Mechanisms of HIV Tat regulation of macrophage gene expression in neuroAIDS

描述（由申请方提供）：HIV在外周感染后2周内进入CNS，尽管进行了cART，但病毒仍然存在。CNS HIV感染导致超过50%的感染人群出现HIV相关神经认知障碍或HAND。HAND至少部分由持续的神经炎症和导致CNS损伤的低水平病毒持续性介导。病毒进入大脑的主要机制是通过HIV感染的单核细胞穿过BBB的迁移。这些单核细胞在CNS实质中分化成巨噬细胞。他们阐述了神经毒性和趋化因子，这些因子招募额外的感染和未感染的单核细胞进入CNS，以及感染血管周围巨噬细胞和小胶质细胞的病毒。因此，慢性炎症在CNS内持续存在。Neuro艾滋病的特征是神经元损伤和丢失，但艾滋病毒不会感染神经元。因此，在神经元中观察到的功能改变和损伤一定是由于HIV感染的间接影响，主要是巨噬细胞和小胶质细胞作为感染的主要靶点，包括神经毒性病毒蛋白达特的加工。达特是病毒的反式激活因子，尽管有cART，它仍然产生。达特还与宿主基因相互作用以改变其表达。我们和其他人证明，达特诱导巨噬细胞分泌细胞因子，包括CCL 2，介导神经炎症，并在HAND的HIV感染者的CNS中高度表达。达特还增加巨噬细胞表面CCR 5和CXCR 4的表达。因此，我们假设HIV达特蛋白与人巨噬细胞宿主基因相互作用，改变其表达，导致巨噬细胞功能障碍、神经炎症、CNS损伤和随后的HAND发展。为了解决这一假设，我们将使用我们从ChIP-seq测定中获得的数据，这些测定鉴定了巨噬细胞宿主基因组中的特异性达特结合位点，并从这67个位点的免疫途径分析（IPA）中获得数据。我们提出三个目标。目的1是确认和表征通过ChIP-seq在THP-1-Tat-Flag细胞系中鉴定的基因。目的2是证明与未感染的巨噬细胞相比，在用含有Tat-Flag的腺病毒和用HIV感染的原代人巨噬细胞中，以及在从有和没有HAND的HIV感染者获得的脑组织切片中，AIM 1中证实的基因表达的改变。目的3是证明达特的氨基酸介导宿主基因的表达。我们的方法将提供有关达特如何改变宿主基因表达以及这些失调基因对HAND发病机制的贡献的新信息。预计这些数据将表明治疗干预的创新策略，以限制HIV CNS感染的后果。

DESCRIPTION (provided by applicant): HIV enters the CNS within 2 weeks after peripheral infection and viral presence persists despite cART. CNS HIV infection results in HIV Associated Neurocognitive Disorders, or HAND, in greater than 50% of the infected population. HAND is mediated, at least in part, by ongoing neuroinflammation and low level viral persistence that causes CNS damage. A major mechanism by which virus enters the brain is by the transmigration of HIV-infected monocytes across the BBB. These monocytes differentiate into macrophages in the CNS parenchyma. They elaborate neurotoxic and chemotactic factors that recruit additional infected and uninfected monocytes into the CNS, as well as virus that infects perivascular macrophages and microglia. Thus, chronic inflammation persists within the CNS. NeuroAIDS is characterized by neuronal damage and loss, but HIV does not infect neurons. Therefore, the altered function and damage seen in neurons must be due to indirect effects of HIV infection, primarily of macrophages and microglia as the major targets of infection, including the elaboration of the neurotoxic viral protein, tat. Tat is the transactivator of the virus and isstill produced despite cART. Tat also interacts with host genes to alter their expression. We and others demonstrated that tat induced cytokine secretion in macrophages, including CCL2, that mediates neuroinflammation and is highly expressed in the CNS of HIV infected people with HAND. Tat also increased the expression of CCR5 and CXCR4 on macrophages. Thus, we hypothesize that the HIV tat protein interacts with human macrophage host genes, altering their expression, resulting in macrophage dysfunction, neuroinflammation, CNS damage and subsequent development of HAND. To address this hypothesis we will use data we obtained from ChIP-seq assays that identified specific tat binding sites in the macrophage host genome and from Ingenuity Pathway Analysis (IPA) of these 67 sites. We propose three Aims. Aim 1 is to confirm and characterize the genes identified by ChIP-seq in THP-1-Tat-Flag cell lines. Aim 2 is to demonstrate alterations in expression of the genes confirmed in AIM 1 in primary human macrophages infected with adenovirus containing Tat-Flag, and with HIV, as compared to uninfected macrophages, and in tissue sections of brains obtained from HIV infected people with and without HAND. Aim 3 is to demonstrate the amino acids of tat that mediate host gene expression. Our approaches will provide new information about how tat alters host gene expression and the contribution of these dysregulated genes to the pathogenesis of HAND. It is anticipated that these data will indicate innovative strategies for therapeutic intervention to limt the consequences of HIV CNS infection.