HCV Core Protein Inhibites Monocytes-derived Macrophages Polarization

Background and aims:Hepatitis C virus (HCV) establishes persistent infection in most infected patients, and eventually causes chronic hepatitis, cirrhosis, and hepatocellular carcinoma in some patients. The ability of HCV to establish persistent infection with great success in human has been attributed, in part, to a variety of strategies to evade host immune and IFN-induced defenses. Epidemiological studies suggest that up to 20% of acutely infected HCV patients can resolve the infection without treatment, which implies that innate and/or adaptive immune responses are indeed capable of controlling the outcome of HCV infection. Monocytes and macrophages provide the first line of defense against pathogens, and play important roles in immune surveillance and immunoregulation depending on their function of powerful phagocytosis and antigen presentation. However, the roles of them in HCV infection remain not fully understood. Our previous work showed that HCV core protein inhibits human blood-derived dendritic cells development. In the present study, we tested whether or not HCV core protein affects human blood-derived monocytes differentiating into macrophages, which would be an important cause of virus persistence.Methods:Peripheral blood mononuclear (PBMCs) cells from healthy donors were separated by gradient centrifugation method, and purified CD14+ monocytes were received by immune magnetic bead separation method. For M1 macrophages polarization, CD14+ monocytes were cultured with granulocyte-macrophage colony stimulating factor (GM-CSF) for 5 days, and replaced by interferon-γ (IFN-γ) and lipopolysaccharide (LPS) with fresh medium for another 24 hours. For M2 macrophages polarization, CD14+ monocytes were cultured with macrophage colony stimulating factor (M-CSF) for 5 days, and replaced by interleukin-4 (IL-4) and IL-13 with fresh medium for another 24 hours. Phenotypes, cytokines production and genes expression were detected by flow cytometry, enzyme-linked immuno sorbent assay (ELISA) and real-time quantitative polymerase chain (RT-qPCR) to verify the successful polarization.We freshly isolated PBMCs from healthy donors and chronic hepatitis C virus (CHC) infected patients before or after direct-acting antivirals (DAAs) treatment. Purified monocytes by immune magnetic bead separation method were polarized to M1 or M2 macrophages with corresponding stimuants and stimulating conditions as above. Analyze the effect of HCV in macrophages polarization at levels of phenotypes expression, cytokines secretion and genes expression. In vitro expreriments, purified CD14+ monocytes from healthy individuals were polarized to M1 or M2 macrophages in the presence or absence of HCV core protein, and the effect of HCV core protein or β-galactosidase in the macrophages polarization were analyzed by flow cytometry, ELISA and RT-qPCR. Moreover, phagocytic, antigen presenting and immuno-modulatory function of polarized macrophages with or without HCV core protein were detected by phagocytic test and T cell proliferation experiment. Signal transducer and activator of transcription (STAT) of polarized macrophages were detected by western blot to test the effect of HCV core protein. In order to test the signal pathway of HCV core protein inhibiting monocytes differentiation to macrophages, toll-like receptor (TLR) 2 ligand (Pam3CSK4), anti-TLR2 antibody and STATs inhibitor were added to the polarization systerm. The effects of TLR2 and STATs in macrophages polarization were analyzed by flow cytometry, ELISA and RT-qPCR as above.Results:(1) Purified CD14+ monocytes cultured with GM-CSF, IFN-y and LPS were successfully polarized to M1 macrophages, and cultured with M-CSF, IL-4 and IL-13 were successfully polarized to M2 macrophages.M1 macrophages highly expressed phenotypes of CD80 and CD86, the inflammatory cytokine of TNF-α and the gene of iNOS. M2 macrophages highly expressed phenotypes of CD163 and CD206, the anti-inflammatory cytokine of IL-10 and the gene of Argl.(2) Peripheral monocytes-derived M1 and M2 macrophages polarization were impaired in chronic HCV infected patients compared with healthy individuals. HCV viral eradication or reduction with direct-acting antivirals (DAAs) drug partially repaired the macrophages polarization, which verified the inhibitory role of HCV on macrophages polarization.(3) In vitro experiment, HCV core protein inhibited monocytes differentiation to both M1 and M2 macrophages, and inhibited phagocytosis of M1 and M2 polarized macrophages. HCV core protein hampered M1 polarized macrophages-induced autologous and allogeneic CD4+ T cells proliferation, but promoted M2 polarized macrophages-induced autologous and allogeneic CD4+T cells proliferation.(4) Pam3CSK4 acted as HCV core protein, which inhibited the polarization of Ml and M2 macrophages in phenotypes, cytokines production and genes expression. Anti-TLR2 antibody could rescue the inhibitory effect of HCV core protein. Moreover, STAT1 and STAT3 phosphorylation were suppressed in HCV core treated M1 and M2 macrophages, respectively. The inhibitors of STAT1 (fludarabine) and STAT3 (stattic) inhibited M1 and M2 macrophages polarization in genes expression level, but not phenotypes and cytokines production.Conclusions:(1) Purified monocytes cultured with GM-CSF, IFN-y and LPS are successfully polarized to M1 macrophages.Other more, monocytes cultured with M-CSF, IL-4 and IL-13 are successfully polarized to M1 macrophages.(2) HCV inhibits monocytes polarizing to both of M1 and M2 macrophages.(3) HCV core protein inhibits monocytes differentiation to macrophages. Moreover, HCV core protein inhibits phagocytosis of M1 and M2 macrophages, and lead to disfunctions of antigen presentation and immuno-modulation of macrophages.(4) In chronic HCV infection, HCV core protein inhibits monocytes-derived macrophages polarization via TLR2/STATs signaling. The hampered polarization of macrophages with disfunction in immune response may contribute to the mechanism of HCV persistent infection, and it may be the reason why virus is difficult to eradicate. The innovation of the work is that through the study of the inhibition of HCV in macrophages differentiation, we put forward that blockade of interaction of HCV core protein and TLR2 may be a novel therapeutic approach to this viral infection.