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

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Abstract
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-β (TRIF)-dependent signaling, which induces IFN-β gene expression downstream of TLR3 and TLR4. First, treatment of monocyte-derived dendritic cells (DC) with wortmannin or LY294002 was found to enhance IFN-β expression upon TLR3 or TLR4 engagement. In the same models of DC activation, PI3K inhibition increased DNA-binding activity of NF-κB, but not interferon response factor (IRF)-3, the key transcription factors required for TLR-mediated IFN-β synthesis. In parallel, wortmannin-treated DC exhibited enhanced levels of IκB kinase (IKK)-α/β phosphorylation and IκB-α degradation with a concomitant increase in NF-κB nuclear translocation. Experiments carried out in HEK 293T cells stably expressing TLR3 or TLR4 confirmed that inhibition of PI3K activity enhances NF-κB-dependent promoters as well as IFN-β promoter activities without interfering with transcription at the positive regulatory domain III-I. Furthermore, wortmannin enhanced NF-κB activity induced by TRIF overexpression in HEK 293T cells, while overexpression of catalytically active PI3K selectively attenuated TRIF-mediated NF-κB transcriptional activity. Finally, in co-immunoprecipitation experiments, we showed that PI3K physically interacted with TRIF. We conclude that inhibition of PI3K activity enhances TRIF-dependent NF-κB activity, and thereby increases IFN-β synthesis elicited by TLR3 or TLR4 ligands.