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

SVGA astrocytes were cultured and seeded in 6 well plates. The cells were 1h pre-treated with chemical inhibitor for MAPK pathway (SB203580 –p38; SP600125 –Jnk and UO126—Erk) 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 48h post-transfection, and protein concentration of secreted IL-6 and IL-8 was determined using BioPlex multi-cytokine assay. (A, C) mRNA expression levels of IL-6 and IL-8 calculated relative to mock-transfected controls in the presence of chemical inhibitors, respectively. (B, D) Protein concentrations for secreted IL-6 and IL-8 with chemical inhibitors, respectively. (E, F) Depicts the effect of SB203580 and SP600125 on phosphorylated p38 and Jnk MAPK’s, respectively. (G, H) represents the effect of Erk-MAPK inhibitor UO126 on the mRNA expression levels of IL-6 and IL-8, respectively. (I, K) portray mRNA levels while (J, L) show protein concentration for IL-6 and IL-8 when individuals p38-MAPK 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, # p < 0.01, ^a p < 0.05 as compared to Vpr transfected cells; ** p < 0.01 and * p < 0.05 compared to mock-transfected controls.