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的、潜伏感染的CD4+T细胞是HIV的耐推车宿主，但很少有人关注替代宿主的位置。使用超灵敏的荧光原位杂交(FISH)技术 我们绘制了HIV在慢性感染、无症状的HIV血清阳性者肺中的分布，发现该病毒在肺泡巨噬细胞(AM)中的存在比在淋巴细胞中更丰富。AM的不同寻常之处在于，它们是来自肺内保留的自我维持细胞谱系的长寿命细胞。最近，我们将我们的研究扩展到接受CART的一组患者，发现尽管有些人已经接受CART长达9年，但那些HIV mRNA阳性的AM仍保持在持续水平。这项建议的目的是评估这些HIV阳性AM作为潜在宿主的重要性，以及在CART停止或失败时重建外周病毒血症的能力，并制定根除策略。1.HIV感染AM的分子特征。我们修改了FISH检测平台，对HIV mRNA阳性的细胞进行了分类，使我们能够执行RNAseq来分析宿主和病毒转录本，并对病毒基因组插入位点进行DeepSeq分析，以绘制病毒种群结构图。我们建议定义来自抑制车的个体的病毒基因组，以与从那些随后治疗失败的个体分离的外周病毒进行比较，以确定它们之间的相关性。2.HIV m RNA阳性AM是否有效感染？我们将确定HIV mRNA阳性的AM是否能产生感染性病毒粒子，这些病毒粒子可以种子CD4+T细胞并重建病毒血症。我们正在重新设计病毒报告细胞系，用于马拉维检测受感染AM产生的传染性病毒粒子。3.非人类灵长类动物(NHP)的肺泡巨噬细胞(AM)是否同样感染了SIV？我们的合作者，匹兹堡大学的Charles Scanga博士和威斯康星大学的Shelby O‘Connor博士，正在进行一项关于SIV/M.tb混合感染的NHP研究。我们将在SIV感染的早期对这些猕猴进行分析，以确定感染SIV的NHP是否显示出有效的AM种群早期播种。我们还将对这些细胞进行FISH和RNAseq，以与人类艾滋病毒转录本进行比较。4.HIV阳性的AM能在体外导致细胞死亡吗？我们将寻求消除这些细胞的新战略。CD4+T细胞和巨噬细胞的关键区别在于巨噬细胞不会因感染而导致细胞死亡。我们将在体外感染的人类单核细胞来源的巨噬细胞(HMDM)和AM中通过化学扰动来操作，为未来的HTS产生初步数据，这些小分子能够驱动HIV感染的AM进入程序性细胞死亡。

 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.