Cell-lineage specific epigenomic determinants of HIV latency in humanized mouse brain and blood

人源化小鼠大脑和血液中HIV潜伏期的细胞谱系特异性表观基因组决定因素

基本信息

批准号：
10747752
负责人：
Schahram Akbarian
金额：
$ 72.93万
依托单位：
ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-07-25 至 2028-05-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10747752
关键词：
3-Dimensional Acute Adult Affect Aftercare Architecture Autopsy Binding Biological Markers Biological Models Blood Brain Cell Culture System Cell Culture Techniques Cell Lineage Cells Central Nervous System Chromatin Chromium Complex Disease Dominant-Negative Mutation Drug Modulation DsRed Encephalitis Epigenetic Process Experimental Models Frequencies Genetic Genetic Engineering Genome Genome Mappings Genomics Goals HIV HIV Infections HIV-1 HIV-associated neurocognitive disorder Histone H3 Histones Human Immune Immune system In Vitro Infection Inflammation Intervention Kinetics Life Cycle Stages Link Long Terminal Repeats Lymphocyte Lysine Methionine Methylation Microglia Molecular Molecular Conformation Mus Myelogenous Myeloid Cells Neurologic Symptoms Neurons Organ Pathogenicity Peripheral Persons Population Productivity Proviruses Regimen Reporter Repression Resolution Retrotransposon Retroviridae SETDB1 gene Shock Site Sorting Spleen Synapses System Techniques Testing Transgenes Viral Viral reservoir Virus Virus Activation Virus Diseases Virus Integration Virus Latency Work Xenograft procedure antiretroviral therapy brain cell cell type chromatin remodeling derepression design epigenomics genetic approach histone methyltransferase humanized mouse in vivo innovation integration site mouse model novel pharmacologic response single-cell RNA sequencing tissue culture transcriptome transcriptomic profiling transcriptomics

项目摘要

Project Summary Human immunodeficiency virus type 1 (HIV-1) infection affects more than 38 million people worldwide and remains incurable due to the early establishment of reservoirs where the virus remains latent. HIV-1 enters the brain within the first two weeks of infection, and neurologic symptoms have been observed with accompanying central nervous system (CNS) biomarkers in acute HIV disease. The seven billion microglial cells in the CNS are the primary cell type infected by HIV in the adult human brain, and in the central nervous system represents a large potential reservoir site. Additionally, the brain is one of the organs with the highest burden of HIV-associated disease. HIV-associated neurocognitive disorder (HAND) affects 20-50% of people with HIV (PWH), with the milder forms of HAND predominating in the era of combined antiretroviral therapy (cART). Importantly, intact HIV proviruses persist in the brain despite viral suppression with cART. Despite this, little is known about the unique regulatory mechanisms governing HIV activation and latency in the brain. According to our recent cell studies in human postmortem brain, inflammation-associated reprogramming of microglial transcriptomes and 3D genomes (chromosomal conformations) is a key factor linked to viral infection and integration in brain cells during advanced stages of infection associated with encephalitis. However, non-encephalitic infected human brain, other than showing transcriptomic signatures indicative for disrupted interactions of microglia with the neuronal synapse, provides little information about the epigenomic and other determinants governing viral activation and latency in the brain. Here, as a first step towards understanding molecular mechanisms governing HIV latency in the humanized mouse brain, we will explore an extremely innovative molecular toolbox differentiating, on the single cell level, infected microglia and other myeloid cells actively expressing HIV, and separating them from infected cells not expressing HIV (latent). We will use this toolbox for advanced experimental approaches to quantitatively test molecular, epigenetic, and pharmacological interventions aimed at reducing the reservoir of humanized HIV+ brain, spleen, and blood. We will employ a novel genetic approach called enhanced HIV- induced lineage tracing (E-HILT) to reveal the frequency and kinetics of the establishment of latency in the CNS at the single cell resolution. We will define, in cell culture, and in humanized mouse brain and spleen/blood at single cell level resolution, the proportional representation of productively infected versus latently infected microglia and lymphocytes and other peripheral myeloid cells subject to genetically or pharmacologically induced disruption of chromatin-bound silencers, including the Human Silencing Hub (HUSH)/CTIP2- KAP1/KMT1E/SETDB1 repressive histone methyltransferase complex and more, broadly, histone H3-lysine 9 methylation (H3K9me)-associated repressive chromatin remodeling. The study will uncover the degree of latency in primary microglial cells in vivo, explore the transcriptome, epigenomic and chromatic 3D architecture that supports latency and explore the effects of chromatin modulating drugs on these cell states.

项目摘要人类免疫缺陷病毒1型（HIV-1）感染影响全球超过3800万人由于早期建立了病毒潜在的水库，因此仍然无法治愈。 HIV-1进入在感染的前两周内，已经观察到神经系统症状急性HIV疾病中的中枢神经系统（CNS）生物标志物。中枢神经系统中的70亿个小胶质细胞是成人人脑中的HIV感染的主要细胞类型，在中枢神经系统中代表庞大的潜在储层站点。此外，大脑是艾滋病毒相关负担最高的器官之一疾病。与HIV相关的神经认知障碍（Hand）影响20-50％的HIV患者（PWH），与在抗逆转录病毒疗法（CART）的时代，较温和的手形式占主导地位。重要的是，完整的艾滋病毒尽管被推车抑制了病毒，但在大脑中，病毒仍在大脑中持续存在。尽管如此，对独特的知之甚少控制艾滋病毒激活和大脑潜伏期的调节机制。根据我们最近的细胞研究人类验尸大脑，小胶质转录组和3D的炎症相关的重编程基因组（染色体构象）是与病毒感染和脑细胞中整合有关的关键因素与脑炎相关的感染的晚期阶段。但是，非脑膜感染的人脑，除了显示转录组特征指示小胶质细胞与神经元相互作用的中断突触，很少提供有关构基因组学和其他决定病毒激活和其他决定因素的信息大脑的潜伏期。在这里，作为了解艾滋病毒潜伏期的分子机制的第一步在人源化的小鼠大脑中，我们将在区分的大脑单细胞水平，被感染的小胶质细胞和其他积极表达HIV的髓样细胞，并将其与未表达HIV的感染细胞（潜在）。我们将使用此工具箱进行高级实验方法定量测试分子，表观遗传学和药理干预措施，旨在减少储层人源化的艾滋病毒+脑，脾和血液。我们将采用一种称为增强HIV-的新型遗传方法诱导的谱系跟踪（E-HILT）以揭示CNS中延迟的频率和动力学在单细胞分辨率下。我们将在细胞培养中定义小鼠脑和脾脏/血液中的定义单细胞水平分辨率，有效感染与潜在感染的比例表示小胶质细胞和淋巴细胞以及其他受遗传学或药理诱导的周围髓细胞细胞染色质结合的消音器的破坏，包括人类沉默枢纽（HUSH）/CTIP2- KAP1/KMT1E/SETDB1抑制组蛋白甲基转移酶复合物等等，广泛的组蛋白H3-赖氨酸9 甲基化（H3K9ME）相关的抑制性染色质重塑。该研究将揭示潜伏期的程度在体内原代小胶质细胞中，探索转录组，表观基因组和色体3D体系结构支持潜伏期并探索染色质调节药物对这些细胞态的影响。