Hepatitis C virus NS4 protein impairs the Th1 polarization of immature dendritic cells

Abstract

Dendritic cells (DCs) in chronic hepatitis C patients display impaired function, although the details remain unclear. To investigate the hepatitis C virus (HCV) protein that has the most impact on DC function, we compared five recombinant proteins and seven HCV protein genes in modulating DC phenotype and function. Immature DCs (iDCs) were established from healthy donor peripheral blood monocytes with granulocyte-macrophage colony stimulating factor (GM-CSF) and IL-4. Lipopolysaccharide was used to establish mature DCs (mDCs). Cells were then pulsed with HCV recombinant proteins or transfected with HCV plasmids and subsequently assayed for cell surface marker expression by flow cytometry. For cytokine and proliferative T-cell response analysis, DCs were cultured with autologous CD4 T cells and tuberculin purified protein derivative (PPD). Mean fluorescent intensity of CD86 was reduced in HCV protein-pulsed iDCs. Proliferative T-cell responses and Th1 cytokine concentrations were reduced with HCV nonstructural proteins (NS), particularly with HCV NS4. HCV nonstructural proteins, particularly NS4, change the iDC phenotype and reduce antigen-specific T-cell stimulatory function with Th1 cytokine reductions.

Fig. 1

MFI of HLA and co-stimulatory molecules on dendritic cells (DCs) was reduced with hepatitis C virus (HCV) protein addition. HCV protein-pulsed DCs are shown as p-core, p-NS3, p-NS4, p-NS5A, p-NS5B. (a) MFI index of HLA class I and II and co-stimulatory molecules CD40 and CD86 expressed on HCV protein-pulsed DCs. CD86 was reduced by addition of HCV NS4 protein compared with HCV NS3 and NS5B. (b) MFI of surface markers of DCs pulsed with HCV recombinant proteins after maturation with LPS. HCV protein-pulsed DCs showed comparable ability to mature in terms of surface expression of co-stimulatory molecule. *P < 0.05 (Student’s t-test).

Fig. 2

MFI of HLA class I and II and co-stimulatory molecules CD40 and CD86 expressed on dendritic cells (DCs) transfected with hepatitis C virus (HCV) protein encoding plasmids. HCV protein encoding plasmids are shown as g-core, g-NS2, g-NS3, g-NS4A, g-NS4B, g-NS5A, g-NS5B. (a) HCV protein encoding plasmid-transduced DCs showed comparable ability to mature in relation to surface expression of co-stimulatory molecules. (b) MFI of surface markers on DCs matured with LPS following transfection with HCV protein encoding plasmids. These DCs showed comparable ability to mature in terms of surface expression of co-stimulatory molecules.

Fig. 4

Effect on CD4 T-cell proliferation with tuberculin purified protein derivative (PPD) following transfection with hepatitis C virus (HCV) protein encoding plasmids. (a) CD4 T-cell proliferation with PPD and iDCs was relatively reduced by exposure to NS4B compared with NS4A. (b) Dendritic cells (DCs) transduced with HCV plasmids and subsequently matured with LPS. Relative reduction in CD4 T-cell stimulatory function by NS4B was overcome by LPS maturation. *P < 0.05 (Student’s t-test).

Fig. 3

CD4 T-cell proliferation with tuberculin purified protein derivative (PPD) and dendritic cells (DCs) was not affected by recombinant hepatitis C virus (HCV) protein addition. (a) CD4 T-cell proliferation with PPD and DCs after addition of HCV core, NS3, NS4, NS5A and NS5B recombinant proteins. No difference was observed in PPD-specific T-cell proliferative responses with HCV recombinant protein addition. (b) DCs pulsed with HCV recombinant proteins and subsequent maturation with LPS. As in the case of iDCs, no difference was observed in PPD-specific T-cell proliferative responses with HCV recombinant protein addition.

Fig. 5

Recombinant NS4 protein addition reduced production of Th1 cytokines in antigen-specific CD4 T-cell responses. (a) Cytokine profile of supernatant from mixed culture of CD4 T cells, PPD and dendritic cells (DCs) after pulsing with hepatitis C virus (HCV) recombinant proteins. NS4 addition reduced production of Th1 cytokines IFNγ and IL-2, whilst Th2 cytokines IL4 and IL6 were unaffected. (b) Maturation of DCs with LPS rescued Th1 cytokine production after pulsing with HCV recombinant proteins. No effect was seen with HCV protein encoding plasmids. *P < 0.05 (Dunnett post-test).

Fig. 6

Cytokine profile in supernatants from mixed cultures of CD4 T cells, tuberculin purified protein derivative (PPD) and dendritic cells (DCs) transduced with hepatitis C virus (HCV) protein encoding plasmids. Maturation of DCs with LPS rescued the Th1 cytokine production. (a) Cytokine profile in supernatants from mixed cultures of CD4 T cells, PPD and iDCs transduced with HCV protein encoding plasmids. Transduction produced no effect. (b) Cytokine profile in supernatant from mixed cultures of CD4 T cells, PPD and DCs transduced with HCV protein encoding plasmids and subsequently matured with LPS (mDCs). As in the case of iDCs, no effect was seen with HCV plasmid-transfected DCs.