Induction of interferon-λ contributes to Toll-like receptor-3-activated hepatic stellate cell-mediated hepatitis C virus inhibition in hepatocytes

Abstract

There is limited information about the role of hepatic stellate cells (HSC) in liver innate immunity against hepatitis C virus (HCV). We thus examined whether HSC can produce antiviral factors that inhibit HCV replication in human hepatocytes. HSC expressed functional Toll-like receptor 3 (TLR-3), which could be activated by its ligand, polyinosine-polycytidylic acid (poly I:C), leading to the induction of interferon-λ (IFN-λ) at both mRNA and protein levels. TLR-3 signalling of HSC also induced the expression of IFN regulatory factor 7 (IRF-7), a key regulator of IFN signalling pathway. When HCV JFH-1-infected Huh7 cells were co-cultured with HSC activated with poly I:C or incubated in media conditioned with supernatant (SN) from poly I:C-activated HSC, HCV replication was significantly suppressed. This HSC SN action on HCV inhibition was mediated through IFN-λ, which was evidenced by the observation that antibody to IFN-λ receptors could neutralize the HSC-mediated anti-HCV effect. The role of IFN-λ in HSC-mediated anti-HCV activity is further supported by the observation that HSC SN treatment induced the expression of IRF-7 and IFN-stimulated genes (ISGs), OAS-1 and MxA in HCV-infected Huh7 cells. These observations indicate that HSC may be a key regulatory bystander, participating in liver innate immunity against HCV infection using an IFN-λ-dependent mechanism.

Fig. 1

Poly I:C-stimulated LX-2 cells or supernatant (SN) from cell culture suppresses HCV replication in Huh7 cells. (a) Effect of poly I:C on HCV JFH-1 replication in Huh7 cells. Huh7 cells were infected with HCV JFH-1 for 48 h, and then stimulated with poly I:C (1μg/mL) and total cellular RNA was extracted from cells for the HCV real-time RT-PCR. The data are expressed as HCV RNA levels relative (%) to control (without poly I:C stimulation). (b) Co-culture of HCV JFH-1-infected Huh7 cells with poly I:C-stimulated LX-2 cells. LX-2 cells were plated in the low compartment of a 24-well plate and stimulated with different doses of poly I:C (0.25, 1 and 4μg/mL) for 16 h, while HCV JFH-1-infected Huh7 cells (day 3 postinfection) were plated in the upper compartment for co-culture. After 48 h co-culture, total cellular RNA extracted from Huh7 cells was subjected to real-time RT-PCR for HCV and GAPDH RNA quantification. (c) Effect of SN of LX-2 cell culture stimulated with poly I:C (1μg/mL) on HCV replication in Huh7 cells. HCV JFH-1-infected Huh7 cells (day 3 postinfection) were cultured in the presence or absence of SN of LX-2 stimulated with poly I:C by transfection at the indicated concentration for 48 h. Total cellular RNA extracted from Huh7 cells was subjected to e real-time RT-PCR for HCV and GAPDH RNA quantification. (d) Suppression of HCV RNA expression by LX-2 SN under different conditions. Huh7 cells were cultured in media conditioned with or without LX-2 SN for either 24 h prior to HCV infection, or simultaneously or 8 h postinfection. The cells were then washed five times to remove input HCV after 6 h incubation with HCV JFH1 and then cultured in the presence or absence of LX-2 SN for 72 h. Intracellular RNA extracted from hepatocytes at 72 h postinfection was subjected to real-time RT-PCR for HCV and GAPDH RNA quantification. The data are expressed as HCV RNA levels relative (%) to control (without poly I:C stimulation or without LX-2 SN treatment, which is defined as 100%). The results are mean ± SD of triplicate cultures, representative of three experiments (* P<0.05, **P<0.01). (e) Effect of LX-2 SN on HCV core protein expression in Huh7 cells. HCV JFH-1-infected Huh7 cells (day 3 postinfection) were cultured in the presence or absence of poly I:C stimulated LX-2 cell culture SN at the indicated concentrations for 48 h. HCV core protein expression was determined by immunofluoresence staining with antibody against HCV core protein (green). The nuclei were stained with Hoechst 33342 (blue). One representative experiment is shown (original magnification 200 X, scale bar: 100μm).

