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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.


NK cell functions were restored after interaction with BILN-2061-treated HCV-infected Huh-7.5 cells.(A) NK cell degranulation was measured as described above. NK cells were seeded in a 96-well round bottom culture plate and treated with 400 nM of BILN-2061 for 18 h and then co-cultivation with K-562 cells at a 1:1 ratio for 4 h. (B) NK cell degranulation after co-cultivation with BILN-2061-treated HCV-infected Huh-7.5 cells. NK cells were pre-incubated with uninfected, HCV-infected, or BILN-2061-treated HCV-infected Huh-7.5 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 BILN-2061-treated HCV-infected Huh-7.5 cells. NK cells were pre-incubated with uninfected, HCV-infected, or BILN-2061-treated HCV-infected Huh-7.5 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 IL-12 and 100 ng/mL IL-15 for 6 h. IFN-γ production was assessed by intracellular staining of IFN-γ. (D) IFN-γ secretion by NK cells after co-cultivation with BILN-2061-treated HCV-infected Huh-7.5 cells. NK cells were pre-incubated with uninfected, HCV-infected, or BILN-2061-treated HCV-infected Huh-7.5 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 IL-12 and 100 ng/mL IL-15 for 18 h. Secreted IFN-γ in the supernatant was measured by ELISA. (A-C) Representative pseudo color plots obtained for three, seven and five independent individuals. (D) Data from seven independent individuals. Bar presents the median value.
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pone.0175793.g003: NK cell functions were restored after interaction with BILN-2061-treated HCV-infected Huh-7.5 cells.(A) NK cell degranulation was measured as described above. NK cells were seeded in a 96-well round bottom culture plate and treated with 400 nM of BILN-2061 for 18 h and then co-cultivation with K-562 cells at a 1:1 ratio for 4 h. (B) NK cell degranulation after co-cultivation with BILN-2061-treated HCV-infected Huh-7.5 cells. NK cells were pre-incubated with uninfected, HCV-infected, or BILN-2061-treated HCV-infected Huh-7.5 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 BILN-2061-treated HCV-infected Huh-7.5 cells. NK cells were pre-incubated with uninfected, HCV-infected, or BILN-2061-treated HCV-infected Huh-7.5 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 IL-12 and 100 ng/mL IL-15 for 6 h. IFN-γ production was assessed by intracellular staining of IFN-γ. (D) IFN-γ secretion by NK cells after co-cultivation with BILN-2061-treated HCV-infected Huh-7.5 cells. NK cells were pre-incubated with uninfected, HCV-infected, or BILN-2061-treated HCV-infected Huh-7.5 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 IL-12 and 100 ng/mL IL-15 for 18 h. Secreted IFN-γ in the supernatant was measured by ELISA. (A-C) Representative pseudo color plots obtained for three, seven and five independent individuals. (D) Data from seven independent individuals. Bar presents the median value.

Mentions: To verify the role of NS3 in the reduced NK cell degranulation and IFN-γ production, HCV-infected Huh-7.5 cells were treated with an NS3 inhibitor, BILN-2061. BILN-2061 (up to 700 nM) did not affect the cell viability of Huh-7.5 and Huh-7 cells (S2A Fig). BILN-2061-treatment decreased the expression levels of HCV Core protein and NS3 protein in a dose-dependent manner (S2B–S2D Fig). We determined the effects of BILN-2061 treatment on NK cells. Treatment with 400 nM of BILN-2061 did not affect NK cell degranulation directly (Fig 3A). After 4 h of HCV infection, Huh-7.5 cells were transferred to fresh medium containing 400 nM BILN-2061. Treatment of HCV-infected Huh-7.5 cells with BILN-2061 restored NK cells degranulation (from 4.86% in the untreated group to 7.10% in the treated group; Fig 3B). This restoration of function by BILN-2061-treatment was also observed in the case of IFN-γ production (Fig 3C) and secretion (Fig 3D). These observations corroborate the above data (Fig 2), connoting that NS3 plays a role in the modulation of NK cell functions.


