Suppression of Interferon Regulatory Factor 8 Modulates Toll Like Receptor 2 and Toll Like Receptor 7/8 Induced Dendritic Cell Activation

Interferon regulatory factor 8 (IRF-8) is a member of the IRF family of transcription factors, which are stimulated through interferon mediated pathways. In mice, IRF-8 seems to play an essential role in the development and maturation of dendritic cells (DCs). However, very limited knowledge is available about the potential role of IRF-8 in the human system. To bridge this gap we analyzed function and activation of human monocyte-derived dendritic cells (mDCs) lacking IRF-8 expression. To knockdown IRF-8 protein levels, we electroporated mDCs with different siRNAs against IRF-8. Additionally, we stimulated the electroporated mDCs with the Toll like receptor (TLR) 2 ligand Pam₃Cys or the TLR 7/8 ligand R848. IRF-8 knockdown in mDCs was verified constantly by Western Blot analysis using an anti-IRF-8 antibody. We found that IRF-8 knockdown clearly diminished the expression of the human lymphocyte antigen molecules HLA-ABC and HLA-DR in Pam₃Cys and R848 stimulated mDCs. To gain functional data, we performed ELISAs to determine cytokine and chemokine secretion. The electroporation of mDCs with IRF-8 specific siRNA resulted in profound inhibition of secretion of the cytokines IL-6, IL-12 and TNF-a as well as the chemokines MIP-1a (CCL3), MCP-1 (CCL2) and RANTES (CCL5). To get additional information on IRF-8 function in human mDCs, the regulation of signal transduction pathways was determined by Western Blot analysis. The suppression of IRF-8 diminished the nuclear translocation of the NF-kB family member’s c-Rel and RelB as well as PU.1 and IRF-3 in activated mDCs. In addition, we showed that the suppression of IRF-8 caused a reduced phosphorylation of ERK and JNK, but had no effect on the expression of STAT3. In summary, the knockdown of IRF-8 reduced the capability of mDCs to develop appropriate phenotype and functions in response to activating stimuli. Our results indicate that these effects are mediated via the ERK, NF-kB and PU.1 signalling pathways. IRF-8 plays an important role in the activation and function of human mDCs.