Interferon-λ4 (IFNL4) transcript expression in human liver tissue samples

Abstract

Eradication of hepatitis C virus (HCV) infection, both spontaneous and treatment-induced, is marked by the wildtype allele C of a single nucleotide polymorphism upstream of the IL28B gene, rs12979860. This favorable allele was recently described to be in linkage disequilibrium with the wildtype allele TT of a dinucleotide polymorphism, ss469415590, located within a new protein-coding gene. While the TT allele introduces a frame-shift and disrupts the open reading frame, only the variant allele, ΔG, creates a novel type III interferon (IFN) protein, IFN-λ4/IFNL4. Absence of IFNL4 is thus supposed to favor resolution of HCV infection. As to date IFNL4 mRNA transcription has only been investigated in polyI:C-stimulated primary human hepatocytes and not yet in HCV infection in vivo, this study analyzed IFNL4 mRNA expression in human liver biopsy specimens. Samples were obtained from patients with a broad panel of disorders including no liver disease, liver diseases of non-viral etiology, chronic hepatitis B and chronic hepatitis C. Hepatic IFNL4 transcripts were detectable exclusively in a subgroup of chronic hepatitis C patients (24/45). Their amounts were positively related to liver HCV RNA copy numbers (p = 0.0023, r = 0.56) suggesting that the hepatic viral load influences IFNL4 transcription irrespective of IFNL4 governing genotype. Both, the IFNL4 creating allele ΔG (p<0.0001) and actual IFNL4 transcription (p = 0.0015) were found to be correlated to the activation of IFN stimulatory genes (ISGs). By contrast, IFNL4 ss469415590 genotypes were not found to be related to IFN-λ2/3/IL28 or IFN-λ1/IL29 gene expression. In conclusion, this study is the first report on intrahepatic transcript levels of the recently discovered IFNL4 gene. Data indicate that HCV infection in particular might activate IFNL4 transcription in the liver. It provides a possible explanation as to why hepatitis C patients show ISG stimulation in their livers in the apparent absence of an induction of other IFN subtypes.

Figure 1. Electropherograms of IFNL4 amplicons.

After completion of PCR amplification, 1 µl of samples was separated and analyzed in the Agilent 2100 bioanalyzer using the DNA 1000 LabChip kit. Electropherograms and gels are shown for two representative samples each of ss469415590 TT homozygotes without any amplification of IFNL4-specific transcripts (IFNL4 nd) (#1, #2) or of ΔG homozygotes (#3, #4) and of TT homozygotes (#5, #6) with detectable amounts of hepatic IFNL4-specific mRNA (IFNL4 d). Analysis confirmed the lack of products in samples #1 and #2, and the presence of products of the expected size (accuracy ±5 bp, as given by the supplier) both in ΔG and TT homozygotes.

Figure 2. IFNL4 transcript expression in human liver samples.

Liver biopsy specimens taken from patients with suspected but later excluded liver diseases (healthy controls) and from patients with non-viral liver diseases, chronic HBV infection or chronic HCV infection were analyzed for IFNL4 gene expression. IFNL4 gene expression was quantified by applying a 5′-nuclease assay using a specific primer-probe set as outlined in the Patients and Methods section. Data were normalised to GAPDH transcripts as a reference.

Figure 3. The number of hepatic IFNL4 transcripts relates to hepatic viral loads.

A linear regression analysis among hepatitis C patients with measurable amounts of IFNL4 mRNA revealed a correlation between hepatic viral loads, which was assessed by the amount of viral nucleic acids in relation to the amount of GAPDH transcripts, and hepatic IFNL4 transcripts, which were also normalised to the amount of GAPDH transcripts. Correlation coefficient and level of significance are given. Symbols indicate patient genotypes.

Figure 4. Hepatic activation of ISGs.

Liver samples from chronic hepatitis C patients were quantified for IFI44 (A, B) and MxA (D, E) transcripts with regard to IFNL4 ss469415590 genotypes and detectability of hepatic IFNL4 transcripts. Medians are indicated by horizontal bars. Carriers of the ΔG allele experienced significantly higher ISG expression than TT homozygotes (A, D), as do ΔG allele carriers (closed symbols) with detectable amounts of hepatic IFNL4 transcripts (IFNL4 d) when compared to those without (IFNL4 nd) (B, E). Open symbols indicate TT homozygotes, data of whom were not included in significance testing. Linear regression analyses revealed positive relationships between IFNL4 gene transcripts and IFI44 (C) and MxA (F) mRNA expression.