Human immunodeficiency virus type 1 viral protein R (Vpr) induces CCL5 expression in astrocytes via PI3K and MAPK signaling pathways

Abstract 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.Peer Reviewe

Multiple Protein Kinases via Activation of Transcription Factors NF-κB, AP-1 and C/EBP-δ Regulate the IL-6/IL-8 Production by HIV-1 Vpr in Astrocytes

<div><p>Neurocognitive impairments affect a substantial population of HIV-1 infected individuals despite the success of anti-retroviral therapy in controlling viral replication. Astrocytes are emerging as a crucial cell type that might be playing a very important role in the persistence of neuroinflammation seen in patients suffering from HIV-1 associated neurocognitive disorders. HIV-1 viral proteins including Vpr exert neurotoxicity through direct and indirect mechanisms. Induction of IL-8 in microglial cells has been shown as one of the indirect mechanism through which Vpr reduces neuronal survival. We show that HIV-1 Vpr induces IL-6 and IL-8 in astrocytes in a time-dependent manner. Additional experiments utilizing chemical inhibitors and siRNA revealed that HIV-1 Vpr activates transcription factors NF-κB, AP-1 and C/EBP-δ via upstream protein kinases PI3K/Akt, p38-MAPK and Jnk-MAPK leading to the induction of IL-6 and IL-8 in astrocytes. We demonstrate that one of the mechanism for neuroinflammation seen in HIV-1 infected individuals involves induction of IL-6 and IL-8 by Vpr in astrocytes. Understanding the molecular pathways involved in the HIV-1 neuroinflammation would be helpful in the design of adjunct therapy to ameliorate some of the symptoms associated with HIV-1 neuropathogenesis.</p></div

HIV-1 Vpr mediated induction of IL-6 and IL-8 in astrocytes involves PI3K/Akt pathway.

<p>SVGA astrocytes were cultured and seeded in 6 well plates. The cells were 1h pre-treated with chemical inhibitor for PI3K/Akt pathway (LY294002) and then transfected with a plasmid encoding HIV-1 Vpr or were mock-transfected. The cells were harvested at 6h post-transfection followed by the determination of IL-6 and IL-8 mRNA expression levels using real-time RT-PCR. The cell culture supernatants were collected at 48 h post-transfection, and protein concentration of secreted IL-6 and IL-8 was determined using BioPlex multi-cytokine assay. <b>(A, C)</b> mRNA expression levels of IL-6 and IL-8 calculated relative to mock-transfected controls in the presence of LY294002, respectively. <b>(B, D)</b> Protein concentrations for secreted IL-6 and IL-8 with LY294002, respectively. <b>(E, F)</b> Depicts the effect of LY294002 on phosphorylated Akt and NF-κB p-65 nuclear translocation, respectively. <b>(G, I)</b> portray mRNA levels while <b>(H, J)</b> show protein concentration for IL-6 and IL-8 when individuals Akt isoforms were silenced, respectively. Every bar represents the mean ± SE of thee independent experiments done in triplicates. Statistical analyses were performed using 1-way ANOVA using post-hoc Tukey HSD test, <b>#</b> p < 0.01, ^<b>a</b> p < 0.05 as compared to Vpr transfected cells; <b>**</b> p < 0.01 and <b>*</b> p < 0.05 compared to mock-transfected controls.</p

HIV-1 Vpr time-dependently induces IL-6 and IL-8 in astrocytes.

<p>SVGA astrocytes were seeded in 6 well plates and transfected with a plasmid encoding HIV-1 Vpr or were mock-transfected. The cells were harvested at 1, 3, 6, 12, 24, 48 and 72h post-transfection followed by the determination of IL-6 and IL-8 mRNA expression levels using real-time RT-PCR. The cell culture supernatants were also collected at 6, 12, 24, 48 and 72h post-transfection, and protein concentration of IL-6 and IL-8 was determined using BioPlex multi-cytokine assay. <b>(A, C)</b> mRNA expression levels of IL-6 and IL-8 calculated relative to mock-transfected controls, respectively. <b>(B, D)</b> protein concentration for secreted IL-6 and IL-8 in cell culture supernatants, respectively. <b>(E)</b> Expression of Vpr in SVGA astrocytes using western blotting. <b>(F)</b> Expression of GFP-Vpr in SVGA and primary astrocytes using fluorescent microscopy. <b>(G, H)</b> dose-dependent induction of IL-6 and IL-8 by HIV-1 Vpr plasmid in SVGA astrocytes. <b>(I, J)</b> depicts the mRNA expression levels, while <b>(K, L)</b> shows the secreted protein levels of IL-6 and IL-8 in human fetal astrocytes by HIV-1 Vpr electroporation, respectively. Every bar represents the mean ± SE of three independent experiments done in triplicates. Statistical analyses were performed using 1-way ANOVA using post-hoc Tukey HSD test, ** p < 0.01 and * p < 0.05.</p

Immunocytochemical staining portraying induction of IL-6 and IL-8 by HIV-1 Vpr in SVGA astrocytes.

<p>SVGA astrocytes were cultured on cover slips and were either mock-transfected <b>(d-f)</b> or were transfected with a plasmid encoding Vpr <b>(g-i)</b>. Non-transfected cells were used as control <b>(a-c)</b>. The cells were stained for nucleus (<i>blue</i>); IL-6 and IL-8 (<i>green</i>) and GFAP (<i>red</i>). The images were captured using a Leica TCS SP5 II on an inverted microscope platform with a 40× zoom oil emersion lens. The Image J software was used to get the merged images, and the Multi Measure tool in Image J was used to quantify the intensities. <b>(A, B)</b> Immunocytochemical staining depicting induction of IL-6 and IL-8 by HIV-1 Vpr in SVGA astrocytes. <b>(C, D)</b> The image intensities for IL-6 and IL-8 are plotted relative to GFAP. Every bar represents the mean ± SE of thee independent experiments done in triplicates. Statistical analyses were performed using 1-way ANOVA using post-hoc Tukey HSD test, ** p < 0.01 and * p < 0.05.</p

Neuronal and astrocytic contributions to Huntington’s disease dissected with zinc finger protein transcriptional repressors

Huntington's disease (HD) is caused by expanded CAG repeats in the huntingtin gene (HTT) resulting in expression of mutant HTT proteins (mHTT) with extended polyglutamine tracts, including in striatal neurons and astrocytes. It is unknown whether pathophysiology in&nbsp;vivo can be attenuated by lowering mHTT in either cell type throughout the brain, and the relative contributions of neurons and astrocytes to HD remain undefined. We use zinc finger protein (ZFP) transcriptional repressors to cell-selectively lower mHTT in&nbsp;vivo. Astrocytes display loss of essential functions such as cholesterol metabolism that are partly driven by greater neuronal dysfunctions, which encompass neuromodulation, synaptic, and intracellular signaling pathways. Using transcriptomics, proteomics, electrophysiology, and behavior, we dissect neuronal and astrocytic contributions to HD pathophysiology. Remarkably, brain-wide delivery of neuronal ZFPs results in strong mHTT lowering, rescue of HD-associated behavioral and molecular phenotypes, and significant extension of lifespan, findings that support translational development