Methamphetamine and Immune Cells in NeuroAIDS

描述（由申请人提供）：甲基苯丙胺（Meth）滥用导致增加暴露于HIV 1 -3风险的行为。NeuroAIDS影响超过40%的HIV+个体，是感染引起神经功能缺损的特别严重的并发症，不能通过不能有效穿过血脑屏障的抗病毒药物降低。据报道，与这种并发症相关的神经艾滋病和外周病变会因Meth 5 6而加重。因此，Meths的作用是主要的，但不限于CNS。我们已经发现，在SIV/恒河猴模型和体外，Meth调节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CCR 5+细胞7。我们将研究甲基对先天免疫细胞的直接作用的假设，引起可能增强HIV+甲基滥用者中病毒触发的组织病理学的改变。在本提案中，我们将关注存在于大脑和外周中的先天免疫细胞，特别是巨噬细胞和小胶质细胞。鉴于此，我们假设甲硫氨酸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抗病毒宿主免疫应答。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的脑靶点是巨噬细胞和小胶质细胞。我们将确定Meth是否调节SIV感染的猕猴中CNS衍生的免疫细胞中的基因表达，并通过比较健康和SIV感染的猕猴中脾和脑冷冻保存的免疫细胞（耗尽神经元和星形胶质细胞/寡胶质细胞）中的基因表达来改变脑中由SIV触发的基因组，用Meth处理或不用Meth处理。将使用综合系统生物学方法比较表达数据并进行验证。我们将优先考虑高分途径，但有利于参与细胞周期，氧化应激和炎症表型的分子，其中已观察到甲基的影响。将使用qRTPCR和蛋白质检测（脑和脾冷冻组织片段的蛋白质印迹、冷冻细胞悬液的FACS和石蜡包埋组织的免疫组织化学-已处理并可用于所有动物）进行验证。同时，我们将评估甲基直接调节巨噬细胞/小胶质细胞基因/蛋白质表达和功能特性的能力，使用巨噬细胞系和人类小胶质细胞原代培养物。基因表达和功能测定评估了Meth对细胞周期、氧化应激、趋化因子受体和炎性分子表达、线粒体功能和抗原加工/呈递的影响。这将允许观察对被感染并在大脑中占主导地位的先天免疫细胞的直接影响。这些研究将有助于了解调节HIV+ Meth使用者大脑和其他组织病理严重程度的机制，并揭示先天免疫细胞上的直接Meth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.