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Interferon-lambda serum levels in hepatitis C
Research Article
Interferon-lambda serum levels in hepatitis C Bettina Langhans1,⇑, Bernd Kupfer2, Ingrid Braunschweiger1, Simone Arndt1, Wibke Schulte1, Hans Dieter Nischalke1, Jacob Nattermann1, Johannes Oldenburg3, Tilman Sauerbruch1, Ulrich Spengler1 1 Department of Internal Medicine I, University of Bonn, Bonn, Germany; 2Institute of Virology, University of Bonn, Bonn, Germany; 3Institute for Experimental Hematology and Transfusion Medicine, University of Bonn, Bonn, GermanyReceived 11 January 2010; received in revised form 29 July 2010;
accepted 4 August 2010
See Editorial, pages 844–847
Background & Aims: Dendritic cells (DCs) trigger adaptive immune responses and are an important source of antiviral cytokines. In hepatitis C virus (HCV) infection DC function is markedly impaired. Thus far, studies have focused on types I and II interferon (IFN). We studied IFN-lambda1 (IL-29) and IFN-lambda2/3 (IL-28A/ B) serum levels in patients with different outcomes of HCV infection. Methods: IFN-lambdas were measured by ELISAs detecting IL-29 or IL-28A and IL-28B, respectively. Results were stratified with respect to the recently discovered rs12979860 T/C polymorphism upstream of the IL-28B gene. Results: In general IL-29 serum levels exceeded IL-28A/B at least twofold, with IL-29 and IL-28A/B levels being significantly higher in carriers of the rs12979860 C allele than in TT homozygous individuals (p <0.02). IL-29 levels were substantially lower in patients with chronic hepatitis C than in healthy controls (p = 0.005) and patients with spontaneously resolved hepatitis (p = 0.001). Patients with acute hepatitis C showed IL-29 levels intermediate between chronic hepatitis C and normal controls; and IL-29 serum levels were higher in patients who spontaneously resolved hepatitis C than in those who became chronic. In vitro HCV proteins NS3 and E2 directly inhibited IL-29 production in poly I:C-stimulated purified DCs. Conclusions: Our data suggest that HCV proteins modify IFNlambda production in DCs. Carriers of the rs12979860 C allele associated with resolution of HCV infection exhibited increased IFN-lambda levels. Moreover, high IFN-lambda levels predisposed to spontaneous resolution of HCV infection. Thus, IFN-lambdas seem to play an important role in the control of hepatitis C. Ó 2010 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.
Keywords: Hepatitis C virus (HCV); IFN-lambdas (IL-29, IL-28A/B); IL-28B rs12979860 T/C polymorphism; Dendritic cells (DCs). Received 11 January 2010; received in revised form 29 July 2010; accepted 4 August 2010; available online 23 October 2010 q DOI of original article: 10.1016/j.jhep.2010.10.008. ⇑ Corresponding author. Address: Department of Internal Medicine I, University of Bonn, Sigmund-Freud-Straße 25, 53105 Bonn, Germany. Tel.: +49 228 287 51416; fax: +49 228 287 51419. E-mail address: [email protected] (B. Langhans).
