Methamphetamine and Immune Cells in NeuroAIDS

描述（由申请人提供）：甲基苯丙胺（冰毒）滥用导致行为增加暴露于艾滋病毒1-3的风险。神经性艾滋病，影响了40%的HIV阳性个体，是一种特别严重的感染并发症，导致神经功能障碍，不能有效穿过血脑屏障的抗病毒药物也不能减轻这种情况。据报道，与这种合并症相关的神经aids和周围病变会因Meth5而加重。因此，甲基苯丙胺的作用是主要的，但并不局限于中枢神经系统。在SIV/恒河猴模型和体外实验中，我们发现甲基苯丙胺可以调节大脑和外周先天免疫细胞的表型，增加细胞因子分泌并富集病毒靶CCR5+细胞7。我们将研究甲基苯丙胺直接作用于先天免疫细胞的假设，在HIV+甲基苯丙胺滥用者中引起的改变可能会增强病毒引发的组织病理。在本提案中，我们将重点关注存在于大脑和外周的先天免疫细胞，特别是巨噬细胞和小胶质细胞。鉴于此，我们假设甲基安非他明可以通过直接影响巨噬细胞/小胶质细胞谱系的先天免疫细胞来干扰宿主的抗病毒免疫反应。这可能是神经病理学增加的一个重要组成部分，特别是在神经艾滋病中，因为HIV的大脑靶点是巨噬细胞和小胶质细胞。我们将通过比较健康和未使用冰毒的SIV感染猕猴的脾脏和大脑冷冻保存的免疫细胞（神经元和星形胶质细胞缺失）的基因表达，来确定冰毒是否会调节SIV感染猕猴中枢神经系统衍生免疫细胞的基因表达，并改变SIV在大脑中触发的一系列基因。表达数据将使用综合系统生物学方法进行比较并验证。我们将优先考虑高分途径，但更倾向于参与细胞周期、氧化应激和炎症表型的分子，在这些方面已经观察到冰毒的影响。将使用qRTPCR和蛋白质检测（脑和脾脏冷冻组织片段的western blot，冷冻细胞悬液的FACS，以及石蜡包埋组织的免疫组织化学-已经处理并可用于所有动物）进行验证。同时，我们将评估甲基安非他明直接调节巨噬细胞/小胶质细胞基因/蛋白表达和功能特征的能力，使用巨噬细胞细胞系和人小胶质细胞原代培养。基因表达和功能分析评估甲基安非他明对细胞周期、氧化应激、趋化因子受体和炎症分子表达、线粒体功能和抗原加工/呈递的影响。这将允许观察对先天免疫细胞的直接影响，这些细胞被感染并在大脑中占主导地位。这些研究将有助于了解调节HIV+冰毒使用者大脑和其他组织病理严重程度的机制，并揭示冰毒对先天免疫细胞的直接靶点。

DESCRIPTION (provided by applicant): Methamphetamine (Meth) abuse leads to behaviors that increase the risk of exposure to HIV1-3. NeuroAIDS, which affects >40% of HIV+ individuals, is a particularly severe complication of the infection causing neurological deficits, not decreased by antiviral drugs that do not cross the blood-brain-barrier efficiently 4. NeuroAIDS and peripheral pathologies associated to this co-morbidity have been reported to be aggravated by Meth5 6. Thus, the effects of Meth are predominant, but not restricted to the CNS. We have found that Meth modulates the phenotype of brain and peripheral innate immune cells in the SIV/Rhesus macaque model, increasing cytokine secretion and enriching virus targets CCR5+ cells7, and in vitro. We will investigate the hypothesis of direct action of Meth on innate immune cells, causing alterations that may enhance virus-triggered tissue-pathologies in HIV+ Meth abusers. In the present proposal, we will focus on innate immune cells existing in the brain and in the periphery, especially macrophages and microglia cells. Given that, we hypothesize that Meth can interfere with the anti-viral host immune response, by directly affecting innate immune cells of the macrophage/ microglia lineage. This can be an important component of increased neuropathology, particularly in neuroAIDS, since HIV brain targets are macrophages and microglia. We will determine if Meth modulates gene expression in CNS-derived immune cells in SIV-infected macaques, and changes sets of genes triggered by SIV in the brain by comparing gene expression in spleen and brain- cryopreserved immune cells (depleted of neurons and astro/oligoglia) in healthy and SIV-infected macaques, treated or not with Meth. Expression data will be compared using an integrative systems biology approach and validated. We will prioritize high score pathways, but favor molecules involved in cell cycle, oxidative stress, and inflammatory phenotype, in which the effects of Meth have been observed. Validation will be performed using qRTPCR and protein detection (western blot on brain and spleen frozen tissue fragments, FACS of frozen cell suspensions, and immunohistochemistry on paraffin-embedded tissue - already processed and available for all animals). In parallel, we will assess the ability of Meth to directly modulate macrophage/ microglia gene/protein expression and functional characteristics, using macrophage cell lines and human microglia primary cultures. Gene expression and functional assays assess the effects of Meth on cell cycle, oxidative stress, chemokine receptor and inflammatory molecule expression, mitochondrial functions and antigen processing/ presentation. This will allow the observation of direct effects on innate immune cells that get infected and predominate in the brain. These studies will help understand mechanisms regulating the severity of brain and other tissues pathology in HIV+ Meth users and reveal direct Meth targets on innate immune cells.