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Hepatitis C virus impairs natural killer cell activity via viral serine protease NS3

View Article: PubMed Central - PubMed

ABSTRACT

Hepatitis C virus (HCV) infection is characterized by a high frequency of chronic cases owing to the impairment of innate and adaptive immune responses. The modulation of natural killer (NK) cell functions by HCV leads to an impaired innate immune response. However, the underling mechanisms and roles of HCV proteins in this immune evasion are controversial, especially in the early phase of HCV infection. To investigate the role of HCV nonstructural proteins especially NS3 in the impairment of NK functions, NK cells were isolated from the PBMCs by negative selection. To assess the direct cytotoxicity and IFN-γ production capability of NK cells, co-cultured with uninfected, HCV-infected, HCV-NS3 DNA-transfected Huh-7.5, or HCV-NS replicon cells. To determine the effect of an NS3 serine protease inhibitor, HCV-infected Huh-7.5 cells were treated with BILN-2061. Then, NK cells were harvested and further co-cultured with K-562 target cells. NK cell functions were analyzed by flow cytometry and enzyme-linked immunosorbent assay. When co-cultured with HCV-infected Huh-7.5 cells, the natural cytotoxicity and IFN-γ production capability of NK cells were significantly reduced. NK cell functions were inhibited to similar levels upon co-culture with HCV-NS replicon cells, NS3-transfected cells, and HCV-infected Huh-7.5 cells. These reductions were restored by BILN-2061-treatment. Furthermore, BILN-2061-treatment significantly increased degranulation against K-562 target cells and IFN-γ productivity in NK cells. Consistent with these findings, the expression levels of activating NK cell receptors, such as NKp46 and NKp30, were also increased. In HCV-infected cells, the serine protease NS3 may play a role in the abrogation of NK cell functions in the early phase of infection through downregulation of NKp46 and NKp30 receptors on NK cells. Together, these results suggest that NS3 represents a novel drug target for the treatment of HCV infections.

No MeSH data available.


Related in: MedlinePlus

Hepatitis C Virus-Non-Structural (HCV-NS) protein-expressing cells reduce Natural Killer (NK) cell cytotoxicity and Interferon (IFN)-γ production.(A) Schematic diagrams of the HCV–NS replicon constructs. (B) Degranulation of NK cells after co-cultivation with HCV-infected Huh-7.5 cells or HCV–NS replicon cells. NK cells were pre-incubated with HCV-infected Huh-7.5 cells or HCV-NS replicon cells for 18 h, and harvested the NK cells then co-cultured with K-562 cells at a 1:1 ratio for 4 h. NK cell degranulation was measured by estimating CD107a expression. (C) IFN-γ production by NK cells after co-cultivation with HCV-infected Huh-7.5 cells or HCV–NS replicon cells. NK cells were pre-incubated with HCV-infected Huh-7.5 cells or HCV-NS replicon cells for 18 h, and harvested the NK cells then co-cultured with K-562 cells at a 1:1 ratio with treatment of 10 ng/mL recombinant human interleukin (IL)-12 and 100 ng/mL IL-15 for 6 h. IFN-γ production was assessed by intracellular staining of IFN-γ followed by flow cytometry. (B-C) Representative pseudo color plots obtained for five independent individuals. Bar presents the median value.
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pone.0175793.g001: Hepatitis C Virus-Non-Structural (HCV-NS) protein-expressing cells reduce Natural Killer (NK) cell cytotoxicity and Interferon (IFN)-γ production.(A) Schematic diagrams of the HCV–NS replicon constructs. (B) Degranulation of NK cells after co-cultivation with HCV-infected Huh-7.5 cells or HCV–NS replicon cells. NK cells were pre-incubated with HCV-infected Huh-7.5 cells or HCV-NS replicon cells for 18 h, and harvested the NK cells then co-cultured with K-562 cells at a 1:1 ratio for 4 h. NK cell degranulation was measured by estimating CD107a expression. (C) IFN-γ production by NK cells after co-cultivation with HCV-infected Huh-7.5 cells or HCV–NS replicon cells. NK cells were pre-incubated with HCV-infected Huh-7.5 cells or HCV-NS replicon cells for 18 h, and harvested the NK cells then co-cultured with K-562 cells at a 1:1 ratio with treatment of 10 ng/mL recombinant human interleukin (IL)-12 and 100 ng/mL IL-15 for 6 h. IFN-γ production was assessed by intracellular staining of IFN-γ followed by flow cytometry. (B-C) Representative pseudo color plots obtained for five independent individuals. Bar presents the median value.

Mentions: Next, to investigate whether the non-structural proteins of HCV can reduce NK cell functions, we co-cultured NK cells with HCV-NS replicon cells (Fig 1A). NK cytotoxicity and IFN-γ productivity were reduced by co-cultivation of NK cells with HCV-NS replicon cells, similar to the co-cultivation of NK cells with HCV-infected Huh-7.5 cells (Fig 1B and 1C). Together, these data demonstrate that HCV-infected cells regulate NK cell functions via cell-to-cell interaction and that HCV-NS proteins might be involved in this modulation.


