Neuroinflammation associated with HIV-1 infection is a problem affecting ϳ50% of HIV-infected individuals. NLR family pyrin domain containing 3 (NLRP3) inflammasome has been implicated in HIV-induced microglial activation, but the mechanism(s) remain unclear. Because HIV-1 Transactivator of Transcription (Tat) protein continues to be present despite antiretroviral therapy and activates NF-kB, we hypothesized that Tat could prime the NLRP3 inflammasome. We found a dose-and time-dependent induction of NLRP3 expression in microglia exposed to Tat compared with control. Tat exposure also time-dependently increased the mature caspase-1 and IL-1␤ levels and enhanced the IL-1␤ secretion. These in vitro findings were validated in archival brain tissues from Simian Immunodeficiency Virus (SIV)-infected and uninfected rhesus macaques. Further validation of NLRP3 priming in vivo involved administration of lipopolysaccharide (LPS) to HIV transgenic (Tg) rats followed by assessment of IL-1␤ mRNA expression and inflammasome activation (ASC oligomers and mature IL-1␤). Intriguingly, LPS potentiated upregulation of IL-1␤ mRNA and inflammasome activation in HIV-Tg rats compared with the wild-type controls. Interestingly, we found an inverse relationship in the expression of NLRP3 and its negative regulator, miR-223, suggesting a miR-223-mediated mechanism for Tat-induced NLRP3 priming. Furthermore, blockade of NLRP3 resulted in decreased IL-1␤ secretion. Collectively, these findings suggest a novel role of Tat in priming and activating the NLRP3 inflammasome. Therefore, NLRP3 can be envisioned as a therapeutic target for ameliorating Tat-mediated neuroinflammation.

BackgroundNeuroinflammation associated with advanced human immunodeficiency virus (HIV)-1 infection is often exacerbated by chronic cocaine abuse. Cocaine exposure has been demonstrated to mediate up-regulation of inflammatory mediators in in vitro cultures of microglia. The molecular mechanisms involved in this process, however, remain poorly understood. In this study, we sought to explore the underlying signaling pathways involved in cocaine-mediated activation of microglial cells.MethodsBV2 microglial cells were exposed to cocaine and assessed for toll-like receptor (TLR2) expression by quantitative polymerase chain reaction (qPCR), western blot, flow cytometry, and immunofluorescence staining. The mRNA and protein levels of cytokines (TNFα, IL-6, MCP-1) were detected by qPCR and ELISA, respectively; level of reactive oxygen species (ROS) production was examined by the Image-iT LIVE Green ROS detection kit; activation of endoplasmic reticulum (ER)-stress pathways were detected by western blot. Chromatin immunoprecipitation (ChIP) assay was employed to discern the binding of activating transcription factor 4 (ATF4) with the TLR2 promoter. Immunoprecipitation followed by western blotting with tyrosine antibody was used to determine phosphorylation of TLR2. Cocaine-mediated up-regulation of TLR2 expression and microglial activation was validated in cocaine-injected mice.ResultsExposure of microglial cells to cocaine resulted in increased expression of TLR2 with a concomitant induction of microglial activation. Furthermore, this effect was mediated by NADPH oxidase-mediated rapid accumulation of ROS with downstream activation of the ER-stress pathways as evidenced by the fact that cocaine exposure led to up-regulation of pPERK/peIF2α/ATF4 and TLR2. The novel role of ATF4 in the regulation of TLR2 expression was confirmed using genetic and pharmacological approaches.ConclusionsxThe current study demonstrates that cocaine-mediated activation of microglia involves up-regulation of TLR2 through the ROS-ER stress-ATF4-TLR2 axis. Understanding the mechanism(s) involved in cocaine-mediated up-regulation of ROS-ER stress/TLR2 expression and microglial activation could have implications for the development of potential therapeutic targets aimed at resolving neuroinflammation in cocaine abusers.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-016-0501-2) contains supplementary material, which is available to authorized users.

