Mechanisms of HIV Tat regulation of macrophage gene expression in neuroAIDS

描述（由申请人提供）：HIV在外周感染后2周内进入中枢神经系统，尽管cART仍然存在病毒。在超过50%的感染人群中，中枢神经系统HIV感染导致HIV相关神经认知障碍（HAND）。HAND至少部分是由持续的神经炎症和引起中枢神经系统损伤的低水平病毒持续性介导的。病毒进入大脑的一个主要机制是通过感染hiv的单核细胞在血脑屏障上的转运。这些单核细胞在中枢神经系统实质中分化为巨噬细胞。他们精心设计了神经毒性因子和趋化因子，这些因子招募了额外的感染和未感染的单核细胞进入中枢神经系统，以及感染血管周围巨噬细胞和小胶质细胞的病毒。因此，慢性炎症在中枢神经系统内持续存在。神经艾滋病的特点是神经元损伤和丧失，但HIV不感染神经元。因此，在神经元中看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目标，包括神经毒性病毒蛋白的细化。这是病毒的反激活子，尽管有cART，它仍然会产生。它还与宿主基因相互作用，改变它们的表达。我们和其他人证明，在巨噬细胞中诱导细胞因子分泌，包括介导神经炎症的CCL2，并在HIV感染的HAND患者的中枢神经系统中高度表达。这也增加了巨噬细胞上CCR5和CXCR4的表达。因此，我们假设HIV - tat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、中枢神经系统损伤和随后的HAND发展。为了证明这一假设，我们将使用我们从ChIP-seq分析中获得的数据，这些数据确定了巨噬细胞宿主基因组中的特定结合位点，并从这67个位点的独创性途径分析（IPA）中获得。我们提出三个目标。目的1是确认THP-1-Tat-Flag细胞系中ChIP-seq鉴定的基因。目的2是证明，与未感染的巨噬细胞相比，感染了含有Tat-Flag的腺病毒和艾滋病毒的原代人巨噬细胞，以及从患有和不患有HAND的艾滋病毒感染者获得的大脑组织切片中，在Aim 1中证实的基因表达发生了变化。目的3是证明tat的氨基酸介导宿主基因表达。我们的方法将提供关于如何改变宿主基因表达以及这些失调基因在HAND发病机制中的作用的新信息。预计这些数据将为限制艾滋病毒中枢神经系统感染后果的治疗干预提供创新策略。

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.