Etude de la signalisation virale de l'induction du gene de l'IL-15 dans les cellules monocytaires THP-1

Respiratory syncytial virus (RSV) is a single-stranded negative sense RNA virus, member of the Paramyxoviridae family. It is the primary cause of hospitalisation in the first year of life for infants in most parts of the world. In vitro and in vivo studies demonstrated that RSV is a powerful inducer of pro-inflammatory cytokines. One of these is interleukin-15 (IL-15), an important anti-viral cytokine. Herpes simplex virus 1 (HSV-1) is a double-stranded DNA virus, member of the Herpesviridae family. It is the most common cause of fatal sporadic viral encephalitis in North America. In vitro and in vivo studies demonstrated that HSV-1 induces type I interferon and IL-15 expression in infected cells and mice. IL-15 plays an important role in NK and T cell development and differentiation; it activates their proliferation, their cytotoxicity and their cytokine and chemokine production. IL-15 also regulates NK cell/macrophage interaction and promotes regulation of monocytes/macrophages and granulocytes. Previous studies from our laboratory have demonstrated that different viruses upregulated IL-15 gene expression in human PBMCs. Our recent results demonstrated that RSV and HSV-1 also induces the expression of IL-15 mRNA in the monocytic cell line THP-1. Signalling pathway involved in such virus-induced up-regulation of IL-15 has not yet been identified. Here, we report a study with RSV- and HSV-1-treated THP-1 cells describing this mechanism. Our hypothesis was that RSV and HSV-1 induced the expression of IL-15 in THP-1 cells through the involvement of the serine/threonine kinase and/or the tyrosine kinase pathway, kinases usually known to be involved in these viral infections. Using RT-PCR, we demonstrate here for the first time that RSV and HSV-1 induce the expression of IL-15 mRNA in THP-1 cells, and show that this induction involves the phosphorylation of PKC alpha/beta by RSV, and the phosphorylation of PKC and tyrosine kinase by HSV-1. Further, inhibition of these kinases by different specific inhibitors was found to prevent the RSV- and HSV-1-induced up-regulation of IL-15. Since previous studies had described the important role of the nuclear transcription factor NF-kappaB in the regulation of IL-15 transcription, we then examined the role of NF-kappaB in the transactivation of the IL-15 gene by RSV- and HSV-1-infected THP-1 cells. We show by electrophoretic mobility shift assay that the activated form of NF-kappaB binds to the IL-15 promoter sequence. We then confirm by an ELISA assay the involvement of NF-kappaB in the transcription of IL-15 in RSV- and HSV-1-infected THP-1 cells.;These studies provide new insights into the signaling mechanism activated by a single-stranded RNA virus, RSV and a double-stranded DNA virus, HSV-1 and allow a better understanding of the cascade of events mediated by different receptors including TLR3, via which the innate immune system may respond to different viral infections.;Keywords. RSV, HSV-1, IL-15, monocytes, PKC, PTK, TLR3, TRIF, TRAF6, NF-kappaB, signaling.;Furthermore, it has been clearly assumed that the innate immune Toll-like Receptor (TLR) 3 plays a fundamental role in the recognition of double stranded RNA virus and contributes in host response to viral infection. Previous studies have reported that RSV enhances the expression of the TLR3, which mediates inflammatory cytokine and chemokine production in RSV-infected epithelial cells. In our study, we sought to investigate whether TLR3 is involved in mediating the RSV-induced IL-15 gene expression in monocytic cells. We present data showing that targeting the expression of TLR3 with specific small interfering RNA (siRNA) reduces the IL-15 mRNA expression in RSV-treated cells. In addition, transfection of THP-1 cells with siRNA targeted to TRIF and TRAF6 also decreases RSV-upregulated IL-15 mRNA comparatively with control siRNA and non-transfected cells. It is known that TRAF6 activates IKK which phosphorylates IkappaB, causing its degradation and the subsequent release of NF-kappaB. We then examined the phosphorylation of IkappaB kinase and the activation of NF-kappaB in RSV infected cells and found that RSV induces the activation of NF-kappaB. We also examined the role of MyD88 in RSV-induced IL-15 expression and found that MyD88 is not involved. These results demonstrate that RSV activates the inflammatory cytokine IL-15 expression through TLR3-mediated pathway and that adapter protein MyD88 is not involved in this mechanism.