Myeloid suppressor cells induced by hepatitis C virus suppress T-cell responses through the production of reactive oxygen species†

Hepatitis C virus (HCV) infection in humans is almost invariably associated with viral persistence leading to chronic hepatitis, which in turn predisposes the infected individual to hepatocellular carcinoma and the necessity of a liver transplant.1 CD8+ T cells play a pivotal role in controlling HCV infection; however, severe CD4+ and CD8+ T-cell dysfunction has been observed in chronic HCV patients.2 This suggests that HCV may employ mechanisms to evade or possibly suppress the host T-cell response. Innate immune cells play a pivotal role in controlling viral infection during the early phase of infection and in shaping adaptive immunity. Because monocytes/macrophages (M/Mϕ) and dendritic cells (DCs) are the major innate immune cell types at the site of viral infection, their interaction with effector T cells is crucial for determining the course of the immune response. However, during chronic viral infection M/Mϕ and DCs exhibit aberrant antigen-presenting cell (APC) activation and function, including abnormally low production of inflammatory cytokines (i.e., interferon-alpha [IFN-α], interleukin [IL]-12).3 Thus, it is possible that HCV actively suppresses the immune response by altering the differentiation of innate immune cells, resulting in an impairment of a subsequent robust antiviral adaptive response. HCV infection and replication mainly occurs in hepatocytes.4 Due to fenestrations in liver endothelial cells, innate immune cells recruited to the liver following HCV infection directly interact with HCV-infected hepatocytes. Intriguingly, HCV core protein (21 kDa) is secreted from HCV-infected hepatocytes and is present extracellularly in the plasma of chronically infected patients.5 Extracellular core exerts an immunomodulatory role in human M/Mϕ and DCs resulting in inhibition of Toll-like receptor (TLR)-induced proinflammatory cytokine production including IFN-α and IL-12.6,7 Furthermore, HCV core activates signal transducer and activator of transcription 3 (STAT3), a transcription factor that is critical for the development of regulatory APCs, through the up-regulation of IL-6.8 These studies suggest that HCV core alters APC activation and differentiation. Thus, T-cell responses against HCV are likely impaired through viral factor-mediated alteration of myeloid cells, allowing the establishment of persistent infection in the liver. Myeloid-derived suppressor cells (MDSCs) are a heterogeneous subset of regulatory APCs that are responsible for the inhibition of T-cell responses. MDSCs have been well described in multiple severe human diseases such as cancer, autoimmune disease, and bacterial infections.9 In the mouse, the MDSC populations have been divided into two groups; polymorphonuclear MDSCs (PMN-MDSC) described as CD11b+Gr-1highLy6G+Ly6Clow/int cells and mononuclear MDSCs (Mo-MDSC) described as CD11b+Gr-1intLy6G−Ly6Chigh cells.10,11 However, the phenotypic markers of MDSCs are less clear in humans. Although MDSCs have been described as CD33+CD11b+HLADRlow/− in some cancer models, the expression level of CD14 is variable in different experimental systems.9,12 Although the precise molecular mechanism for the differentiation of MDSCs is yet to be defined, the expansion and accumulation of these cells are mediated by tumor-derived factors including M-CSF, granulocyte-macrophage colony-stimulating factor (GM-CSF), transforming growth factor beta (TGF-β), vascular endothelial cell growth (VEGF), and IL-6.13 The vast majority of these factors activate STAT3, underscoring STAT3 as an important transcription factor in MDSC differentiation. Indeed, ablation of STAT3 using conditional knockout mice reduced the expansion of MDSCs and improved T-cell responses in tumor-bearing mice.14 MDSCs have been shown to suppress T-cell responses by way of numerous mechanisms including expression of inhibitory cell surface molecules, production of regulatory cytokines, the metabolism of arginine through activation of arginase-1, production of nitric oxide, and the up-regulation of reactive oxygen species (ROS).9 Arginase-1 inhibits T-cell responses through depletion of nonessential amino acid, L-arginine, resulting in down-regulation of CD3-ζ and inhibition of T-cell proliferation.15,16 Nitric oxide (NO) production in MDSCs is induced through up-regulation of inducible nitric oxide synthase (iNOS), NO down-regulates MHC class II in APCs and leads to T-cell apoptosis.17,18 In leukocytes, ROS is primarily generated through NADPH oxidase. The oxidase is a multicomponent enzyme consisting of two membrane proteins, gp91 and p22, and at least four cytosolic components: p47phox, p67phox, p40phox, and a small G protein Rac.19 In MDSCs a number of these components have been shown to be up-regulated, including p47phox and gp91.20 Notably, the regulation of these proteins was shown to be dependent on STAT3 activation, which provides further evidence for the importance of this transcription factor.20 Here we show that HCV induces the accumulation of MDSC through extracellular core protein. Human CD33+ cells cocultured with HCV-infected hepatocytes, or treated with HCV core, suppress the activation of autologous T cells. Additionally, the suppression of T cells by HCV core-treated MDSCs is ROS-dependent. Core-treated CD33+ cells were CD14+-CD11blow/+ and HLADR−/low. Further, HCV core treatment up-regulated NOX2 component, p47phox. Lastly, CD33+ cells from chronically infected patients were CD11b+CD14+ and HLADR−/low; these cells also up-regulated p47phox compared with healthy donors. These data provide evidence that HCV core induces the accumulation of ROS producing MDSCs, thereby inhibiting host T-cell responses. Therefore, this study describes a novel mechanism for HCV-mediated immune regulation, and suggests that regulation of the MDSC population may be an attractive target for future HCV therapies.