Introduction Hepatitis C virus (HCV) infection is a major cause of chronic liver disease worldwide [1]. Chronic infection is characterized by weak and functionally impaired cellular immune responses against HCV proteins. Recent evidence suggests a pivotal role of dendritic cell (DC) dysfunction for the outcome of HCV infection [2,3]. DCs are important for the initiation of innate and adaptive immune responses, because they can secrete interferons (IFNs) and trigger activation of T-lymphocytes. Although some studies described normal DCs function [4–7], others demonstrated poor allostimulatory DC activity in chronic hepatitis C [8]. DCs maintain an immature phenotype and do not respond to maturation stimuli [9]. Importantly, DCs of HCV-infected patients produce reduced levels of IFN-alpha and tumor necrosis factor alpha (TNF-alpha) so that antiviral cytokines may not reach sufficient levels for viral elimination [10,11]. IFNs consist of several protein classes, among which type I IFNs, such as IFN-alpha and IFN-beta [12] play a pivotal role in the first line defense against viruses. Recently, a novel IFN class, designated type III IFNs, has been discovered comprising the closely related proteins IL-29 (IFN-lambda1), IL-28A (IFN-lambda2), and IL-28B (IFN-lambda3) [13,14]. IFN-lambdas interact with shared unique cell surface receptors composed of the specific ligand-binding chain IFN-lR (also known as IL-28R) and the accessory receptor chain IL-10R2, which differ from the type I IFN receptors. IFNlambda receptors are preferentially expressed on hepatocytes and B cells, but not on other haematopoietic cells [14]. IFN-lambdas are mainly produced by DCs in response to viral proteins or toll-like receptor (TLR) agonists [15–19]. IFN-lambdas trigger the same signalling pathways as type I IFNs ultimately resulting in phosphorylation and nuclear translocation of IFNstimulated regulatory factor 3 (IRF-3) to induce expression of IFN-responsive genes [18,20,21]. Thus, IFN-lambdas can inhibit the replication of various viruses [13,14,22] including hepatitis B virus and HCV [23–26]. In particular, IFN-lambdas may play a pivotal role in the antiviral response against HCV, because polymorphisms in close proximity to the IL-28B gene have recently been linked to the outcome of HCV infection during spontaneous and treatment induced elimination of HCV [27–31]. However, the
Journal of Hepatology 2011 vol. 54 j 859–865
Research Article mechanisms, how polymorphisms in the region of the IFN-lambda genes affect antiviral host responses have remained elusive. To better understand the role of IFN-lambdas in HCV infection we performed a cross-sectional analysis of IFN-lambda serum levels in HCV infected patients with different outcomes of HCV infection and studied the in vitro effects of HCV viral proteins on IFN-lambda production by DCs. Materials and methods Patients Sera had been collected from the blood of 19, 60, and 29 patients with acute, chronic persistent, and previous self-limited hepatitis C infection, respectively. Furthermore, 26 HCV-seronegative un-infected subjects served as a reference population. All persons in the study had Caucasian ethnicity. Sera were immediately collected, aliquoted, and frozen at 20 °C. HCV viremia was quantified by Roche Amplicor HCV Monitor or TaqMan HCV (Roche Diagnostics, Grenzach, Germany). Serum from patients with acute hepatitis C was collected at the earliest possible date after discovery of HCV-RNA by PCR. The study protocol was approved by the local ethics committee and conformed to the ethical guidelines of the 1975 Declaration of Helsinki, and informed consent had been obtained from each individual included in the study. IL-28B genotyping Several highly correlated single nucleotide polymorphisms (SNPs) in close proximity to the IL-28B gene are linked to the outcome of treatment-induced and spontaneous viral clearance [27–31], with the closest correlation being described for the rs12979860 T/C polymorphism. Thus, we determined rs12979860 genotypes in our patients analogous to the study of Thomas et al. [29] and Ge et al. [30]. Genotyping was performed using the commercially available LightSNiP rs12979860 hu IL28B assay (TIB MOLBIOL, Berlin, Germany) on a Light Cycler instrument according to the manufacturer’s instructions. Analysis of IL-29 and IL-28 serum levels IL-29 was determined with the human IL-29 ELISA Ready-SET-Go (NatuTec, Frankfurt, Germany). IL-28 was detected by ELISA using R&D IL-28 DuoSet antibodies (R&D Systems Wiesbaden, Germany). The capture antibody in this kit (clone 248512) is directed against IL-28A but also significantly cross-reacts with IL-28B (96% amino acid identity). For this reason combined IL-28A and IL-28B immunoreactivity measured with this ELISA is referred to as IL-28 throughout this manuscript. ELISAs were carried out according to the manufacturer’s instruc-
