Do HIV-infected Alveolar Macrophages represent a cART-resistant Reservoir?

 描述（由申请人提供）：尽管人们普遍认为静息、潜伏感染的CD 4 + T细胞是HIV的cART耐药储库，但很少有人关注替代储库位点。使用基于超灵敏荧光原位杂交（FISH）的 系统检测HIV mRNA的流式细胞术，我们绘制了分布的HIV在肺的慢性感染，无症状的HIV血清阳性个体，并发现该病毒是目前在更大的丰度在肺泡巨噬细胞（AM）比淋巴细胞。AM是不寻常的，因为它们是来自保留在肺内的自我维持细胞谱系的长寿细胞。最近，我们将我们的研究扩展到了一组接受cART的患者，发现尽管一些人接受了长达9年的cART，但那些HIV mRNA阳性的AM仍保持在持续水平。 本提案的目的是评估这些HIV阳性AM作为潜在储库的重要性，这些储库能够在cART停止或失败后重建外周病毒血症，并制定根除策略。1. HIV感染的AM的分子表征。我们已经修改了我们的FISH检测平台，以分选HIV mRNA阳性的细胞，使我们能够执行RNASeq来分析宿主和病毒转录组，并对病毒基因组插入位点进行DeepSeq分析，以绘制病毒群体结构。我们建议定义来自抑制性cART个体的病毒基因组，以与从随后治疗失败的个体中分离的外周病毒进行比较，以确定其相关性。2. HIV mRNA阳性的AM是否会被感染？我们将确定HIV mRNA阳性AM是否可以产生感染性病毒体，这些病毒体可以接种CD 4 + T细胞并重建病毒血症。我们正在重新设计用于马拉维的病毒报告细胞系，以检测受感染AM的感染性病毒体的产生。3.非人灵长类动物（NHP）中的AM是否同样感染SIV？我们的合作者，匹兹堡大学的Charles Scanga博士和威斯康星州大学的Shelby奥康纳博士，正在进行SIV/M.tb合并感染的NHP研究。我们将分析这些猕猴在早期SIV感染，以确定是否NHP感染SIV表现出有效的早期接种的AM人口。我们还将对这些细胞进行FISH和RNASeq，以与人类HIV转录组进行比较。4. HIV阳性AM能否在体外导致细胞死亡？我们将寻求消除这些细胞的新策略。CD 4 + T细胞和巨噬细胞之间的关键区别是巨噬细胞不会被感染驱动细胞死亡。我们将通过化学扰动在体外操纵感染的人单核细胞衍生的巨噬细胞（HMDM）和AM，以产生能够驱动HIV感染的AM进入程序性细胞死亡的小分子的未来HTS的初步数据。

 DESCRIPTION (provided by applicant): Although it is widely accepted that resting, latently-infected CD4+ T-cells are a cART-resistant reservoir for HIV, little attention has been devoted to alternative reservoir sites. Using an ultrasensitive Fluorescent in situ Hybridization (FISH)-based system for detecting HIV mRNA by flow cytometry we mapped the distribution of HIV in the lung of chronically infected, asymptomatic HIV-seropositive individuals and found the virus to be present at greater abundance in alveolar macrophages (AMs) than in lymphocytes. AMs are unusual in that they are long-lived cells derived from a self-sustaining cell lineage retained within the lung. Recently, we extended our study to a cohort of patients on cART and found that those AMs positive for HIV mRNA persisted at sustained levels despite some individuals having been on cART for up to 9 years. The goal of this proposal is to assess the significance of these HIV-positive AMs as a potential reservoir with the capacity to reconstitute peripheral viremia upon cessation or failure of cART, and to develop strategies for its eradication. 1. Molecular Characterization of HIV-infected AMs. We have modified our FISH detection platform to sort cells positive for HIV mRNA, enabling us to perform RNASeq to profile both host and viral transcriptomes, and DeepSeq analysis of viral genome insertion sites to map viral population structure. We propose defining the viral genomes from individuals on suppressive cART to compare with peripheral virus isolated from those individuals who subsequently go on to fail therapy to establish their relatedness. 2. Are HIV mRNA-positive AMs productively infected? We will determine whether HIV mRNA-positive AMs can generate infectious virions that could seed CD4+ T-cells and reconstitute viremia. We are re-engineering viral reporter cell lines for use in Malawi to detect production of infectious virions from infected AMs. 3. Are AMs in non-human primates (NHP) similarly infected with SIV? Our collaborators, Dr. Charles Scanga, University of Pittsburgh and Dr. Shelby O'Connor, University of Wisconsin, are conducting a NHP study of SIV/M.tb co-infection. We will analyze these macaques during early SIV infection to determine whether NHP infected with SIV exhibit efficient early seeding of the AM population. We will also perform FISH and RNASeq on these cells to compare with the human HIV transcriptomes. 4. Can HIV-positive AMs be driven to cell death in vitro? We will pursue novel strategies towards elimination of these cells. The critical difference between CD4+ T-cells and macrophages is that macrophages are not driven to cell death by the infection. We will manipulate in vitro-infected human monocyte-derived macrophages (HMDMs) and AMs by chemical perturbation to generate preliminary data for a future HTS for small molecules capable of driving HIV-infected AMs into programmed cell death.