Does the IFN-λ rather than the IFN-α pathway determine the outcome of

hepatitis C virus infection?

Sabine Mihm1, Ulrich Spengler

2, Ahmad Amanzada

1, Giuliano Ramadori

1

1Department of Gastroenterology and Endocrinology, University Medical Center

Goettingen; 2Department of General Internal Medicine, University Hospital Bonn

E-mail addresses:

Sabine Mihm smihm@med.uni-goettingen.de

Ulrich Spengler Ulrich.Spengler@ukb.uni-bonn.de

Ahmad Amanzada ahmad.amanzada@med.uni-goettingen.de

Giuliano Ramadori gramado@med.uni-goettingen.de

Keywords

Hepatitis C virus (HCV), IFN-λ receptor subunit 1 (IFN-λR1), interferon-λ4 (IFNL4), rs12979860,

ss469415590

This article has been accepted for publication and undergone full peer review but has not beethrough the copyediting, typesetting, pagination and proofreading process which may lead todifferences between this version and the Version of Record. Please cite this article asdoi: 10.1002/hep.27345

2

Correspondence to:

Sabine Mihm, Prof. Dr. rer. nat.

University Medical Center Goettingen

Department of Gastroenterology and Endocrinology

Robert-Koch-Straße 40

37075 Goettingen, Germany

phone: 49-551-398946

fax: 49-551-397855

E-mail: smihm@med.uni-goettingen.de

Page 2 of 8

Hepatology

Hepatology

3

Duong FH, Trincucci G, Boldanova T, Calabrese D, Campana B, Krol I, Durand SC, et al. IFN-λ

receptor 1 expression is induced in chronic hepatitis C and correlates with the IFN-λ3

genotype and with nonresponsiveness to IFN-α therapies. J Exp Med 2014;211:857-868.

Abstract (reprint)

The molecular mechanisms that link IFN-λ3 genotypes to differential induction of

interferon (IFN)-stimulated genes (ISGs) in the liver of patients with chronic hepatitis C

(CHC) are not known. We measured the expression of IFN-λ and of the specific IFN-λ

receptor chain (IFN-λR1) in 122 liver biopsies of patients with CHC and 53 control samples.

The IFN-λ3 genotype was not associated with differential expression of IFN-λ, but rather

IFN-λR1. In a series of 30 primary human hepatocyte (PHH) samples, IFN-λR1 expression

was low but could be induced with IFN-α. IFN-α-induced IFN-λR1 expression was

significantly stronger in PHHs carrying the minor IFN-λ3 allele. The analysis of liver biopsies

of patients with CHC revealed a strong association of high IFN-λR1 expression with

elevated ISG expression, with IFN-λ3 minor alleles, and with nonresponse to pegylated

IFN-α and ribavirin. The findings provide a missing link between the IFN-λ3 genotype and

the associated phenotype of treatment nonresponse.

Page 3 of 8

Hepatology

Hepatology

4

Comment

In 2009, genome-wide association studies revealed an impressive correlation between genetic

variations within the region of interferon-λ (IFN-λ) genes, e.g. IFN-λ rs12979860, and clearance of a

hepatitis C virus (HCV) infection (1). This was true for both spontaneous remission and virus

eradication in response to an IFN-α -based antiviral treatment. As IFN-λ subtypes 1-3 were not

found to be differentially expressed among liver samples from patients with favorable and

unfavorable genotypes (2-4), the functional relevance of these loci remained elusive; the link

between IFN-λ genotypes and viral clearance was missing.

Likewise puzzling, the transcriptional baseline activity of the IFN effectors - the IFN stimulated

genes (ISGs) - appeared to be inversely linked to the outcome of infection, meaning that patients

with high pretreatment ISG expression were poor responders to IFN-based therapy. The minor

allele T of IFN-λ rs12979860 thus associates with an unfavorable outcome of infection and with

increased hepatic ISG expression.

In a recent study published in the Journal of Experimental Medicine, Duong et al. addressed - in

addition to hepatic expression of IFN-λ1 and of IFN-λ2/3 - hepatic expression of the IFN-λ receptor

subunit 1 (IFN-λR1). In liver biopsy specimens from chronic hepatitis C patients carrying the IFN-λ

rs12979860 unfavorable allele T the authors found a moderately yet significantly higher expression

of IFN-λR1 when compared to those from patients homozygous for the favorable allele C (5).

