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

项目总结： 世界上三分之一的人口感染结核分枝杆菌(Mtb)，这种细菌会导致 结核病(TB)。人类免疫系统在控制结核分枝杆菌和免疫活性方面非常成功。 被感染的人一生中有5-10%的机会经历活动性(症状)结核病。这一级别 在肉芽肿中，保护作用是由基于细胞因子的细胞-细胞通讯介导的，与 结核病。失调保护性Th1反应的因素，包括下调巨噬细胞激活- ING细胞因子干扰素γ和肿瘤坏死因子，可促进活动性肺结核的发生。艾滋病毒显著增加了患结核病的风险，并 艾滋病毒-结核分枝杆菌混合感染者每年患活动性结核病的风险为5%-15%，结核病是头号杀手 在这群人中。这种增加的风险发生在外周血中CD4+T细胞数量正常的个体中 病毒载量控制得很好。最好的SIV-Mtb混合感染非人灵长类研究 现有的HIV-结核分枝杆菌混合感染模型显示，混合感染和仅感染结核分枝杆菌的动物的肉芽肿含有 数量相似的结核分枝杆菌特异性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+人群中推广结核病。巨噬细胞可能是了解HIV-Mtb病理的关键 混合感染。巨噬细胞可以支持结核分枝杆菌复制，但也是粒细胞白血病中主要的抗结核分枝杆菌效应细胞。 玛斯。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，混合感染的肉芽肿 增加了1型干扰素(IFN1)转录本的表达。IFN1是由病毒感染引起的，但 也与恶化的结核病有关，根据这些数据，我们假设病毒感染诱导了IFN1- 调节肉芽肿巨噬细胞的免疫，从而抑制巨噬细胞的抗分枝杆菌免疫。 从而促进结核病的发生。这一假设不能在人类身上检验，而小鼠模型在实验上是容易处理的。 但免疫生物学、艾滋病毒易感性和结核病表现与人类显著不同。为了过度- 面对这些障碍，我们将使用高度多元化的循环IHC和RNAScope治疗FFPE NHP肉芽肿 确定在SIV/Mtb混合感染中，IFN1、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NHP肉芽肿上进行，这些肉芽肿具有已知的结核分枝杆菌和SIV负载，我们将使用 计算机图像分析和机器学习以识别SIV+/-细胞，SIV如何影响细胞组织， IFN1的表达和巨噬细胞的激活。为了检验我们在这些研究中的发现，我们将使用肺肉芽肿 由结核分枝杆菌感染的猕猴肺组织组成的有机类化合物，与荧光蛋白EX结合。 按Mtb、SIV和IFN1抑制。这些研究将确定SIV调节的IFN1前-病毒之间的关系 合并感染的肉芽肿的抑制率和结核分枝杆菌存活率。我们的长期目标是确定宿主指导的治疗方法 改善艾滋病毒-结核分枝杆菌混合感染的结果和我们提出的机制研究，通过这种创新和 翻译系统将产生有关艾滋病毒-结核病发病机制的新信息，有助于实现这一目标。

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.