tions. Briefly, serum samples were used undiluted and analysed in duplicates. After 2 h incubation in anti-cytokine-coated plates, avidin-coupled horseradish peroxidase was added and the plates were developed with tetramethylbenzidine (TMB; Roth, Karlsruhe, Germany). Plates were measured in an ELISA-Reader (Orgentec, Mainz, Germany) at 450 nm. The IL-29 ELISA Ready-SET-Go assay has a detection limit of 8 pg/ml and has been validated in the range between 8.0 and 1000 pg/ml with 7.0% and 9.6% intraand inter-assay variations, respectively. The detection limit was 15.0 pg/ml in the IL-28 DuoSet ELISA with a validated detection range between 15.0 and 2000 pg/ ml. Intra- and inter-assay variations were 3.0% and 29.2%, respectively. To further validate the IL-28 DuoSet ELISA regarding analysis of serum samples, spiking experiments were performed. For this purpose increasing amounts of IL-28A (62.5–500 pg/ml; taken from the kit standard) were added to representative serum samples from both patients with chronic hepatitis C (n = 3) and healthy controls (n = 3). Sera were analysed by ELISA as described above. Reagents Synthetic polyinosinic-polycytidylic acid (poly I:C; ALEXIS Biochemicals; Lörrach, Germany) was used to stimulate IFN production in DCs via triggering TLR3 and RIG-I. Recombinant HCV proteins (core, NS3, NS4, and NS5B; <4.0 pg/lg LPS contamination) corresponding to HCV genotype 1 were purchased from Mikrogen GmbH (Martinsried, Germany). HCV E2 protein was kindly provided by Chiron Corporation (Emeryville, California, USA). Viral surface proteins (HBsurface Ag; kindly provided from Rhein Biotec GmbH, Düsseldorf, Germany; recombinant HIV-1 IIIB gp120 from NIH AIDS Research & Reference Reagent Program, MD, USA) and cytomegalovirus (CMV) glycoprotein p65 (AUSTRAL Biologicals, San Ramon, California, USA) were used as controls to check antigen-specificity. RPMI 1640 medium, supplemented with 10% human AB serum, glutamine (200 mg/ml; all PAA Laboratories GmbH) was used for cell culture. In vitro modulation of IL-29 production by HCV-antigens DCs were separated from freshly isolated peripheral blood mononuclear cells (PBMC) of healthy blood donors using the human blood dendritic cell isolation kit II (Miltenyi Biotec, Bergisch Gladbach, Germany). This immunomagnetic separation kit enables the concurrent isolation of pDCs and mDCs from human blood at physiological ratios. Isolation of DCs was performed in a two-step procedure first using a ‘‘Non-Dendritic Cell Depletion Cocktail’’ followed by a ‘‘Dendritic Cell Enrichment Cocktail’’ according to the manufacturer’s instructions. Then, 50,000 DCs were plated into 96-well plates and stimulated with poly I:C (10–50 lg/ml) at 37 °C in 5% CO2. To test modulation of IL-29 production by HCV proteins, poly I:C-stimulated DCs were co-stimulated with recombinant HCV core, NS3, NS4, and NS5B and E2 protein (5 lg/ml). Specificity was checked with a CMV control protein (5 lg/ml). Supernatants were collected after 20 h and IL-29 measured by ELISA.
Table 1. Demographic and virological data of the study groups.
Acute hepatitis C
Chronic hepatitis C
Self-limited hepatitis C
Healthy Controls
Numbers
19
60
29
26
Sex (% males) a Age (years)
53 33 (25-63)
60 49 (25-79)
96 35 (23 - 76)
42 30 (22-52)
HCV load (IU/ml) a
7.046.068
503.924
<10
n.d.
HCV genotypes
(681 - 61.038.462) 69, 10, 17, 4
(3.648 - 5.306.449) 86, 0, 14, 0
n.a.
n.a.
(%; GT1, GT2, GT3, GT4) rs12979860 genotypes
16, 37, 47
11, 44, 45
7, 39, 54
5, 36, 59
(%; TT, CT, CC) a
Median (range). n.d. = not done. n.a. = not applicable.
860
Journal of Hepatology 2011 vol. 54 j 859–865
JOURNAL OF HEPATOLOGY To identify putatively involved HCV binding receptors, we used azide-free antibodies and isotype controls (5 lg/ml) to block interactions via CD81 (clone JS-81; BD Pharmingen) and TLR2 (clone TL2.1; eBioscience) (5 lg/ml each), respectively. Statistics All calculations were performed on a personal computer with the Prism software package 4.0 (Graph Pad Software, San Diego California, USA). Differences between experiments were compared by t-test and Wilcoxon test as appropriate. A p-value <0.05 was considered to indicate significant differences.
A
p = 0.019
160
n.s.
