Type III interferons are expressed by Coxsackievirus-infected human primary hepatocytes and regulate hepatocyte permissiveness to infection

Abstract

Hepatitis is a common and potentially fatal manifestation of severe Coxsackievirus infections, particularly in newborn children. Little is known of the immune-mediated mechanisms regulating permissiveness to liver infection. It is well established that type I interferons (IFNs) play an important role in the host innate immune response to Coxsackievirus infections. Recent studies have highlighted a role for another IFN family, the type III IFNs (also called IFN-λ), in anti-viral defence. Whether type III IFNs are produced by hepatocytes during a Coxsackievirus infection remains unknown. Moreover, whether or not type III IFNs protects hepatocytes from a Coxsackievirus infection has not been addressed. In this study, we show that primary human hepatocytes respond to a Coxsackievirus B3 (CVB3) infection by up-regulating the expression of type III IFNs. We also demonstrate that type III IFNs induce an anti-viral state in hepatocytes characterized by the up-regulated expression of IFN-stimulated genes, including IFN-stimulated gene (ISG15), 2'-5'-oligoadenylate synthetase 2 (OAS2), protein kinase regulated by dsRNA (PKR) and myxovirus resistance protein 1 (Mx1). Furthermore, our study reveals that type III IFNs attenuate CVB3 replication both in hepatocyte cell lines and primary human hepatocytes. Our studies suggest that human hepatocytes express type III IFNs in response to a Coxsackievirus infection and highlight a novel role for type III IFNs in regulating hepatocyte permissiveness to this clinically relevant type of virus.

Keywords: Coxsackievirus; anti-viral defence; enterovirus; hepatocytes; interferons.

Figure 1

Coxsackievirus-infected primary human hepatocytes express type III interferons (IFNs). Primary hepatocytes from four donors were infected with mock (RPMI-1640) or CVB3 Nancy at a multiplicity of infection (MOI) of 100 for 3, 6 or 24 h. (a) At the indicated time-points, supernatant was collected and the amount of infectious virus particles that had accumulated in the tissue culture medium was determined by a standard plaque assay. Data are presented as log₁₀ [plaque-forming units (PFU)/ml]. (b) At the indicated time-points, the mRNA expression of IFN-λ1 and IFN-λ2 was measured using real-time reverse transcription–polymerase chain reaction (RT–PCR). The expression level of each gene was first normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (ΔCt) and then the expression in CVB3-infected cells was compared to that of mock treated cells from the same donor (ΔΔCt). Data are presented as fold induction: 2^∧−ΔΔCt, mean ± standard deviation; *P < 0·05 versus mock treated cells, paired t-test.

Figure 2

Type III interferons (IFNs) protect cell lines of hepatic origin from a Coxsackievirus infection. (a) HepG2 cells were treated with mock [phosphate-buffered saline (PBS) containing 0·1% bovine serum albumin (BSA)], IFN-λ1 (10, 50 or 500 ng/ml) or IFN-λ2 (10, 50 or 500 ng/ml) for 24 h. After 24 h the mRNA expression of IFN-stimulated gene (ISG15) was measured using real-time reverse transcription–polymerase chain reaction (RT–PCR). The mRNA expression of the gene was first normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (ΔCt) and then the expression in IFN-λ-treated cells was compared to that of mock treated cells from the same donor (ΔΔCt). Data are presented as fold induction: 2^∧−ΔΔCt, mean ± standard deviation (s.d.) for two–three independent experiments. (b) HepG2 (n = 4), Huh7·5 (n = 3) and HeLa (n = 2) cells were treated with mock (PBS containing 0·1% BSA), IFN-λ1 (100 ng/ml) or IFN-λ2 (100 ng/ml) for 24 h prior to infection with Coxsackievirus group B3 (CVB3) at a multiplicity of infection (MOI) of 30 (HepG2), 10 (Huh7·5) and 20 (HeLa). At 6 h post-infection (p.i.) (HepG2 and Huh7·5) or 4 h p.i. (HeLa), intracellular viral protein-1 (VP-1) expression was measured using flow cytometry. Results are presented as the percentage of cells that were positively stained by the VP-1 antibody. Data from HepG2 and Huh7·5 cells is presented as mean ± s.d.; **P < 0·01; ***P < 0·001, one-way analysis of variance (anova) with Bonferroni correction. Data from HeLa cells is shown as mean from two independent experiments. (c) HepG2 cells were treated with mock (PBS containing 0·1% BSA) or increasing concentrations of IFN-λ1 (0·1–100 ng/ml) or IFN-λ2 (0·1–100 ng/ml) for 24 h prior to infection with CVB3 at an MOI of 10. At 48 h p.i., cells were fixed and stained with crystal violet followed by optical density (OD) measurements at 595 nm to estimate the degree of lysed cells [cytopathic effect (CPE)]. Data shown are the mean ± s.d. of four independent experiments, in which each condition was performed at least in triplicate.

Figure 3

Primary human hepatocytes enter an anti-viral state following exposure to type III interferons (IFNs). (a) Primary human hepatocytes were treated with mock [phosphate-buffered saline (PBS) containing 0·1% bovine serum albumin (BSA)], IFN-λ1 (100 ng/ml) or IFN-λ2 (100 ng/ml) for 6 or 24 h. At the indicated time-points, the mRNA expression of IFN-stimulated gene (ISG15), 2′-5′-oligoadenylate synthetase 2 (OAS2), protein kinase regulated by dsRNA (PKR) and myxovirus resistance protein 1 (Mx1) was measured using real-time reverse transcription–polymerase chain reaction (RT–PCR). The expression level of each gene was first normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (ΔCt) and then the expression in IFN-λ-treated cells was compared to that of mock treated cells from the same donor (ΔΔCt). Data are shown as fold induction: 2^∧−ΔΔCt. One representative donor out of two is shown. (b) Human hepatocytes from five donors were treated with mock (PBS containing 0·1% BSA), IFN-λ1 (100 ng/ml) or IFN-λ2 (100 ng/ml) for 24 h prior to infection with Coxsackievirus group B3 (CVB3) at a multiplicity of infection (MOI) of 100. At 24 h post-infection (p.i.), the supernatant was collected and the amount of virus that had accumulated in the tissue culture medium was determined by a standard plaque assay. Data are presented as log₁₀ [plaque-forming units (PFU)/ml]. *P < 0·05; one-way analysis of variance (anova) (Kruskal–Wallis test) with Dunn's correction. All data are presented as mean ± standard deviation.