Methamphetamine and Immune Cells in NeuroAIDS

描述(由申请人提供)：滥用甲基苯丙胺(Meth)会导致增加接触HIV1-3病毒的风险的行为。神经艾滋病，影响40%的HIV+个体，是一种引起神经功能障碍的感染的特别严重的并发症，不能通过不能有效地穿过血脑屏障的抗病毒药物来减少4。据报道，与这种共同发病相关的神经艾滋病和外周病理因方法5 6而加重。因此，MEH的影响是主要的，但不限于中枢神经系统。我们发现，在SIV/恒河猴模型中，Meth调节脑和外周天然免疫细胞的表型，增加细胞因子的分泌，并在体外丰富病毒靶点CCR5+细胞7。我们将研究Meth对先天免疫细胞直接作用的假设，这种改变可能会增强HIV+Meth滥用者的病毒触发的组织病理。在目前的提案中，我们将重点关注存在于大脑和外周的先天性免疫细胞，特别是巨噬细胞和小胶质细胞。鉴于此，我们假设Meth可以通过直接影响巨噬细胞/小胶质细胞谱系的固有免疫细胞来干扰抗病毒宿主的免疫反应。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患者，因为艾滋病毒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猕猴和SIV感染猕猴的脾和脑冷冻保存的免疫细胞(耗尽神经元和星形/寡神经节)的基因表达，确定Meth是否调节SIV感染猕猴中枢神经系统来源的免疫细胞中的基因表达，并改变SIV在大脑中触发的基因集。表达数据将使用综合系统生物学方法进行比较和验证。我们将优先考虑高分途径，但倾向于参与细胞周期、氧化应激和炎症表型的分子，在这些分子中已经观察到了甲硫氨酸的影响。验证将使用qRTPCR和蛋白质检测(脑和脾冰冻组织片段的蛋白质印迹，冰冻细胞悬液的流式细胞仪，以及石蜡包埋组织的免疫组织化学-已经处理并对所有动物可用)。同时，我们将使用巨噬细胞系和人小胶质细胞原代培养来评估Meth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。基因表达和功能分析评估Meth对细胞周期、氧化应激、趋化因子受体和炎症分子表达、线粒体功能和抗原处理/提呈的影响。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HIV+Meth使用者大脑和其他组织病理严重程度的调节机制，并揭示Meth针对先天性免疫细胞的直接靶点。 公共卫生相关性：在甲基苯丙胺(Meth)使用者中，与艾滋病毒感染有很大重叠，是神经艾滋病的一个重要并发症，据报道这种药物加剧了这种并发症，尽管其对病理的影响并不局限于中枢神经系统。鉴于该药物加重病理和系统作用的机制尚不清楚，目前还没有治疗艾滋病毒或其他病毒感染中使用Meth的病理后果的药物。为了帮助了解HIV+Meth中调节大脑和其他组织病理严重程度的机制，本探索性研究将调查对先天免疫细胞的直接影响，这些细胞主要在大脑中感染，也存在于其他炎症条件下，旨在为治疗干预提供分子基础，并为药物滥用中免疫介导的病理学提供新的范式。

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. PUBLIC HEALTH RELEVANCE: Among Methamphetamine (Meth) users, there is a large overlap with HIV infection, being NeuroAIDS an important complication reported to be aggravated by this drug, although its effects on pathology are not restricted to the CNS. Given that the mechanisms by which the drug aggravates pathology and operates systemically are not well understood, there are no medications for treating the pathological consequences of Meth use in HIV or in other viral infections. To help understand mechanisms regulating the severity of brain and other tissue pathologies in HIV+ Meth, the present exploratory study will investigate direct effects on innate immune cells, which are predominantly infected in the brain and present also in other inflammatory conditions, aiming a molecular basis for therapeutic intervention, and for a new paradigm of immune-mediated pathology in drug abuse.