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Background: Neurocognitive impairments remain prevalent in HIV-1 infected individuals despite current antiretroviral therapies. It is increasingly becoming evident that astrocytes play a critical role in HIV-1 neuropathogenesis through the production of proinflammatory cytokines/chemokines. HIV-1 viral protein R (Vpr) plays an important role in neuronal dysfunction; however, its role in neuroinflammation is not well characterized. The major objective of this study was to determine the effect of Vpr in induction of proinflammatory chemokine CCL5 in astrocytes and to define the underlying mechanism(s)., Methods: SVGA astrocytes were either mock transfected or were transfected with a plasmid encoding HIV-1 Vpr, and the cells were harvested at different time intervals. The mRNA level of CCL5 expression was quantified using real-time RT-PCR, and cell culture supernatants were assayed for CCL5 protein concentration. Immunocytochemistry was performed on HIV-1 Vpr transfected astrocytes to check CCL5 expression. Various signaling mechanisms such as p38 MAPK, PI3K/Akt, NF-κB and AP-1 were explored using specific chemical inhibitors and siRNAs., Results: HIV-1 Vpr transfected astrocytes exhibited time-dependent induction of CCL5 as compared to mock-transfected astrocytes at both the mRNA and protein level. Immunostained images of astrocytes transfected with HIV-1 Vpr also showed much higher accumulation of CCL5 in comparison to untransfected and mock-transfected astrocytes. Pre-treatment with NF-κB (SC514) and PI3K/Akt (LY294002) inhibitor partially abrogated CCL5 mRNA and protein expression levels as opposed to untreated controls after HIV-1 Vpr transfection. Specific siRNAs against p50 and p65 subunits of NF-κB, p38δ MAPK, Akt-2 and Akt-3, and AP-1 transcription factor substantially inhibited the production of CCL5 in HIV-1 Vpr transfected astrocytes., Conclusion: These results demonstrate the ability of HIV-1 Vpr to induce CCL5 in astrocytes in a time-dependent manner. Furthermore, this effect was observed to be mediated by transcription factors NF-κB and AP-1 and involved the p38-MAPK and PI3K/Akt pathway.

Methamphetamine (MA) is one of the commonly used illicit drugs and the central nervous system toxicity of MA is well documented. The mechanisms contributing to this toxicity have not been fully elucidated. In this study, we investigated the effect of MA on the expression levels of the proinflammatory cytokines/chemokines, IL-6 and IL-8 in an astrocytic cell line. The IL-6 and IL-8 RNA levels were found to increase by 4.6 ± 0.2 fold and 3.5 ± 0.2 fold, respectively, after exposure to MA for three days. Exposure of astrocytes to MA for 24 hours also caused increased expression of IL-6 and IL-8 at the level of both RNA and protein. The potential involvement of the nuclear factor-Kappa B (NF-κB) pathway was explored as one of the possible mechanism(s) responsible for the increased induction of IL-6 and IL-8 by MA. The MA-mediated increases in IL-6 and IL-8 were significantly abrogated by SC514. We also found that exposure of astrocytes to MA results in activation of NF-κB through the phosphorylation of IκB-α, followed by translocation of active NF-κB from the cytoplasm to the nucleus. In addition, treatment of cells with a specific inhibitor of metabotropic glutamate receptor-5 (mGluR5) revealed that MA-mediated expression levels of IL-6 and IL-8 were abrogated by this treatment by 42.6 ± 5.8% and 65.5 ± 3.5%, respectively. Also, LY294002, an inhibitor of the Akt/PI3K pathway, abrogated the MA-mediated induction of IL-6 and IL-8 by 77.9 ± 6.6% and 81.4 ± 2.6%, respectively. Thus, our study demonstrates the involvement of an NF-κB-mediated signaling mechanism in the induction of IL-6 and IL-8 by MA. Furthermore, we showed that blockade of mGluR5 can protect astrocytes from MA-mediated increases of proinflammatory cytokines/chemokines suggesting mGluR5 as a potential therapeutic target in treating MA-mediated neurotoxicity.

Background: The exact mechanism underlying HIV-associated neurocognitive disorders still remains largely unresolved. However, viral genes (for example gp120 and tat) and their effect on cytokine/chemokine expressions have been linked with neuroinflammation. Conversely, interlekin-8 (IL-8) is a known proinflammatory chemokine and is known to be over-expressed in human brain microvascular endothelial cells in response to gp120. In this study, we sought to address whether HIV-1gp120 could affect IL-8 expression in astrocytes and whether the NF-κB pathway is involved in this phenomenon., Methods: SVGA astrocytes were transfected with a plasmid expressing HIV-1 pSyn gp120 JR-FL using Lipofectamine2000. The cells were harvested at different time points after transfection, and total cellular RNA was used for quantification of IL-8 using a real time PCR. IL-8 protein expression was also determined in supernatants collected at different time points after transfection. Involvement of the NF-κB pathway was addressed using both pharmacological inhibitors and an siRNA approach. In order to explore gene specificity, gp120-specific siRNAs were designed and IL-8 expression was monitored at both mRNA and protein levels., Results: Gp120 increased IL-8 expression both at mRNA and protein levels by 7.1 ± 1.04 and 2.41 ± 0.35 fold at 6 and 48 hours post-transfection, respectively. This increase was time-dependent and was abrogated by use of gp120-specific siRNA. We have also shown that the NF-κB pathway is involved in gp120-mediated IL-8 overexpression as IKK-2 and IKKβ inhibitors inhibited IL-8 expression by 63.5% and 57.5%, respectively at the mRNA level, and by 67.3% and 58.6% at the protein level. These results were also confirmed with use of NF-κB-specific siRNA., Conclusion: These results indicate that gp120 can modulate expression of a pro-inflammatory chemokine (IL-8) in astrocytes in a time-dependent manner with significant up-regulation at different times. This phenomenon is specific and is mediated by the NF-κB pathway.