Inhibition of phosphoinositide 3-kinase enhances TRIF-dependent NF-κB activation and IFN-β synthesis downstream of Toll-like receptor 3 and 4

Phosphoinositide 3-kinases (PI3K) are known to regulate Toll-like receptor (TLR)mediated inflammatory responses, but their impact on the different pathways of TLR signaling remains to be clarified. Here, we investigated the consequences of pharmacological inhibition of PI3K on Toll-IL-1 receptor domain-containing adapter-inducing IFN-beta (TRIF) -dependent signaling, which induces IFN-beta gene expression downstream of TLR3 and TLR4. First, treatment of monocyte-derived dendritic cells (DC) with wortmannin or LY294002 was found to enhance IFN-beta expression upon TLR3 or TLR4 engagement. In the same models of DC activation, PI3K inhibition increased DNA-binding activity of NF-kappa B, but not interferon response factor (IRF)-3, the key transcription factors required for TLR-mediated IFN-beta synthesis. In parallel, wortmannin-treated DC exhibited enhanced levels Of I kappa B kinase (IKK)-alpha/beta phosphorylation and I kappa B-alpha degradation with a concomitant increase in NF-kappa B nuclear translocation. Experiments carried out in HEK 293T cells stably expressing TLR3 or TLR4 confirmed that inhibition of PI3K activity enhances NF-kappa B-dependent promoters as well as IFN-beta promoter activities without interfering with transcription at the positive regulatory domain III-I. Furthermore, wortmannin enhanced NF-kappa B activity induced by TRIF overexpression in HEK 293T cells, while overexpression of catalytically active PI3K selectively attenuated TRIF-mediated NF-kappa B transcriptional activity. Finally, in coimmunoprecipitation experiments, we showed that PI3K physically interacted with TRIF. We conclude that inhibition of PI3K activity enhances TRIF-dependent NF-kappa B activity, and thereby increases IFN-beta synthesis elicited by TLR3 or TLR4 ligands.