Hepatitis C virus impairs natural killer cell activity via viral serine protease NS3
NK cell functions were restored after interaction with BILN-2061-treated HCV-infected Huh-7.5 cells.(A) NK cell degranulation was measured as described above. NK cells were seeded in a 96-well round bottom culture plate and treated with 400 nM of BILN-2061 for 18 h and then co-cultivation with K-562 cells at a 1:1 ratio for 4 h. (B) NK cell degranulation after co-cultivation with BILN-2061-treated HCV-infected Huh-7.5 cells. NK cells were pre-incubated with uninfected, HCV-infected, or BILN-2061-treated HCV-infected Huh-7.5 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 BILN-2061-treated HCV-infected Huh-7.5 cells. NK cells were pre-incubated with uninfected, HCV-infected, or BILN-2061-treated HCV-infected Huh-7.5 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 IL-12 and 100 ng/mL IL-15 for 6 h. IFN-γ production was assessed by intracellular staining of IFN-γ. (D) IFN-γ secretion by NK cells after co-cultivation with BILN-2061-treated HCV-infected Huh-7.5 cells. NK cells were pre-incubated with uninfected, HCV-infected, or BILN-2061-treated HCV-infected Huh-7.5 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 IL-12 and 100 ng/mL IL-15 for 18 h. Secreted IFN-γ in the supernatant was measured by ELISA. (A-C) Representative pseudo color plots obtained for three, seven and five independent individuals. (D) Data from seven independent individuals. Bar presents the median value.
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pone.0175793.g003: NK cell functions were restored after interaction with BILN-2061-treated HCV-infected Huh-7.5 cells.(A) NK cell degranulation was measured as described above. NK cells were seeded in a 96-well round bottom culture plate and treated with 400 nM of BILN-2061 for 18 h and then co-cultivation with K-562 cells at a 1:1 ratio for 4 h. (B) NK cell degranulation after co-cultivation with BILN-2061-treated HCV-infected Huh-7.5 cells. NK cells were pre-incubated with uninfected, HCV-infected, or BILN-2061-treated HCV-infected Huh-7.5 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 BILN-2061-treated HCV-infected Huh-7.5 cells. NK cells were pre-incubated with uninfected, HCV-infected, or BILN-2061-treated HCV-infected Huh-7.5 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 IL-12 and 100 ng/mL IL-15 for 6 h. IFN-γ production was assessed by intracellular staining of IFN-γ. (D) IFN-γ secretion by NK cells after co-cultivation with BILN-2061-treated HCV-infected Huh-7.5 cells. NK cells were pre-incubated with uninfected, HCV-infected, or BILN-2061-treated HCV-infected Huh-7.5 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 IL-12 and 100 ng/mL IL-15 for 18 h. Secreted IFN-γ in the supernatant was measured by ELISA. (A-C) Representative pseudo color plots obtained for three, seven and five independent individuals. (D) Data from seven independent individuals. Bar presents the median value.
Mentions: To verify the role of NS3 in the reduced NK cell degranulation and IFN-γ production, HCV-infected Huh-7.5 cells were treated with an NS3 inhibitor, BILN-2061. BILN-2061 (up to 700 nM) did not affect the cell viability of Huh-7.5 and Huh-7 cells (S2A Fig). BILN-2061-treatment decreased the expression levels of HCV Core protein and NS3 protein in a dose-dependent manner (S2B–S2D Fig). We determined the effects of BILN-2061 treatment on NK cells. Treatment with 400 nM of BILN-2061 did not affect NK cell degranulation directly (Fig 3A). After 4 h of HCV infection, Huh-7.5 cells were transferred to fresh medium containing 400 nM BILN-2061. Treatment of HCV-infected Huh-7.5 cells with BILN-2061 restored NK cells degranulation (from 4.86% in the untreated group to 7.10% in the treated group; Fig 3B). This restoration of function by BILN-2061-treatment was also observed in the case of IFN-γ production (Fig 3C) and secretion (Fig 3D). These observations corroborate the above data (Fig 2), connoting that NS3 plays a role in the modulation of NK cell functions.

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.