HIV-1 gp120-mediated increases in IL-8 production in astrocytes are mediated through the NF-kappaB pathway and can be silenced by gp120-specific siRNA

Abstract

Abstract

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.