of evidence, and understanding the mechanisms of resistance will be critical to decrease the burden of TB in HIV infected people. Mtb infection leads to latent TB infection (LTBI) which is inferred from T- cell immune assays. Since the absence of acquired anti-mycobacterial immunity defines resistance, innate mechanisms are critical to prevent Mtb infection and the establishment of LTBI. The hypotheses of our application are that alveolar macrophages (AMs), the first pulmonary cells that encounter inhaled Mtb bacilli, differ in their innate ability to resist establishment of Mtb infection, and tat inter-individual differences in innate AM resistance are controlled by host genetic factors. Hence, we propose to complement current methods of LTBI detection (TST, IGRA) with a cellular assay of infection resistance in AMs that will be used to optimize the resistance phenotype investigated in genetic studies. In aim 1 of our study, we propose to screen a large number of HIV-infected persons in South Africa (discovery sample) and Haiti (replication sample), and identify age-stratified groups of persons who are either TST/IGRA double positive or double negative. In aim 2, we will obtain AMs by broncho-alveolar lavage and derive monocyte-derived and induced pluripotent stem cells (iPSCs)-derived AM-like cells from blood of healthy and HIV-infected subjects. We will map Mtb-specific epigenetic modifications in the genome in presence and absence of HIV. In aim 3, we will derive induced pluripotent stem cells (iPSCs) from all enrolled South African subjects and use these iPSC AM-like cells for an assay that quantifies their innate resistance to Mtb infection by measuring Mtb uptake, intracellular growth, host cell apoptosis and production of cytokines as well as transcriptional and epigenetic changes. Next, we will conduct a quantitative trait locus (QTL) analysis and map the genetic variants that impact on gene expression levels and the quantitative read-outs of the functional AM assays. In aim 4, we will perform whole genome sequencing of the South African samples and engage in a search for rare variants with strong individual effects influencing a stringently defined infection resistance phenotype, employing both patient-centric and cohort-based designs. Genetic variants will be prioritized by a number of criteria, including their location in i) AM-specific epigenetic segments characteristic for HIV-infected subjects and either naive or infected AMs (aim 2), and (ii) regions known to be involved in infection resistance in HIV-negative subjects. Identified genetic resistance factors that replicate in the Haiti sample will be subjected to functional validation employing both subject PBMCs and subject iPSC AM-like cells. These experiments offer a powerful integrated approach to identify the molecular bases of Mtb infection resistance in HIV infected people, and point to a natural path forward through boosting of resistance factors in iPSCs cells by small molecule screening.