Regulation of IFN Regulatory Factor-7 and IFN-α Production by Enveloped Virus and Lipopolysaccharide in Human Plasmacytoid Dendritic Cells

Abstract

Human plasmacytoid dendritic cells (PDC) are a major source of IFN-α upon exposure to enveloped viruses and TLR-7 and TLR-9 ligands. Although IFN regulatory factor-7 (IRF-7) is known to play an essential role in virus-activated transcription of IFN-α genes, the molecular mechanisms of IFN-α production in human PDC remain poorly understood. We and others have recently reported high constitutive levels of IRF-7 expression in PDC as compared with other PBMC. In this study, we demonstrate that both LPS and HSV up-regulate the expression of IRF-7 in PDC, and that this enhancement of IRF-7 is dependent on NF-κB activation. The NF-κB inhibitors MG132 and pyrrolidinedithiocarbamate efficiently inhibited the induction of IRF-7 by HSV or LPS, and also down-regulated the constitutive expression of IRF-7 in PDC and blocked the HSV-induced production of IFN-α. In addition, we found that nuclear translocation of IRF-7 occurred rapidly in response to HSV stimulation, but not in response to LPS, which is consistent with the stimulation of IFN-α production by virus and not by LPS. Although LPS by itself was not able to induce IFN-α production, it led to rapid up-regulation of TLR-4 on PDC and increased the magnitude and accelerated the kinetics of HSV-induced IFN-α production in PDC, providing a mechanism that might be operative in a scenario of mixed infection. In contrast to the current concept of IFN-α regulation established in cell lines, this study strongly supports the immediate availability of high constitutive levels of IRF-7 expression in PDC, and suggests an activation required for IRF-7 that contributes to IFN-α production in virus-stimulated PDC.

Receptor Cross-Linking on Human Plasmacytoid Dendritic Cells Leads to the Regulation of IFN-α Production

“…In PDC, IRF-7 is translocated to the nucleus within 2 h of stimulation with HSV, as shown by fluorescence microscopy (29). Preliminary experiments using this technique showed that cross-linking CD4 on PDC inhibited the nuclear translocation of IRF-7 in response to stimulation with HSV (data not shown).…”

“…We postulate that these high levels of constitutive IRF-7 allow for the rapid expression of large quantities of IFN-␣ by PDC without the need for the aforementioned autocrine feedback loop. As in other cells, after viral stimulation, IRF-7 is phosphorylated, forms homodimers, and is translocated from the cytoplasm to the nucleus where it acts as a transcription factor for IFN-␣ genes (26,29,39). Because IRF-7 plays a large part in the production of IFN-␣, it may also play a pivotal role in the regulation of this cytokine.…”

“…In PDC, IRF-7 is expressed at high constitutive levels and is further up-regulated in response to HSV and IFN-␣ stimulation (26,29,39) (Fig. 7A).…”

“…The PBMC were then stimulated with HSV (MOI ϭ 1) for 6 h at 37°C. Cells were surface stained for PDC-defining markers CD123 PE and HLA-DR allophycocyanin (29,30) or BDCA-2 PE and BDCA-4 allophycocyanin, and intracellular flow cytometry for IFN-␣ was performed. As shown in Fig.…”

TLR signaling

“…[64][65][66][67] Unlike other types of DCs, pDCs constitutively express high levels of IRF7. 68,69 Furthermore, treatment of pDCs with TLR9 ligands causes nuclear translocation of IRF7, and pDCs derived from IRF7-deficient mice are incapable of producing type I IFN in response to TLR7/8 and TLR9 ligands. 64,65 Thus, IRF7 is an essential transcription factor that regulates type I IFN induction in pDCs.…”