Ewa Terczyńska-Dyla

Interferon lambda 4 signals via the IFNλ receptor to regulate antiviral activity against HCV and coronaviruses

The IFNL4 gene is a recently discovered type III interferon, which in a significant fraction of the human population harbours a frameshift mutation abolishing the IFNλ4 ORF. The expression of IFNλ4 is correlated with both poor spontaneous clearance of hepatitis C virus (HCV) and poor response to treatment with type I interferon. Here, we show that the IFNL4 gene encodes an active type III interferon, named IFNλ4, which signals through the IFNλR1 and IL‐10R2 receptor chains. Recombinant IFNλ4 is antiviral against both HCV and coronaviruses at levels comparable to IFNλ3. However, the secretion of IFNλ4 is impaired compared to that of IFNλ3, and this impairment is not due to a weak signal peptide, which was previously believed. We found that IFNλ4 gets N‐linked glycosylated and that this glycosylation is required for secretion. Nevertheless, this glycosylation is not required for activity. Together, these findings result in the paradox that IFNλ4 is strongly antiviral but a disadvantage during HCV infection.

Guarding the frontiers: the biology of type III interferons.

Type III interferons (IFNs) or IFN-λs regulate a similar set of genes as type I IFNs, but whereas type I IFNs act globally, IFN-λs primarily target mucosal epithelial cells and protect them against the frequent viral attacks that are typical for barrier tissues. IFN-λs thereby help to maintain healthy mucosal surfaces through immune protection, without the significant immune-related pathogenic risk associated with type I IFN responses. Type III IFNs also target the human liver, with dual effects: they induce an antiviral state in hepatocytes, but specific IFN-λ4 action impairs the clearance of hepatitis C virus and could influence inflammatory responses. This constitutes a paradox that has yet to be resolved.

Reduced IFNλ4 activity is associated with improved HCV clearance and reduced expression of interferon-stimulated genes

Hepatitis C virus (HCV) infections are the major cause of chronic liver disease, cirrhosis and hepatocellular carcinoma worldwide. Both spontaneous and treatment-induced clearance of HCV depend on genetic variation within the interferon-lambda locus, but until now no clear causal relationship has been established. Here we demonstrate that an amino-acid substitution in the IFNλ4 protein changing a proline at position 70 to a serine (P70S) substantially alters its antiviral activity. Patients harbouring the impaired IFNλ4-S70 variant display lower interferon-stimulated gene (ISG) expression levels, better treatment response rates and better spontaneous clearance rates, compared with patients coding for the fully active IFNλ4-P70 variant. Altogether, these data provide evidence supporting a role for the active IFNλ4 protein as the driver of high hepatic ISG expression as well as the cause of poor HCV clearance.

Transcriptome analysis reveals a classical interferon signature induced by IFNλ4 in human primary cells

The IFNL4 gene is negatively associated with spontaneous and treatment-induced clearance of hepatitis C virus infection. The activity of IFNλ4 has an important causal role in the pathogenesis, but the molecular details are not fully understood. One possible reason for the detrimental effect of IFNλ4 could be a tissue-specific regulation of an unknown subset of genes. To address both tissue and subtype specificity in the interferon response, we treated primary human hepatocytes and airway epithelial cells with IFNα, IFNλ3 or IFNλ4 and assessed interferon mediated gene regulation using transcriptome sequencing. Our data show a surprisingly similar response to all three subtypes of interferon. We also addressed the tissue specificity of the response, and identified a subset of tissue-specific genes. However, the interferon response is robust in both tissues with the majority of the identified genes being regulated in hepatocytes as well as airway epithelial cells. Thus we provide an in-depth analysis of the liver interferon response seen over an array of interferon subtypes and compare it to the response in the lung epithelium.

Frequently used bioinformatics tools overestimate the damaging effect of allelic variants

We selected two sets of naturally occurring human missense allelic variants within innate immune genes. The first set represented eleven non-synonymous variants in six different genes involved in interferon (IFN) induction, present in a cohort of patients suffering from herpes simplex encephalitis (HSE) and the second set represented sixteen allelic variants of the IFNLR1 gene. We recreated the variants in vitro and tested their effect on protein function in a HEK293T cell based assay. We then used an array of 14 available bioinformatics tools to predict the effect of these variants upon protein function. To our surprise two of the most commonly used tools, CADD and SIFT, produced a high rate of false positives, whereas SNPs&GO exhibited the lowest rate of false positives in our test. As the problem in our test in general was false positive variants, inclusion of mutation significance cutoff (MSC) did not improve accuracy.

The role of IFN in the development of NAFLD and NASH

Non-alcoholic fatty liver disease (NAFLD) and its progressive inflammatory form non-alcoholic steatohepatitis (NASH) are major health challenges due to a significant increase in their incidence and prevalence. While NAFLD is largely benign, the chronic liver inflammation in NASH patients may cause progression to liver cirrhosis and hepatocellular carcinoma. There is an urgent need for a better understanding of the factors, which drive the progression from NAFLD to NASH and how to use this information both to improve diagnostic and to develop new treatment strategies. Increasing evidence points to interferons (IFNs) as key players in NAFLD and particular in the progression to NASH. IFNs crucial role in disease development is supported by both genetic evidence and animal studies. In this review, we describe the involvement of both type I and type III IFNs in the development and progression of NAFLD and NASH.

ID: 101: Investigating the role of interferon λ4 in hepatitis C virus infection

IFNL4, a recently discovered member of the interferon λ family (IFNλs or type III IFNs), is a pseudogene in a significant fraction of the human population. A single nucleotide polymorphism located in IFNL4, which determines the ability to express IFNλ4, has been correlated with poor spontaneous and treatment-induced clearance of hepatitis C virus (HCV) infections. We show that IFNλ4 is an active type III IFN that induces a typical subset of ISGs, signals through the classical type III IFN receptor complex and is antiviral against HCV and coronaviruses. However, its secretion is impaired and this impairment is caused by a yet unknown molecular determinant, but appears to be partially caused by a weak signal peptide and inefficient N-linked glycosylation. This glycosylation is not required for antiviral activity and secretion of IFNλ4, but seems to improve its processing. The impaired secretion of IFNλ4 appears to be a recently acquired feature of primates. A single amino acid substitution in IFNλ4 changing a proline at position 70 to a serine (P70S) alters its activity. We demonstrate that the IFNλ4-S70 variant has a significantly lower antiviral activity compared to IFNλ4-P70. Our subsequent genetic study on a cohort of patients infected with HCV shows that individuals, who encode IFNλ4-S70, display lower hepatic ISG expression, better treatment response rates and better spontaneous clearance rates than patients encoding IFNλ4-P70. This study provides important evidence supporting a role for active IFNλ4 as the driver of high hepatic ISG expression as well as the cause of poor HCV clearance.