IntroductionThe eradication of hepatitis C virus (HCV) in Ͼ 50% of chronically infected patients by treatment with IFN-␣ 1,2 suggests that production of endogenous IFN-␣ plays an important role in the control of HCV infection. Plasmacytoid dendritic cells (pDCs) are a highly specialized subset of dendritic cells that function as sentinels for viral infection and are responsible for production of type I (IFN-␣ and ␤) and type III (IFN-/IL-28/29) IFNs, proinflammatory cytokines, and antigen presentation during viral infection. [3][4][5][6] pDCs are able to detect genetic material of viruses with a subset of Toll-like receptors (TLRs) localized to the endosomal compartment. 7 These nucleotide-sensing TLRs include TLR7, which recognizes single-stranded RNA, and TLR9, which recognizes DNA.In addition to nucleotide-sensing TLRs, pDCs also recognize pathogens through a battery of cell surface regulatory receptors, including Fc (FcR) and C-type lectin receptors (CLRs). The principal function of these regulatory receptors on pDCs is to facilitate antigen capture and to prevent aberrant immune responses by modulating production of type I IFNs and proinflammatory cytokines. 7 Some of these receptors, such as a member of CLR family, blood DC antigen 2 (BDCA-2), associate with the ␥-chain of Fc⑀RI receptor, which contains an immunoreceptor tyrosinebased activation motif. In pDCs, triggering of these receptors initiates a signaling pathway similar to the one that occurs downstream of the B-cell receptor. 8 Other CLRs of pDC, such as DC-immunoreceptor (DCIR), 9 contain an immunoreceptor tyrosinebased inhibitory motif. Triggering of BDCA-2 8,10 or DCIR 9 inhibits TLR9-and in some cases TLR7-induced production of type I IFNs and proinflammatory cytokines.Several reports have shown that exposure of pDCs from healthy donors to HCV particles results in no or only weak production of type I IFN. [11][12][13][14][15] However, in addition to this passive role, HCV particles also actively impair production of IFN-␣ triggered in pDCs by synthetic agonists of TLR9. 11,13 In contrast to HCV particles, a recent report has shown that HCV-infected hepatoma cells induce in pDCs a robust TLR7-mediated production of type I IFN. 14 Here we investigated whether HCV particles suppress production of IFN-␣ induced in pDCs by HCV-infected hepatoma cells. Furthermore, we investigated the mechanism of the impairment of pDC functions by HCV particles. We demonstrate that, in addition to type I IFNs, exposure of pDCs to HCV-infected hepatoma cells resulted in the production of type III IFNs and that HCV particles or HCV envelope glycoprotein E2 inhibited the production of both types of IFNs by binding to CLRs, BDCA-2 and DCIR. We further show that HCV particles induce in pDCs a rapid phosphorylation of Akt and Erk1/2, in a manner similar to the crosslinking of BDCA-2 or DCIR. Blocking of BDCA-2 and DCIR with Fab fragments of mAbs protects pDCs against the inhibition of IFN production induced by HCV particles. Thus, negative interference of CLR signaling t...

Crosslinking of regulatory immunoreceptors (RR), such as BDCA-2 (CD303) or ILT7 (CD85g), of plasmacytoid dendritic cells (pDCs) efficiently suppresses production of type-I interferon (IFN)-α/β and other cytokines in response to Toll-like receptor (TLR) 7/9 ligands. This cytokine-inhibitory pathway is mediated by spleen tyrosine kinase (Syk) associated with the ITAM-containing adapter of RR. Here we demonstrate by pharmacological targeting of Syk that in addition to the negative regulation of TLR7/9 signaling via RR, Syk also positively regulates the TLR7/9 pathway in human pDCs. Novel highly specific Syk inhibitor AB8779 suppressed IFN-α, TNF-α and IL-6 production induced by TLR7/9 agonists in primary pDCs and in the pDC cell line GEN2.2. Triggering of TLR9 or RR signaling induced a differential kinetics of phosphorylation at Y352 and Y525/526 of Syk and a differential sensitivity to AB8779. Consistent with the different roles of Syk in TLR7/9 and RR signaling, a concentration of AB8779 insufficient to block TLR7/9 signaling still released the block of IFN-α production triggered via the RR pathway, including that induced by hepatitis B and C viruses. Thus, pharmacological targeting of Syk partially restored the main pDC function—IFN-α production. Opposing roles of Syk in TLR7/9 and RR pathways may regulate the innate immune response to weaken inflammation reaction.