Cocaine is known to activate microglia both in vitro and in vivo. High expression of microglial Toll-like receptors (TLRs) and their downstream signal transducers play critical roles in determining microglial activation status. Emerging reports have also demonstrated that cocaine can enhance the strength of TLR signaling. Detailed molecular mechanisms underlying this phenomenon, however, remain elusive. In this study, we investigated the role(s) of miR-124 in regulating microglial TLR4 signaling in the context of cocaine. Herein, we found a dose- and time-dependent upregulation of KLF4 in cocaine-exposed BV-2 cells and rat primary microglial cells (rPMs). KLF4 also identified as a novel 3'-UTR target directly regulated by miR-124. In parallel, miR-124 regulated multiple TLR4 signaling molecules including TLR4, MyD88, TRAF6, and IRAK1. Repeated doses of cocaine (20 mg/kg; i.p.) administration in mice for 7 days further validated the in vitro key findings. Also, miR-124 overexpression significantly blocked the cocaine-mediated upregulation of pro-inflammatory cytokines. In contrast, miR-124 overexpression notably increased the expression of anti-inflammatory mediators in cocaine-exposed microglial cells. Intriguingly, stereotactic administration of lentivirus-miR-124 in the striatum significantly inhibited cocaine-mediated microglial activation and locomotor hyperactivity in vivo. In summary, these findings implicate the role of miR-124 in regulating TLR4 signaling, thereby indicating a new pathway responsible for cocaine-mediated microglial activation.

Cocaine is known to induce inflammation, thereby contributing in part, to the pathogenesis of neurodegeneration. A recent study from our lab has revealed a link between macroautophagy/autophagy and microglial activation. The current study was aimed at investigating whether cocaine could also mediate activation of astrocytes and, whether this process involved induction of autophagy. Our findings demonstrated that cocaine mediated the activation of astrocytes by altering the levels of autophagy markers, such as BECN1, ATG5, MAP1LC3B-II, and SQSTM1 in both human A172 astrocytoma cells and primary human astrocytes. Furthermore, cocaine treatment resulted in increased formation of endogenous MAP1LC3B puncta in human astrocytes. Additionally, astrocytes transfected with the GFP-MAP1LC3B plasmid also demonstrated cocaine-mediated upregulation of the green fluorescent MAP1LC3B puncta. Cocaine-mediated induction of autophagy involved upstream activation of ER stress proteins such as EIF2AK3, ERN1, ATF6 since blockage of autophagy using either pharmacological or gene-silencing approaches, had no effect on cocaine-mediated induction of ER stress. Using both pharmacological and gene-silencing approaches to block either ER stress or autophagy, our findings demonstrated that cocaine-induced activation of astrocytes (measured by increased levels of GFAP) involved sequential activation of ER stress and autophagy. Cocaine-mediated-increased upregulation of GFAP correlated with increased expression of proinflammatory mediators such as TNF, IL1B, and IL6. In conclusion, these findings reveal an association between ER stress-mediated autophagy and astrogliosis in cocaine-treated astrocytes. Intervention of ER stress and/or autophagy signaling would thus be promising therapeutic targets for abrogating cocaine-mediated neuroinflammation.

A large-scale assay was performed by transfecting 29,910 individual cDNA clones derived from human placenta, fetus, and normal liver tissues into human hepatoma cells and 22,926 cDNA clones into mouse NIH 3T3 cells. Based on the results of colony formation in hepatoma cells and foci formation in NIH 3T3 cells, 3,806 cDNA species (8,237 clones) were found to possess the ability of either stimulating or inhibiting cell growth. Among them, 2,836 (6,958 clones) were known genes, 372 (384 clones) were previously unrecognized genes, and 598 (895 clones) were unigenes of uncharacterized structure and function. A comprehensive analysis of the genes and the potential mechanisms for their involvement in the regulation of cell growth is provided. The genes were classified into four categories: I, genes related to the basic cellular mechanism for growth and survival; II, genes related to the cellular microenvironment; III, genes related to host-cell systemic regulation; and IV, genes of miscellaneous function. The extensive growth-regulatory activity of genes with such highly diversified functions suggests that cancer may be related to multiple levels of cellular and systemic controls. The present assay provides a direct genomewide functional screening method. It offers a better understanding of the basic machinery of oncogenesis, including previously undescribed systemic regulatory mechanisms, and also provides a tool for gene discovery with potential clinical applications