HIV-1 Nef induces CCL5 production in astrocytes through p38-MAPK and PI3K/Akt pathway and utilizes NF-kB, CEBP and AP-1 transcription factors

Abstract

The prevalence of HIV-associated neurocognitive disorders (HAND) remains high in patients infected with HIV-1. The production of pro-inflammatory cytokines by astrocytes/microglia exposed to viral proteins is thought to be one of the mechanisms leading to HIV-1- mediated neurotoxicity. In the present study we examined the effects of Nef on CCL5 induction in astrocytes. The results demonstrate that CCL5 is significantly induced in Nef-transfected SVGA astrocytes. To determine the mechanisms responsible for the increased CCL5 caused by Nef, we employed siRNA and chemical antagonists. Antagonists of NF-κB, PI3K, and p38 significantly reduced the expression levels of CCL5 induced by Nef transfection. Furthermore, specific siRNAs demonstrated that the Akt, p38MAPK, NF-κB, CEBP, and AP-1 pathways play a role in Nef-mediated CCL5 expression. The results demonstrated that the PI3K/Akt and p38 MAPK pathways, along with the transcription factors NF-κB, CEBP, and AP-1, are involved in Nef-induced CCL5 production in astrocytes.

Figure 1. HIV-1 Nef induces CCL5 in SVGA astrocytes in a time-dependent manner.

7 × 10⁵ SVGA astrocytes were seeded in a 6-well plate and were transfected with a plasmid encoding HIV-1 Nef for 5 h using Lipofectamine 2000™. (A) The cells were harvested at 1 h, 3 h, 6 h, 12 h and 24 h for total RNA isolation and the expression of the CCL5 mRNA were determined by real time RT-PCR. Data in figures show fold-change relative to the mock-transfected wells at each time point. (B) CCL5 concentrations released in the supernatants were measured at 6 h, 12 h, 24 h, 48 h and 72 h by multiplex cytokine assay. Each of the bars represents mean ± SE of three independent experiments in triplicates Statistical analyses were performed using a one way ANOVA with an LSD post-hoc test. Treatments that are significantly different from each other (i.e. p < 0.05) are labeled with different letters.

Figure 2. Confocal microscopy of CCL5 induced by HIV-1 Nef in astrocytes.

7 × 10⁵ SVGA astrocytes were seeded on a cover slip in a 6-well plate and were either mock-transfected (D–F) or transfected with a plasmid encoding HIV-1 Nef (G–I) for 5 h using Lipofectamine 2000™. Untreated controls (A–C) were used to visualize basal expression of CCL5 in astrocytes. 12 h later, cells were incubated with primary antibodies against CCL5 and GFAP and appropriate secondary antibodies labeled with Alexafluor 488 (CCL5) and Alexafluor 555 (GFAP). As illustrated in the figure, cells were stained for nucleus (blue); CCL5 (green) and GFAP (red) and the images were captured using Leica TCS SP5 II fluorescent microscope with 40× zoom oil emersion lens.

Figure 3. Involvement of NF-κB pathway in Nef-mediated increase of CCL5 expression.

SVGA cells were treated with 20 μM SC-514 or 10 μM Bay11-7082 prior to the transfection. CCL5 mRNA (A and C) as well as protein (B and D) levels were determined at 6 and 48 h post transfection, respectively. For knockdown of p50 and p65, the cells were transfected with the siRNA followed by transfection of nef-encoding plasmid as mentioned in the materials and methods. The expression of CCL5 at the mRNA and protein levels were determined at 6 h and 48 h post-transfection by real-time RT-PCR (E) and multiplex cytokine assay (F), respectively. The values presented for mRNA are relative to the mock-transfected controls. Each of the bars represents mean ± SE of three independent experiments in triplicates. Statistical analysis was performed using one way ANOVA with the LSD post-hoc test in which * represents p-value ≤0.05 and ** represents p-value ≤0.01.

Figure 4. HIV-1 Nef induces CCL5 expression through the PI3K-Akt pathway.

SVGA astrocytes were pretreated with 10 μM of specific PI3K inhibitor LY294002 (A–B) or transfected with siRNA against Akt1/2/3 (C–E) prior to transfection with a nef-encoding plasmid. The expression of CCL5 at the mRNA and protein levels were determined at 6 h and 48 h post-transfection by real-time RT-PCR (A and D) and multiplex cytokine assay (B and E), respectively. The values represented for mRNA are relative to the mock-transfected controls. Each of the bars represents mean ± SE of three independent experiments in triplicates. Statistical analysis was performed using a one way ANOVA with the LSD post-hoc test t in which * represents p-value ≤0.05 and ** represents p-value ≤0.01. RT-PCR for Akt1, Akt2 and Akt3 was performed to amplify the mock and scrambled controls as well as the targeted RNA. The products were then resolved by electrophoresis on a 1.5% agarose gel, stained with ethidium bromide and visualized under UV light (C).

Figure 5. HIV-1 Nef induces CCL5 expression through the p38 MAPK pathway.

SVGA astrocytes were pretreated with 10 μM of specific p38α and p38β inhibitor SB203580 (A–B) or transfected with siRNA against p38 isoforms (C–E) prior to transfection with nef-encoding plasmid. The expression of CCL5 at the mRNA and protein levels were determined at 6 h and 48 h post-transfection by real-time RT-PCR (A and D) and multiplex cytokine assay (B and E), respectively. The values represented for mRNA are relative to the mock-transfected controls. Each of the bars represents mean ± SE of three independent experiments in triplicates. Statistical analysis was performed using a one way ANOVA with LSD post-hoc test t in which * represents p-value ≤0.05 and ** represents p-value ≤0.01. RT-PCR for p38α, p38β, p38γ and p38δ was performed to amplify the mock and scrambled controls as well as silenced RNA. The products were then resolved by electrophoresis on a 1.5% agarose gel, stained with ethidium bromide and visualized under UV light (C).

Figure 6. Involvement of transcriptional factors C/EBP and AP-1 in Nef-mediated increase of CCL5 expression.

For knockdown of C/EBPα, C/EBPγ and AP-1, the cells were transfected with the siRNA followed by transfection of nef-encoding plasmid as mentioned in the materials and methods. The expression of CCL5 at the mRNA and protein levels were determined at 6 h and 48 h post-transfection by real-time RT-PCR (A) and multiplex cytokine assay (B), respectively. The values represented for mRNA are relative to the mock-transfected controls. Each of the bars represents mean ± SE of three independent experiments in triplicates. Statistical analysis was performed using a one way ANOVA with the LSD post-hoc test in which * represents p-value ≤0.05 and ** represents p-value ≤0.01.

Figure 7. Schematic of the signaling pathways involved in CCL5 up-regulation caused by HIV-1 Nef in astrocytes.

HIV-1 Nef activates PI3K-Akt and p38 MAPK pathways utilizing different subunits indicated in green (the involvement of a specific isoform is shown in brighter color and the absence is shown in pale color). These signaling molecules differentially regulate the induction of CCL5 by activating various downstream transcription factors, including NF-κB, C/EBPα, C/EBPγ and AP-1.