Evaluating macrophage antiviral immunity as a suppressive factor in SIV-M. tuberculosis co-infection

项目概要： 世界上三分之一的人口感染了结核分枝杆菌（Mtb）， 结核病（TB）。人类免疫系统在控制Mtb和免疫活性方面非常成功。 受感染的个体有5-10%的终生机会经历活动性（症状性）TB。这个水平 在肉芽肿中，这种保护作用是由基于精氨酸的细胞间通讯介导的， TB.保护性Th 1应答失调的因素，包括巨噬细胞活化因子的下调， 细胞因子IFNγ和TNF可促进活动性TB。艾滋病毒大大增加了患结核病的风险， 艾滋病毒-结核分枝杆菌合并感染者每年发生活动性结核病的风险为5-15%，结核病是主要杀手 在这个人群中。这种高风险发生在具有正常范围外周CD 4 + T细胞数量的个体中 病毒载量得到很好的控制在SIV-Mtb共感染的非人灵长类动物（NHP）中进行的研究， 现有的HIV-Mtb共感染模型显示，来自共感染和仅Mtb动物的肉芽肿含有 类似数量的Mtb特异性T细胞。这些数据表明，病毒相关的T细胞非依赖性因子 在SIV/HIV+个体中推广结核病。巨噬细胞可能是理解HIV-Mtb病理学的关键 合并感染巨噬细胞可以支持Mtb复制，但也是颗粒细胞中的主要抗Mtb效应细胞。 马斯我们使用RNAscope的初步数据表明，NHP肉芽肿中的巨噬细胞感染了 对SIV+和SIV-猴肉芽肿的SIV和转录分析表明， 1型干扰素（IFN 1）转录物的表达增加。IFN 1由病毒感染诱导，但 也与肺结核恶化有关，基于这些数据，我们假设病毒感染诱导IFN 1- 在肉芽肿巨噬细胞中调节免疫并且这抑制巨噬细胞抗分枝杆菌免疫， 从而促进结核病。这一假设无法在人类中检验，而小鼠模型在实验上是易于处理的。 但在免疫生物学、HIV易感性和结核病表现方面与人类有显著不同。敬过度- 面对这些障碍，我们将在FFPE NHP肉芽肿上使用高度多重循环IHC和RNAscope， 确定在SIV/Mtb共感染中IFN 1、SIV感染和巨噬细胞抗Mtb活性之间的关系。 这些研究将在一组独特的已知Mtb和SIV载量的NHP肉芽肿上进行，我们将使用 计算图像分析和机器学习来识别SIV+/-细胞，SIV如何影响细胞组织， IFN 1表达和巨噬细胞活化。为了验证我们的研究结果，我们将使用肺肉芽肿 由结核杆菌感染猕猴的肺组织组成的类器官，与荧光蛋白ex-peptide结合， 抑制Mtb、SIV和IFN 1的抑制。这些研究将确定SIV调节的IFN 1前， 表达和Mtb存活。我们的长期目标是确定宿主导向疗法， 改善艾滋病毒-结核分枝杆菌合并感染的结果，以及我们提出的这种创新和 翻译系统将产生新的信息，艾滋病毒结核病的发病机制，有助于这一目标。

PROJECT SUMMARY: A third of the world’s population is infected with Mycobacterium tuberculosis (Mtb), the bacterium that causes tuberculosis (TB). The human immune system is highly successful at controlling Mtb and immunocompetent individuals who are infected have a 5-10% lifetime chance of experiencing active (symptomatic) TB. This level of protection is mediated by cytokine-based cell-cell communication in granulomas, the lesions associated with TB. Factors that dysregulate the protective Th1 responses, including downregulation of the macrophage activat- ing cytokines IFNγ and TNF, can promote active TB. HIV significantly increases the risk of developing TB and HIV-Mtb coinfected individuals experience a 5-15% annual risk of developing active TB and TB is a leading killer in this population. This elevated risk occurs in individuals who have normal range peripheral CD4+ T cell numbers and well controlled virus loads. Studies in SIV-Mtb coinfected nonhuman primates (NHPs), which are the best available model of HIV-Mtb coinfection, show that granulomas from coinfected and Mtb-only animals contain similar numbers of Mtb-specific T cells. These data suggest that virus-associated T cell-independent factors promote TB in SIV/HIV+ individuals. Macrophages may be the key to understanding the pathology of HIV-Mtb coinfection. Macrophages can support Mtb replication but are also the primary anti-Mtb effector cell in granulo- mas. Our preliminary data using RNAscope indicates that macrophages in NHP granulomas are infected with SIV and transcriptional analysis of granulomas from SIV+ and SIV- monkeys shows that coinfected granulomas have increased expression of type 1 interferon (IFN1) transcripts. IFN1s are induced by viral infection but are also associated with exacerbated TB, and based on these data, we hypothesize that viral infection induces IFN1- regulated immunity in granuloma macrophages and this suppresses macrophage antimycobacterial immunity, thus promoting TB. This hypothesis cannot be tested in humans and murine models are experimentally tractable but have significantly different immunobiology, HIV susceptibility, and TB presentation than humans. To over- come these obstacles, we will use highly multiplexed cyclic IHC and RNAscope on FFPE NHP granulomas to identify the relationship between IFN1, SIV infection, and macrophage anti-Mtb activity in SIV/Mtb coinfection. These studies will be done on a unique set of NHP granulomas with known Mtb and SIV loads, and we will use computational image analysis and machine learning to identify SIV+/- cells, how SIV affects cellular organization, IFN1 expression, and macrophage activation. To test our findings from these studies, we will use lung granuloma organoids composed of lung tissue from Mtb-infected macaques, in combination with fluorescent protein ex- pressing Mtb, SIV and IFN1 inhibition. These studies will identify relationships between SIV-regulated IFN1 ex- pression and Mtb survival in coinfected granulomas. Our long-term goal is to identify host-directed therapies to improve outcomes in HIV-Mtb coinfection and the mechanistic studies we are proposing with this innovative and translational system will produce new information on HIV-TB pathogenesis that contributes to this objective.