HCV core protein inhibits polarization and activity of both M1 and M2 macrophages through the TLR2 signaling pathway - PubMed (original) (raw)

Qianqian Zhang 1 2 3, Naicui Zhai 1, Hongxiao Song 1, Haijun Li 1, Yang Yang 1, Tianyang Li 1, Xiaolin Guo 2, Baorong Chi 2, Junqi Niu 2, Ian Nicholas Crispe 1 4, Lishan Su 1 5, Zhengkun Tu 1 2

Affiliations

HCV core protein inhibits polarization and activity of both M1 and M2 macrophages through the TLR2 signaling pathway

Qianqian Zhang et al. Sci Rep. 2016.

Abstract

Hepatitis C virus (HCV) establishes persistent infection in most infected patients, and eventually causes chronic hepatitis, cirrhosis, and hepatocellular carcinoma in some patients. Monocytes and macrophages provide the first line of defense against pathogens, but their roles in HCV infection remains unclear. We have reported that HCV core protein (HCVc) manipulates human blood-derived dendritic cell development. In the present study, we tested whether HCVc affects human blood-derived monocyte differentiating into macrophages. Results showed that HCVc inhibits monocyte differentiation to either M1 or M2 macrophages through TLR2, associated with impaired STATs signaling pathway. Moreover, HCVc inhibits phagocytosis activity of M1 and M2 macrophages, M1 macrophage-induced autologous and allogeneic CD4+ T cell activation, but promotes M2 macrophage-induced autologous and allogeneic CD4+ T cell activation. In conclusion, HCVc inhibits monocyte-derived macrophage polarization via TLR2 signaling, leading to dysfunctions of both M1 and M2 macrophages in chronic HCV infected patients. This may contribute to the mechanism of HCV persistent infection, and suggest that blockade of HCVc might be a novel therapeutic approach to treating HCV infection.

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Figures

Figure 1

Figure 1. Human peripheral monocytes differentiation into M1 or M2 macrophages are impaired in chronic HCV infected patients.

Purified monocytes from 17 chronic HCV patients (CHC) and 17 healthy controls (HC) were polarized to M1 and M2 macrophages. (a–d) The expression level of CD80 and CD86 on M1 macrophages, CD163 and CD206 on M2 macrophages in chronic HCV infected patients are significantly lower than healthy controls. (e,f) TNF-α production of M1 macrophages and IL-10 production of M2 macrophages in chronic HCV infected patients are significantly lower than healthy controls. (g,h) The mRNA expression level of iNOS by M1 macrophages and Arg1 by M2 macrophages in chronic HCV infected patients are significantly lower than healthy controls. (i) The protein level of iNOS of M1 macrophages and Arg1 of M2 macrophages in chronic HCV infected patients are significantly lower than healthy controls. (j) The total number of macrophage is significantly reduced in CHC patients after polarization. (n = 17, *P < 0.05, **P < 0.01, ***P < 0.001).

Figure 2

Figure 2. Direct-acting antivirals (DAAs) partially rescues the differentiation of macrophages.

Eight HCV infected patients administrated with DAAs (NS5A inhibitor) were enrolled. Venous bloods were collected before and after treatment. Purified monocytes were polarized to M1 and M2 macrophages. (a,b) The expression level of CD86 on M1 macrophages, and CD163 on M2 macrophages after treatment are significantly higher than before treatment. (c,d) TNF-α production of M1 macrophages, and IL-10 production of M2 macrophages after treatment are significantly higher than before treatment. (e,f) The mRNA expression level of iNOS by M1 macrophages and Arg1 by M2 macrophages after treatment are significantly higher than before treatment. (n = 8, *P < 0.05, **P < 0.01, ***P < 0.001).

Figure 3

Figure 3. HCVc inhibits monocyte differentiation to both M1 and M2 macrophage.

Purified monocytes from health individuals were polarized to M1 and M2 macrophages in the presence or absence of HCV core protein (HCVc) and β-galactosidase (β-gal, negative control). (a–d) HCVc inhibits the expression level of CD80 and CD86 on M1 macrophages, CD163 and CD206 on M2 macrophages, compared with β-gal and medium. (e,f) HCVc inhibits TNF-α production of M1 macrophages, and IL-10 production of M2 macrophages. (g,h) HCVc inhibits the mRNA expression level of iNOS by M1 macrophages, and Arg1 by M2 macrophages. (n = 5, *P < 0.05, **P < 0.01, ***P < 0.001).

Figure 4

Figure 4. HCVc inhibits macrophage polarization through engagement with TLR2.

Purified monocytes from healthy individuals were polarized to M1 and M2 macrophages in the presence or absence of HCVc and Pam3CSK4, and purified monocytes pre-treated with anti-TLR2 antibody or transfected with siRNAs targeting TLR2 were polarized to M1 and M2 macrophages in the presence of HCVc. Pam3CSK4 as well as HCVc inhibits the expression of CD80 (a) and CD86 (b), TNF-α production (e), and mRNA expression level of iNOS (g) by M1 macrophages, the expression of CD163 (c) and CD206 (d), IL-10 production (f), and mRNA expression level of Arg-1 (h) byM2 macrophages. (a–h) The inhibitions of HCVc-induced macrophages polarization were restored by anti-TLR2 antibody and TLR2-siRNAs. (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 5

Figure 5. HCVc inhibits macrophage polarization correlated with inhibiton of STAT1/STAT3 activation.

(a) Purified monocytes from healthy individuals were polarized to M1 and M2 macrophages in the presence or absence of HCVc, pSTAT1, pSTAT1, total STAT1 and STAT3 were detected by western blot. Shown is one of three representative experiments. (b,c) Statistical analysis of three experiments of western blot. (d,e) The inhibitors of STAT1 (Fludarabine) and STAT3 (Stattic) inhibit the mRNA expression levels of iNOS and Arg1 in M1 and M2 macrophages, respectively. (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 6

Figure 6. HCVc inhibits the functions of M1 and M2 macrophages.

(a) Purified monocytes from healthy individuals were polarized to M1 and M2 macrophages in the presence or absence of HCVc or Pam3CSG4, FITC-microbeads were then added to culture for 1h. Phagocytosis was detected by flow cytometry. (b,c) Polarized macrophages in the presence or absence of HCVc or Pam3CSG4 were co-cultured with autologous activated CD4+ T cells by OKT3 and CD28. CD4+ T cells were stained with CFSE before co-cultured, CD4+ T cell proliferation were examined by flow cytometry, and IFN-γ secretion were assayed by ELISA. (d,e) Polarized macrophages in the presence or absence of HCVc or Pam3CSG4 were co-cultured with allogeneic CD4+ T cells. CD4+ T cells were stained with CFSE before co-cultured, CD4+ T cell proliferation were examined by flow cytometry, and IFN-γ secretion were assayed by ELISA. Shown is one of three representative experiments and statistical analysis. (*P < 0.05, **P < 0.01, ***P < 0.001).

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