HIV-1 glycoprotein 120 (gp120) plays an essential role in viral attachment and entry into host cells. In this study, we showed that gp120 induces IL-8 expression at both RNA and protein level in a time-dependent manner in astrocytes. Several earlier reports from different laboratories have shown the neurotoxic potential of gp120 during HIV-1 infection [25, 26]. This phenomenon has also been shown to be mediated via oxidative stress [13, 27], and has also been shown to correlate with increased production of TNF-α, IL-1β and IL-6 [9, 28, 29]. Recently, an elegant report by Li and co-workers showed indirect evidence for a role for gp120 in IL-8 over-expression, wherein they showed reduced IL-8 production in astrocytes when these were infected with gp120-deleted virion compared to those cells that were infected with wild-type .
In this study we have for the first time shown that gp120 is involved in IL-8 up-regulation in time-dependent manner, and that this effect is specific. This was confirmed by using gp120-specific siRNA. The siRNA approach has evolved into an important tool for gene-silencing studies in mammalian systems during recent years, and a variety of siRNAs have been used to knockdown various genes like gag, tat, pol and integrase, and to inhibit HIV replication [30–34]. However, no study has reported inhibition of IL-8 or any other cytokine/chemokine using the siRNA approach. In order to assess whether IL-8 induction is indeed a result of gp120 introduction into cells, we used various siRNAs in order to knockdown gp120 expression. In view of significant inhibition observed both at RNA and protein levels, we conclude that IL-8 induction observed in gp120-transfected cells is gene specific.
In this study, we have shown that gp120-mediated increases in IL-8 expression could be inhibited by specific NF-κB pathway inhibitors, implying role for this pathway in IL-8 production. Earlier studies have already shown a role for NF-κB activation in IL-8 production in asthmatic patients . Using various mutational and deletion analysis it has been proved that certain promoter elements in NF-κB complex play a potential role in inducing the IL-8 promoter, which emphasizes the important role of NF-κB in IL-8 transcription [36, 37]. SC514 is a novel inhibitor of NF-κB, which targets IKK-2 , whereas BAY11-7082 blocks NF-κB activation by inhibiting TNF-α-induced phosphorylation of IKKβ . IkKβ acts as a negative regulator of the NF-κB pathway and prevents NF-κB activation. Phosphorylation of IkKβ leads to activation of NF-κB, and activated NF-κB later incorporates into the nucleus and leads to a downstream cascade. In our study, we observed that inhibition of NF-κB activation by targeting IkKβ leads to partial abrogation of gp120-mediated IL-8 expression. Involvement of the NF-κB pathway was also confirmed by silencing the NF-κB gene, which resulted in partial-to-near-complete restoration of gp120-mediated IL-8 expression to basal level in astrocytes.
In summary, we have shown that gp120 induces up-regulation of IL-8 in astrocytes, and that the NF-kB pathway appears to be predominantly responsible for this since direct interference with this pathway disrupts gp120-dependent induction of IL-8. We have also shown that gp120-specific siRNA abrogates this effect, suggesting that IL-8 over-expression is gp120 specific. These data suggest that IL-8 may be a potential target for intervention to reduce or ameliorate neuroinflammation, and could also become an important adjunct therapeutic strategy for future consideration.