Fig. 2

Effect of TLR-3 activation on IFN-γ expression in LX-2 cells. LX-2 cells were stimulated with different concentrations of poly I:C (0.25, 1 and 4μg/mL) for 48 h. Total RNA extracted from cells was subjected to real-time RT-PCR for the mRNA levels IFN-γ1 (a) and γ2/3 (b). SN was collected for ELISA to measure the protein levels of IFN-γ1 (c) and γ2/3 (d). (e and f) Effect of HCV on poly I:C-induced IFN-γ expression in LX-2 cells. LX-2 cells were plated in the low compartment of a 24-well plate and stimulated with poly I:C (1μg/mL), while HCV JFH-1-infected Huh7 cells (day 3 postinfection) were plated in the upper compartment for co-culture. After 48 h co-culture, total cellular RNA extracted from LX-2 cells was subjected to the real-time RT-PCR for IFN-γ RNA quantification. The data (a, b, e and f) are expressed as mRNA levels for relative (fold) to the control (without stimulation, which is defined as 1). The results are mean ± SD of three different experiments (*P < 0.05, **P < 0.01).

Fig. 3

Effect of poly I:C on TLR-3 and IRF expression. LX-2 cells were stimulated with poly I:C (1μg/mL) by transfection for 48 h. Total RNA extracted from cells was subjected to the real-time RT-PCR for the mRNA levels of TLR-3 (a), IRF-3 (b) and IRF-7 (c). The data are expressed as mRNA levels relative (fold) to the control (without poly I:C stimulation, which is defined as 1). The results are mean ± SD of three different experiments (**P < 0.01, poly I:C vs control).

Fig. 4

Blocking TLR-3 signaling pathway counteracts poly I:C effect. LX-2 cells were pretreated with or without Bafilomycin A1 (100nM) for 1 h prior to poly I:C transfection. Total RNA extracted from cells was subjected to real-time RT-PCR for mRNA levels of IRF-7 (a), IFN-γ1 (b) and γ2/3 (c). The data are expressed as mRNA levels stimulated relative (fold) to the control (without stimulation, which is defined as 1). The results are mean ± SD of three different experiments (**P < 0.01, poly I:C vs control, or poly I:C vs poly I:C with Bafilomycin A1).

Fig. 5

Effect of antibody to IL-10Rβ on LX-2 supernatant (SN)-mediated anti-HCV activity. HCV JFH-1-infected Huh7 cells (day 3 post infection) were pretreated with antibody to IL-10Rβ or control IgG at the concentration of 5μg/mL for 1 h followed with SN from poly I:C-stimulated (10%, v/v) LX-2 cells for 48 h. Recombinant human IFN-γ3 (10ng/mL) was used as a positive control. HCV JFH-1 replication was measured by real-time RT-PCR for HCV RNA 48 h post treatment. Value is mean ± SD of three different cultures (*P < 0.05).

Fig. 6

Effect of LX2 SN on the expression of ISG56, OAS-1, PKR and MxA in HCV JFH-1- infected Huh7 cells. HCV JFH-1-infected Huh7 cells (day 3 postinfection) were cultured in the presence or absence of SN from LX-2 cells stimulated with poly I:C (1 μg/mL) for 48 h at indicated concentrations for 48 h. Total cellular RNA extracted from Huh7 cells was subjected to real-time RT-PCR for ISG56, OAS-1, PKR and MxA mRNA quantification. The data are expressed as RNA levels relative (fold) to control (without SN treatment, which is defined as 1). The results are mean ± SD of three different experiments (*P < 0.05; **P < 0.01).

Fig. 7

Schematic diagram of the mechanisms involved in HSC-mediated suppression of HCV replication in hepatocytes. HCV disrupts TLR-3 signaling by cleaving the TRIF adaptor protein and suppresses IFN-β expression in hepatocytes. HSC possess functional TLR-3 signaling and can be activated, leading to production of IFN-γ, which binds to IFN-γ receptors expressed in HCV-infected hepatocytes, and induces ISGs expression that can suppress HCV replication.