Functional in vitro analyses on IFN-α stimulated primary human hepatocytes (PHHs) supplemented

these findings, since they revealed a difference in the capacity to induce IFN-λR1 up-regulation

depending on the IFN-λ genotypes; samples from carriers of the T allele presented higher IFN-λR1

expression than samples from C homozygous donors. Beyond that, the authors applied an ex vivo

stimulation protocol on patients’ liver biopsy pieces in order to address pharmacological

responsiveness to IFN-α and to IFN-λ2. They examined the activation of the Jak-STAT pathway as a

Page 4 of 8

Hepatology

Hepatology

5

read out parameter, i.e. the number of nuclei with phospho-STAT1 expression 15 min after drug

administration. Then, they compared phospho-STAT1 signals to the individual expression of IFN-

λR1 mRNA. Duong et al. could demonstrate better responsiveness to IFN-α in liver specimens

exhibiting low IFN-λR1 expression than in those with high IFN-λR1 expression levels. In contrast,

responsiveness to IFN-λ2 was positively related to IFN-λR1 expression. The authors proposed IFN-

λR1 expression to be a missing link between the IFN-λ rs12979860 genotype, ISG activation, and

treatment outcomes, providing new insights on the potential role of the IFN-λ pathway for

clearance of HCV infection.

Considering the decreased responsiveness to IFN-α in linkage with increased responsiveness to

IFN-λ2 in their liver specimens with high baseline IFN-λR1 expression, the authors proposed that

peg-IFN-λ1 might be a particularly promising treatment option in those patients with high hepatic

IFN-λR1 expression. They further proposed to quantify hepatic IFN-λR1 mRNA in liver biopsy

specimens as direct and immediate approach to assess pharmacological responsiveness to both

IFNs. However, measuring IFN-λR1 mRNA expression as a predictive marker of treatment success

still awaits confirmation in prospective studies.

Duong et al. also checked the amounts of type III IFN receptor ligands IFN-λ1 and IFN-λ2/3 in their

sample of 122 hepatitis C liver specimens. In line with several other groups, IFN-λ1 to -3 steady

state mRNA levels did not differ between HCV-infected patients with favorable and unfavorable

rs12979860 genotypes. However, in the discussion of their manuscript Duong et al. also addressed

the possibility that the recently discovered ss469415590 frame-shift variation upstream of the IFN-

λ3 gene - giving rise to a novel type III interferon termed IFN-λ4 - might likewise explain the

enigmatic discrepancy between up-regulated ISGs at baseline and impaired resolution of HCV

infection with IFN-based therapy (6). IFN-λ4 signals through IFN-λ receptors and induces an

antiviral state against HCV in vitro analogous to other type III interferons, albeit being

disadvantageous during HCV infection (7, 8). Importantly, Duong et al. drew attention to the

possibility that the exclusive production of IFN-λ4 in carriers of the minor ss469415590 ∆G allele

Page 5 of 8

Hepatology

Hepatology

6

might up-regulate intrahepatic IFN-λR1 expression in hepatitis C patients carrying one or two

minor alleles and thus could provide an attractive alternative explanation why high intrahepatic

ISG expression was associated with these patients. However, they abandoned this idea because

thus far they were unable to detect IFN-λ4 mRNA in their liver biopsies.

A recent study, however, had succeeded in quantitatively measuring IFN-λ4 mRNA in human liver

biopsy samples (9). IFN-λ4 mRNA was detected exclusively in samples from patients with chronic

hepatitis C, and ISG expression was several times higher in HCV-infected patients with the

ss469415590 ΔG allele coding for IFN-λ4 than in the carriers of the homozygous ss469415590 TT

allele disrupting the IFNL4 gene. Moreover, a close positive correlation between IFN-λ4 mRNA

expression and up-regulation of ISG genes, e.g. IFI-44 and MxA, was found in carriers of the IFN-λ4

ΔG allele; further supporting a role of IFN-λ4 in the activation of ISGs.