Hepatitis C virus impairs natural killer cell activity via viral serine protease NS3
Hepatitis C Virus-Non-Structural (HCV-NS) protein-expressing cells reduce Natural Killer (NK) cell cytotoxicity and Interferon (IFN)-γ production.(A) Schematic diagrams of the HCV–NS replicon constructs. (B) Degranulation of NK cells after co-cultivation with HCV-infected Huh-7.5 cells or HCV–NS replicon cells. NK cells were pre-incubated with HCV-infected Huh-7.5 cells or HCV-NS replicon cells for 18 h, and harvested the NK cells then co-cultured with K-562 cells at a 1:1 ratio for 4 h. NK cell degranulation was measured by estimating CD107a expression. (C) IFN-γ production by NK cells after co-cultivation with HCV-infected Huh-7.5 cells or HCV–NS replicon cells. NK cells were pre-incubated with HCV-infected Huh-7.5 cells or HCV-NS replicon cells for 18 h, and harvested the NK cells then co-cultured with K-562 cells at a 1:1 ratio with treatment of 10 ng/mL recombinant human interleukin (IL)-12 and 100 ng/mL IL-15 for 6 h. IFN-γ production was assessed by intracellular staining of IFN-γ followed by flow cytometry. (B-C) Representative pseudo color plots obtained for five independent individuals. Bar presents the median value.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5391949&req=5

pone.0175793.g001: Hepatitis C Virus-Non-Structural (HCV-NS) protein-expressing cells reduce Natural Killer (NK) cell cytotoxicity and Interferon (IFN)-γ production.(A) Schematic diagrams of the HCV–NS replicon constructs. (B) Degranulation of NK cells after co-cultivation with HCV-infected Huh-7.5 cells or HCV–NS replicon cells. NK cells were pre-incubated with HCV-infected Huh-7.5 cells or HCV-NS replicon cells for 18 h, and harvested the NK cells then co-cultured with K-562 cells at a 1:1 ratio for 4 h. NK cell degranulation was measured by estimating CD107a expression. (C) IFN-γ production by NK cells after co-cultivation with HCV-infected Huh-7.5 cells or HCV–NS replicon cells. NK cells were pre-incubated with HCV-infected Huh-7.5 cells or HCV-NS replicon cells for 18 h, and harvested the NK cells then co-cultured with K-562 cells at a 1:1 ratio with treatment of 10 ng/mL recombinant human interleukin (IL)-12 and 100 ng/mL IL-15 for 6 h. IFN-γ production was assessed by intracellular staining of IFN-γ followed by flow cytometry. (B-C) Representative pseudo color plots obtained for five independent individuals. Bar presents the median value.
Mentions: Next, to investigate whether the non-structural proteins of HCV can reduce NK cell functions, we co-cultured NK cells with HCV-NS replicon cells (Fig 1A). NK cytotoxicity and IFN-γ productivity were reduced by co-cultivation of NK cells with HCV-NS replicon cells, similar to the co-cultivation of NK cells with HCV-infected Huh-7.5 cells (Fig 1B and 1C). Together, these data demonstrate that HCV-infected cells regulate NK cell functions via cell-to-cell interaction and that HCV-NS proteins might be involved in this modulation.

View Article: PubMed Central - PubMed

ABSTRACT

Hepatitis C virus (HCV) infection is characterized by a high frequency of chronic cases owing to the impairment of innate and adaptive immune responses. The modulation of natural killer (NK) cell functions by HCV leads to an impaired innate immune response. However, the underling mechanisms and roles of HCV proteins in this immune evasion are controversial, especially in the early phase of HCV infection. To investigate the role of HCV nonstructural proteins especially NS3 in the impairment of NK functions, NK cells were isolated from the PBMCs by negative selection. To assess the direct cytotoxicity and IFN-γ production capability of NK cells, co-cultured with uninfected, HCV-infected, HCV-NS3 DNA-transfected Huh-7.5, or HCV-NS replicon cells. To determine the effect of an NS3 serine protease inhibitor, HCV-infected Huh-7.5 cells were treated with BILN-2061. Then, NK cells were harvested and further co-cultured with K-562 target cells. NK cell functions were analyzed by flow cytometry and enzyme-linked immunosorbent assay. When co-cultured with HCV-infected Huh-7.5 cells, the natural cytotoxicity and IFN-γ production capability of NK cells were significantly reduced. NK cell functions were inhibited to similar levels upon co-culture with HCV-NS replicon cells, NS3-transfected cells, and HCV-infected Huh-7.5 cells. These reductions were restored by BILN-2061-treatment. Furthermore, BILN-2061-treatment significantly increased degranulation against K-562 target cells and IFN-γ productivity in NK cells. Consistent with these findings, the expression levels of activating NK cell receptors, such as NKp46 and NKp30, were also increased. In HCV-infected cells, the serine protease NS3 may play a role in the abrogation of NK cell functions in the early phase of infection through downregulation of NKp46 and NKp30 receptors on NK cells. Together, these results suggest that NS3 represents a novel drug target for the treatment of HCV infections.

No MeSH data available.


Related in: MedlinePlus