The innate immune system is an important mechanism that protects the host from infection. Viral and bacterial infection triggers activation of the transcription factors interferon response factor (IRF) 3 and nuclear factor (NF)-kB. These transcription factors collaborate to induce transcription of type I interferons (IFNs) cytokines and the interleukin (IL)-12 family of cytokines. Type I IFN and the IL-12 family of cytokines play a critical role in establishing innate immune responses as well as initiating and directing adaptive responses. Our study focused on the role of protein kinase C (PKC) isoforms in Toll-like (TLR)-dependent and –independent activation of IRF-3 and NF-kB and their subsequent regulation of IFN-beta and the IL-12 family of cytokines.
TLR3, TLR4 and retinoic acid-inducible gene 1 (RIG-1)/melanoma differentiation associated gene 5 (MDA-5) activation by double stranded (ds) RNA mimic polyinosine-polycytidylic acid (poly(I:C)), lipopolysaccharide (LPS) and synthetic ds-B-DNA respectively, mediated IFN-beta as well as TNF-alpha and IL-8 synthesis in monocyte-derived DCs. Using the pharmacological inhibitor of conventional PKCs (cPKCs), Gö6976, we demonstrated that this family of kinases was involved in TLR3, TLR4 and RIG-1/ MDA-5 signaling pathways leading to the production of IFN-beta but not of TNF-alpha and IL-8. Further analysis with the use of specific kinase inactive cPKC isoforms and siRNA targeted to PKCalpha, we established that PKCalpha was the isoform involved in the TLR3 signaling pathway. In the case of TLR3, we show that PKCalphaexerts its effect downstream of TRIF and TBK1. Moreover, we show that inactivation of PKCalpha specifically inhibits the activation of IRF-3 and not that of NF-kB. Through biochemical analysis, we assessed the contribution of PKCalpha in the critical events of IRF-3 activation: a) phosphorylation b) homodimerization c) nuclear translocation d) DNA-binding and e) recruitment of creb-binding protein (CBP). We conclude that inhibition of cPKCs severely hinders the association of IRF-3 with CBP. Overall, these data revealed the critical role of cPKCs in TLR-dependent and -independent pathways leading to IFN-beta synthesis.
The selective targeting of IRF-3 by cPKCs prompted us to study the possible implications of cPKCs in the transcriptional control of IL-12 family members, some of which are regulated by IRF3. Indeed, recent studies have emerged demonstrating the essential role of IRF-3 in IL-12p35 and IL-27p28 gene expression (1;2). Likewise, we investigated the role of cPKCs in the regulation of LPS- and poly(I:C)-induced expression of IL-12(p40/p35), IL-23(p40/p19) and IL-27(p28/EBI3) in monocyte-derived DCs. Treatment of monocyte-derived DCs with Gö6976 down-regulated LPS- and poly(I:C)-induced IL-12 and IL-27 synthesis while it did not alter IL-23 production. Next, we showed that impaired IL-12 and IL-27 synthesis was due to repressed IL-12p35 and IL-27p28 gene expression downstream of TLR3 and TLR4 whereas IL-23p19 and IL-27EBI3 gene expression were not modified. Reporter gene assays demonstrated that cPKCs are involved in LPS- and poly(I:C)-induced IL-12p35 and IL-27p28 promoter activity. Finally, experiments in bone marrow-derived DCs from IRF-3-/- and wild type mice showed that IL-23 synthesis does not require IRF-3 activation. We conclude that cPKCs through the control of IRF-3 activity are critically involved in the regulation of IL-12 and IL-27 synthesis downstream of TLR3 and TLR4 while they do not participate in IRF-3-independent IL-23 synthesis.
On whole, we demonstrated a novel function for cPKCs in the regulation of IRF-3 and IRF-3 dependent gene expression, specifically IFN-beta, IL-12 and IL-27. In light of the important and divergent roles of IFN-beta and IL-12 family of cytokines on the development of T helper (Th) Th1, Th2, Th17-mediated immune responses, cPKCs represent a potential target for therapeutic immunomodulation. This modulation needs to be carefully administered due to the complex interplay of the IL-12 family members in immunity.
| Identifer | oai:union.ndltd.org:BICfB/oai:ulb.ac.be:ETDULB:ULBetd-01072008-190712 |
| Date | 27 November 2007 |
| Creators | Johnson, Jolyn |
| Contributors | Leo, Oberdan, Trottein, Francois, Bergmann, Pierre, Goldman, Michel, Bruyns, Catherine, Communi, Didier, Erneux, Christophe, Willems, Fabienne |
| Publisher | Universite Libre de Bruxelles |
| Source Sets | Bibliothèque interuniversitaire de la Communauté française de Belgique |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/vnd.ms-powerpoint, application/pdf |
| Source | http://theses.ulb.ac.be/ETD-db/collection/available/ULBetd-01072008-190712/ |
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