Characterize replication competent myeloid reservoirs in the central nervous system

表征中枢神经系统中具有复制能力的骨髓库

• 批准号: 10327112
• 负责人: Guochun Jiang
• 金额: $ 24.76万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10327112
• 关键词: Address; Animal Model; Animals; Autopsy; Biological Assay; Blood; Brain; Brain region; CCAAT-Enhancer-Binding Proteins; CD4 Positive T Lymphocytes; Cells; Cessation of life; DNA; Data; Disease remission; Enrollment; Environment; Epigenetic Process; Exposure to; Frequencies; Generations; Genes; Gifts; Goals; HIV; HIV Infections; HIV-1; Histone Deacetylase Inhibitor; Histone Deacetylation; Human; Immune; Immune system; In Vitro; Infection; Interruption; Knowledge; Macaca; Macaca mulatta; Measurable; Measures; Microglia; Modification; Molecular; Myelogenous; Myeloid Cells; Neuraxis; Pathway interactions; Penetration; Peripheral; Play; Population; Primates; Proteins; Protocols documentation; RNA; Regulation; Resources; Rest; Role; SIV; Sampling; Source; Study models; T-Lymphocyte; TLR3 gene; Techniques; Technology; Testing; Tissues; Universities; Viral; Viral reservoir; Viremia; Virus; Virus Latency; antiretroviral therapy; brain cell; brain tissue; cohort; epigenetic regulation; histone methylation; histone methyltransferase; in vivo; in vivo imaging; inhibitor/antagonist; latent HIV reservoir; latent infection; macrophage; monocyte; nonhuman primate; novel strategies; peripheral blood; viral rebound; virology

SUMMARY The eradication of HIV-1(HIV) infection must address all tissues where infection persists. Clearance of infection in the central nervous system (CNS) is not achieved by antiretroviral therapy (ART) alone, just as in the periphery. Latent HIV reservoirs in the CNS may allow viral rebound upon discontinuation of ART, as HIV can egress from the brain into the peripheral blood. However, studies are needed to elucidate the contributions of subsets of latently infected CNS cells to ongoing HIV persistence in the CNS. Circulating T cells have been well characterized as the major HIV reservoirs in the peripheral blood, and may circulate into the CNS, contributing to HIV persistence in the brain. Nevertheless, it is still not known whether T cells are the only major viral reservoir in the CNS. Myeloid cells are a major cellular compartment of the immune system infected by HIV in the brain. In vivo imaging and immunostaining studies have revealed that brain myeloid cells (BMCs) harbor HIV DNA and produce HIV RNA and proteins. However, it is not clear whether BMCs, and especially long-lived microglia, are latently infected, and if they are true HIV reservoirs encoding replication-competent HIV despite durable, successful ART. Our team at the UNC HIV Cure Center has recently established a protocol to isolate highly pure myeloid cells from the brain of SIV-infected macaques, in whom ART was interrupted. These SIV containing BMCs can be cultured for many generations ex vivo. Further application of this technology to other fully ART-suppressed animals found that BMCs contain proviral SIV DNA, and that SIV RNA can be effectively induced by latency reversal agents. This platform to study myeloid cells ex vivo allows rigorous characterization of brain myeloid cells to address their role as true HIV reservoirs that can produce replication- competent viruses. Our preliminary data highlights that epigenetic regulation, such as histone deacetylation and histone methylation, may play an essential role in the modulation of HIV latency in the CNS. We hypothesize that BMCs are latently infected by HIV and harbor replication competent HIV. In this proposal, we will isolate highly pure brain myeloid cells from different regions of the brain in both people living with HIV (PLWH) enrolled in the “Last Gift” cohort and fully ART-suppressed SIV-infected rhesus macaques. We will examine whether BMCs harbor replication-competent HIV or SIV using the intact proviral DNA assay (IPDA) and quantitative viral outgrowth assay (QVOA) (Aim1). We will examine whether latent HIV in myeloid cells responds to the currently available latency reversal agents related to epigenetic regulation of HIV latency (Aim 2), which may provide clues to the mechanisms that allow HIV latency in the brain. Our study will address critical gaps in knowledge to better understand whether BMCs serve as true HIV reservoirs and how HIV persists in these CNS cells.

总结 根除HIV-1（HIV）感染必须针对感染持续存在的所有组织。清除 中枢神经系统（CNS）中的感染不能仅通过抗逆转录病毒疗法（ART）实现，正如在 外围。CNS中的潜伏HIV储库可能会在停止ART后允许病毒反弹，因为HIV 可以从大脑进入外周血然而，需要研究来阐明这些贡献 潜伏感染的CNS细胞亚群与CNS中持续的HIV持续存在之间的关系。循环T细胞已经 作为外周血中的主要HIV储库被充分表征，并且可以循环到CNS中， 导致艾滋病病毒在大脑中持续存在。然而，目前还不清楚T细胞是否是唯一的 中枢神经系统中的主要病毒储存库。 髓样细胞是大脑中被HIV感染的免疫系统的主要细胞区室。在 体内成像和免疫染色研究显示，脑髓样细胞（BMC）携带HIV DNA， 产生HIV RNA和蛋白质。然而，目前尚不清楚BMC，特别是长寿的小胶质细胞，是否 潜伏感染，如果他们是真正的艾滋病毒储存库编码复制能力的艾滋病毒，尽管持久， 我们的艾滋病治疗中心的团队最近建立了一个协议， 从SIV感染的猕猴脑中提取的纯骨髓细胞，在这些猕猴中，ART被中断。这些SIV 含有BMC的细胞可以离体培养许多代。将这项技术进一步应用于其他 完全ART抑制的动物发现，BMCs含有前病毒SIV DNA，SIV RNA可以有效地 由潜伏期逆转剂诱导。这种离体研究骨髓细胞的平台允许严格的 脑骨髓细胞的特征，以解决其作为真正的艾滋病毒水库，可以产生复制的作用- 有能力的病毒我们的初步数据强调，表观遗传调控，如组蛋白去乙酰化， 和组蛋白甲基化，可能在调节HIV在CNS中的潜伏期中起重要作用。我们 假设BMC潜伏性感染HIV并携带有复制能力的HIV。 在这个提议中，我们将从两个脑中的不同区域分离高度纯的脑髓样细胞， 参加“最后礼物”队列的艾滋病毒感染者（PLWH）和完全ART抑制的SIV感染恒河猴 猕猴我们将使用完整的前病毒检测BMCs是否具有复制能力的HIV或SIV。 DNA测定（IPDA）和定量病毒生长测定（QVOA）（Aim 1）。我们将检查潜伏的艾滋病毒 在骨髓细胞中，对目前可用的与表观遗传调节相关的潜伏期逆转剂有反应， HIV潜伏期（Aim 2），这可能为HIV在大脑中潜伏的机制提供线索。我们的研究 将解决知识方面的关键差距，以更好地了解BMC是否是真正的艾滋病毒储存库， 艾滋病毒如何在这些中枢神经系统细胞中持续存在。