IFN-α production by plasmacytoid dendritic cells (PDCs) is critical in antiviral immunity. In the present study, we evaluated the IFN-α-producing capacity of PDCs of patients with chronic hepatitis C virus (HCV) infection in treatment-naive, sustained responder, and nonresponder patients. IFN-α production was tested in PBMCs or isolated PDCs after TLR9 stimulation. Treatment-naive patients with chronic HCV infection had reduced frequency of circulating PDCs due to increased apoptosis and showed diminished IFN-α production after stimulation with TLR9 ligands. These PDC defects correlated with the presence of HCV and were in contrast with normal PDC functions of sustained responders. HCV core protein, which was detectable in the plasma of infected patients, reduced TLR9-triggered IFN-α and increased TNF-α and IL-10 production in PBMCs but not in isolated PDCs, suggesting HCV core induced PDC defects. Indeed, addition of rTNF-α and IL-10 induced apoptosis and inhibited IFN-α production in PDCs. Neutralization of TNF-α and/or IL-10 prevented HCV core-induced inhibition of IFN-α production. We identified CD14+ monocytes as the source of TNF-α and IL-10 in the HCV core-induced inhibition of PDC IFN-α production. Anti-TLR2-, not anti-TLR4-, blocking Ab prevented the HCV core-induced inhibition of IFN-α production. In conclusion, our results suggest that HCV interferes with antiviral immunity through TLR2-mediated monocyte activation triggered by the HCV core protein to induce cytokines that in turn lead to PDC apoptosis and inhibit IFN-α production. These mechanisms are likely to contribute to HCV viral escape from immune responses.

Hepatitis C virus (HCV) is a leading cause of end-stage liver disease through sustained inflammation of the liver produced by the host's immune system. The mechanism for HCV evasion or activation of the immune system is not clear. TLRs are cellular activators of the innate immune system. We recently reported that TLR2-mediated innate immune signaling pathways are activated by HCV core and NS3 proteins. TLR2 activation requires homo- or heterodimerization with TLR1 or TLR6. Here, we aimed to determine whether TLR2 coreceptors participated in cellular activation by HCV core or NS3 proteins. By designing small interfering RNAs targeted to TLR2, TLR1, and TLR6, we showed that knockdown of each of these receptors impairs pro- and anti-inflammatory cytokine activation by TLR-specific ligands as well as by HCV core and NS3 proteins in human embryonic kidney-TLR2 cells and in primary human macrophages. We found that HCV core and NS3 proteins induced TNF-alpha and IL-10 production in human monocyte-derived macrophages, which was impaired by TLR2, TLR1, and TLR6 knockdown. Contrary to human data, results from TLR2, TLR1, or TLR6 knockout mice indicated that the absence of TLR2 and its coreceptor TLR6, but not TLR1, prevented the HCV core and NS3 protein-induced peritoneal macrophage activation. In conclusion, TLR2 may use TLR1 and TLR6 coreceptors for HCV core- and NS3-mediated activation of macrophages and innate immunity in humans. These results imply that multiple pattern recognition receptors could participate in cellular activation by HCV proteins.

Hepatitis C virus (HCV) interferes with interferon (IFN)-mediated innate immune defenses. Toll-like receptor (TLR) 7 agonists robustly inhibit HCV infection. We hypothesize thatHCV is a positive single-stranded RNA virus. 2 Some HCV-derived products, including HCV RNA and several HCV proteins, trigger host defense. 3,4 Immune responses, including cell-mediated immunity and type 1 IFNs, are vital in controlling and clearing HCV infection. 5 A number of host receptors, including TLR3, TLR7, TLR8, and TLR9 and helicases RIG-I and MDA-5, lead to a type 1 IFN-mediated response upon stimulation with viral RNA. 6,7 HCV has developed mechanisms to bypass the immune defense and facilitate viral persistence: NS3/4A protein actively disrupts TLR3-and RIG-I/MDA-5-mediated signaling pathways, thus interfering with the IFN production and bypassing the immunomediated response. 8,9 The effect of HCV on other IFN-inducing pathways is currently unknown.Viral recognition receptors, including TLR7, activate IRF7 through a series of phosphorylation events starting with recruitment of MyD88, the common TLR adaptor protein. Once the receptor is engaged, a complex is formed between MyD88, TRAF6, IRAK4, and IRAK1 that allows for activation of IRF7. Both TRAF6 and IRAK1 have been reported to be responsible for phosphorylating IRF7, leading to its homodimerization and subsequent nuclear translocation. 10,11 Recent reports have indicated that robust TLR7 agonists decrease HCV RNA in HCV-infected patients 12 and HCV RNA and NS5A protein in HCV-replicating hepatoma cells. 13 TLR7 has also been reported to recognize other single-stranded RNA viruses such as influenza 14 and dengue. 15 Based on these reports, we postulated that TLR7 may play a role in Abbreviations: GAPDH, HCV, hepatitis C virus; IFN, interferon; mRNA, messenger RNA; TLR,

H epatitis C virus (HCV) is a uniquely successful hepatitis virus in its ability to establish chronic infection in more than two-thirds of those who contract it. The inability of the innate and adaptive immune responses to control HCV invasion and replication contribute to development and persistence of chronic infection. Multiple components have been identified in this process, including pathways by which HCV subverts innate immune recognition and activation, delayed organization of an effective adaptive immune response, the tolerogenic liver environment, and the persistently high levels of viral antigens. We summarize recent findings on the interaction between HCV infection and innate immune responses. Hepatitis C Virus, Immune Responses and Hepatocytes-Brief OverviewA critical role for innate and adaptive immunity has been identified in HCV persistence and liver injury. During acute infection, type I interferon (IFN) is induced in the liver; however, HCV viral load does not decrease, suggesting that HCV interferes with antiviral mechanisms. Innate immune cells, including natural killer cells and dendritic cells shape innate immunity and determine adaptive immune activation. Vigorous, multi-epitopespecific, T helper 1-type and sustained CD4ϩ and CD8ϩ T cell responses were found in resolved acute HCV infections. [1][2][3][4] In contrast, in cases that progress to chronic infection, CD4ϩ T cell responses are weak, short-lived, and targeted to a narrow range of epitopes. 1

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