Since the IFN-λ rs12979860 allele T, which was shown by Duong and colleagues to be associated

with high IFN-λR1 expression, and the IFN-λ4 ss469415590 ΔG allele, which gives rise to IFN-λ4, are

in a close linkage disequilibrium, the rs12979860 T/ ss469415590 ΔG haplotype should lead to

robust IFN-λ4-mediated signaling and strong ISG induction.

Further studies are needed to unravel how far the polymorphic IFN-λ genes contribute to IFN-λR1

gene expression in detail. As proposed by the work of Duong et al., IFN-λR1 could be considered as

an ISG itself. This idea is supported by a binding site for the IFN-α signal transducer STAT1 within

the promoter region of the IFN-λR1 gene. Furthermore, Duong and colleagues demonstrated

induction of IFN-λR1 in PHHs at the transcription and protein level within hours after IFN-α

stimulation. Thus, it would be intriguing to know, if IFN-λ4 actually induces substantial up-

regulation of IFN-λR1. Thus far, there exists indirect circumstantial evidence from Prokunina-Olsson

et al. who showed that plasmid-driven expression of IFN-λ4 in PHHs activates STAT1 (6) and from

observations by Hamming et al. who reported that recombinant IFN-λ4 activated a panel of ISGs in

hepatoma cells (7).

Page 6 of 8

Hepatology

Hepatology

7

Taken together, Duong et al. have provided novel insights how IFN-λ genotypes might translate

into ISG activation and how interactions between the IFN-α or the IFN-λ pathway might take place.

It remains an open issue, nevertheless, whether type I or type III IFNs dominate in determining the

baseline expression of ISGs and the host’s capability to clear an HCV infection.

Page 7 of 8

Hepatology

Hepatology

8

References

1. Hayes CN, Imamura M, Aikata H, Chayama K. Genetics of IL28B and HCV--response to

infection and treatment. Nat Rev Gastroenterol Hepatol 2012;9:406-417.

2. Honda M, Sakai A, Yamashita T, Nakamoto Y, Mizukoshi E, Sakai Y, Yamashita T, et al.

Hepatic ISG expression is associated with genetic variation in interleukin 28B and the

outcome of IFN therapy for chronic hepatitis C. Gastroenterology 2010;139:499-509.

3. Urban TJ, Thompson AJ, Bradrick SS, Fellay J, Schuppan D, Cronin KD, Hong L, et al.

IL28B genotype is associated with differential expression of intrahepatic interferon-

stimulated genes in patients with chronic hepatitis C. Hepatology 2010;52:1888-1896.

4. Asahina Y, Tsuchiya K, Muraoka M, Tanaka K, Suzuki Y, Tamaki N, Hoshioka Y, et al.

Association of gene expression involving innate immunity and genetic variation in

interleukin 28B with antiviral response. Hepatology 2012;55:20-29.

5. Duong FH, Trincucci G, Boldanova T, Calabrese D, Campana B, Krol I, Durand SC, et al.

IFN-lambda receptor 1 expression is induced in chronic hepatitis C and correlates with

the IFN-lambda3 genotype and with nonresponsiveness to IFN-alpha therapies. J Exp

Med 2014;211:857-868.

6. Prokunina-Olsson L, Muchmore B, Tang W, Pfeiffer RM, Park H, Dickensheets H,

Hergott D, et al. A variant upstream of IFNL3 (IL28B) creating a new interferon gene

IFNL4 is associated with impaired clearance of hepatitis C virus. Nat Genet

2013;45:164-171.

7. Hamming OJ, Terczynska-Dyla E, Vieyres G, Dijkman R, Jorgensen SE, Akhtar H, Siupka

P, et al. Interferon lambda 4 signals via the IFNlambda receptor to regulate antiviral

activity against HCV and coronaviruses. EMBO J 2013;32:3055-3065.

8. Booth D, George J. Loss of function of the new interferon IFN-lambda4 may confer

protection from hepatitis C. Nat Genet 2013;45:119-120.

9. Amanzada A, Kopp W, Spengler U, Ramadori G, Mihm S. Interferon-lambda4 (IFNL4)

transcript expression in human liver tissue samples. PLoS One 2013;8:e84026.

Page 8 of 8

Hepatology

Hepatology