Neuronal apoptosis by HIV-1 Vpr: contribution of proinflammatory molecular networks from infected target cells.
Neuronal apoptosis by HIV-1 Vpr: contribution of proinflammatory molecular networks from infected target cells.
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DOI：
10.1186/1742-2094-9-138
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发表时间：
2012-06-22
影响因子：
9.3
通讯作者：
Ayyavoo V
中科院分区：
文献类型：
--
作者：
Guha D;Nagilla P;Redinger C;Srinivasan A;Schatten GP;Ayyavoo V
人类免疫缺陷病毒1型(HIV-1)通过宿主细胞因子和感染的巨噬细胞/小胶质细胞释放的病毒蛋白R(VPR)等病毒蛋白诱导神经元功能障碍。VPR对巨噬细胞等终末分化细胞的感染很重要。本研究的目的是评估感染性病毒颗粒背景下的VPR通过巨噬细胞释放的促炎细胞因子对神经元死亡的影响。用HIV-1野生型(HIV1wt)、vpr缺失突变体(HIV1vpr)或模拟∆感染单核细胞来源的巨噬细胞。用定量逆转录-聚合酶链式反应和酶联免疫吸附试验分别检测MDM细胞裂解物和培养上清液中促炎症细胞因子的表达和释放。免疫印迹法检测活化的MDM中丝裂原活化蛋白激酶(MAPK)的表达。应用Annexin-V染色、四甲基偶氮唑盐比色法和半胱氨酸天冬氨酸氨基转移酶3/7(∆-Glo®3/7)比色法检测VPR对原代培养神经元凋亡的影响。在有无中和抗体的情况下，观察IL-1β、IL-8和肿瘤坏死因子-α在神经细胞凋亡中的作用。与感染∆的MDM相比，感染HIV-1VPR的MDM随着时间的推移表现出感染的减少，特别是在转录和/或蛋白水平上显著下调IL-1β、IL-8和α的表达。这种下调是由于HIV-1∆vpr感染的MDM中p38和应激激活蛋白激酶/c-jun氨基末端激酶的激活受损所致。通过阻断MAPK信号通路，证实SAPK/jnk和p38与IL-1β和IL-8的产生有关。在HIV-1WT中抑制IL-1β和IL-8的升高比在感染HIV-1∆vpr的培养中更明显。∆VPR感染MDM的培养上清液中含有较低浓度的IL-1β、IL-8和α以及病毒蛋白，与感染HIV-1WT的MDM上清液相比，神经毒性降低。仅在HIV-1WT感染的培养中，抗IL-1β和抗IL-8抗体才能减少神经元死亡，这表明vpr对神经元死亡的影响部分是通过释放促炎因子来实现的。总之，这些结果证明了∆-1vpr通过直接或间接地通过抑制病毒复制而在受感染的靶细胞中失调多种促炎细胞因子来限制神经元凋亡的能力。
Human immunodeficiency virus type 1 (HIV-1) induces neuronal dysfunction through host cellular factors and viral proteins including viral protein R (Vpr) released from infected macrophages/microglia. Vpr is important for infection of terminally differentiated cells such as macrophages. The objective of this study was to assess the effect of Vpr in the context of infectious virus particles on neuronal death through proinflammatory cytokines released from macrophages. Monocyte-derived macrophages (MDM) were infected with either HIV-1 wild type (HIV-1wt), Vpr deleted mutant (HIV-1∆Vpr) or mock. Cell lysates and culture supernatants from MDMs were analyzed for the expression and release of proinflammatory cytokines by quantitative reverse transcription-PCR and enzyme-linked immunosorbent assay respectively. Mitogen-activated protein kinases (MAPK) were analyzed in activated MDMs by western blots. Further, the effect of Vpr on neuronal apoptosis was examined using primary neurons exposed to culture supernatants from HIV-1wt, HIV-1∆Vpr or mock-infected MDMs by Annexin-V staining, MTT and Caspase - Glo® 3/7 assays. The role of interleukin (IL)-1β, IL-8 and tumor necrosis factor (TNF)-α on neuronal apoptosis was also evaluated in the presence or absence of neutralizing antibodies against these cytokines. HIV-1∆Vpr-infected MDMs exhibited reduced infection over time and specifically a significant downregulation of IL-1β, IL-8 and TNF-α at the transcriptional and/or protein levels compared to HIV-1wt-infected cultures. This downregulation was due to impaired activation of p38 and stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK) in HIV-1∆Vpr-infected MDMs. The association of SAPK/JNK and p38 to IL-1β and IL-8 production was confirmed by blocking MAPKs that prevented the elevation of IL-1β and IL-8 in HIV-1wt more than in HIV-1∆Vpr-infected cultures. Supernatants from HIV-1∆Vpr-infected MDMs containing lower concentrations of IL-1β, IL-8 and TNF-α as well as viral proteins showed a reduced neurotoxicity compared to HIV-1wt-infected MDM supernatants. Reduction of neuronal death in the presence of anti-IL-1β and anti-IL-8 antibodies only in HIV-1wt-infected culture implies that the effect of Vpr on neuronal death is in part mediated through released proinflammatory factors. Collectively, these results demonstrate the ability of HIV-1∆Vpr to restrict neuronal apoptosis through dysregulation of multiple proinflammatory cytokines in the infected target cells either directly or indirectly by suppressing viral replication.
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