To exclude a potential interference of cytokine-binding proteins regarding quantification of IL-28A/B in serum samples by IL-28 DuoSet ELISA we performed spiking experiments. Of note, the IL-28 DuoSet ELISA showed excellent linearity in serum samples with similar IL-28A recovery rates in spiked serum samples from both patients with chronic hepatitis C (85 ± 4%) and healthy controls (79 ± 11%). Next, we performed a cross-sectional analysis of IL-29 and IL28A/B random serum levels in 60 untreated patients with chronic replicative hepatitis C (frequency of the rs12979860 C allele: 67%), 29 patients who had spontaneously resolved their HCV infection (frequency of the rs12979860 C allele: 73%) and 26
n.s.
120 100
p = 0.009
A
80 60 40 20 0 TT
CT
CC
Genotype
B
p = 0.03
300 p = 0.005
n.s.
100 IL-28A/B (pg/ml)
IL-29 (pg/ml)
140
Results
80
n.s.
60 40 20
p = 0.001
0 TT
CT
CC
200
B 100
0 Healthy control
C
400
Acute
Chronic
Recovered
Hepatitis C
IL-28A/B (pg/ml)
IL-29 (pg/ml)
Genotype 300
200
100
Chronic hepatitis C Resolved hepatitis C
0 Healthy control
Acute
Chronic
Recovered
Hepatitis C
p = 0.054
C p = 0.007
400
100 0 TT
CT Genotype
CC
Fig. 1. IL-29 serum levels of patients with different outcomes of HCV infection. IL-29 serum levels were significantly lower in carriers of a rs12979860 T allele (A). Analysis of serum levels between the study groups shows that IL-29 serum levels were markedly lower in patients with chronic hepatitis C (n = 60) than in individuals who spontaneously resolved from HCV infection (n = 29) and healthy controls (n = 26) (B). Patients with acute hepatitis C (n = 19) showed IL-29 levels intermediate between chronic hepatitis C and normal controls. Differences in IL-29 serum levels between patients with chronic hepatitis C and subjects who had spontaneously resolved their HCV infection were also evident when patients were stratified with respect to rs12979860 genotypes (C). Columns represent means ± SEM.
IL-28A/B (pg/ml)
IL-29 (pg/ml)
p = 0.081
300 200
n.s.
Chronic hepatitis C Resolved hepatitis C
n.s.
300 200 100 0 TT
CT Genotype
CC
Fig. 2. IL-28A/B serum levels of patients with different outcomes of HCV infection. Overall, IL-28A/B serum levels were rather low but still showed increased levels in carriers of the rs12979860 C allele (A). However, differences were not significant when analysis of IL-28A/B serum levels was stratified with respect to study groups (B) or rs12979860 genotypes in patients with resolved and chronic hepatitis C (C). Columns represent means ± SEM.
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Research Article un-infected healthy controls (frequency of the rs12979860 C allele: 67%). The distribution of alleles was in accordance with the Hardy–Weinberg equilibrium in each group. Further demographic and viral data are summarised in Table 1. Overall, IL-29 was detectable in all but two sera from healthy controls and exceeded IL-28A/B at least twofold, so that IL-28 serum levels were above the detection limit in only 27% of subjects. If all study participants were analysed together, IL-29 and IL-28A/B levels were significantly higher in carriers of the rs12979860 C allele than in TT homozygous individuals (Fig. 1A: IL-29; p = 0.019, and Fig. 2A: IL-28A/B; p = 0.009). When allocated to the study group, IL-29 serum levels were markedly lower in patients with chronic hepatitis C than in individuals who spontaneously resolved from HCV infection (p = 0.001) and healthy controls (p = 0.005) (Fig. 1B). Patients with acute hepatitis C showed IL-29 levels intermediate between chronic hepatitis C and normal controls. When IL-29 serum levels in anti-HCV-positive subjects were stratified with respect to rs12979860 alleles, individuals with resolved hepatitis C had consistently higher IL-29 serum levels than patients with chronic hepatitis C (Fig. 1C). Furthermore, there was a non-significant trend for increased IL-29 levels in carriers of the rs12979860 C allele (Fig. 1C). IL-28A/B serum levels were rather low, and differences between patient subgroups were only minor in our analysis (Fig. 2B and C). Thus, further studies were focused exclusively on IL-29. Next, we prospectively studied serial serum levels in 19 patients with acute hepatitis C, among whom 12 patients resolved
A
Acute Acute
chronic hepatitis C resolved hepatitis C p = 0.051
IL-29 (pg/ml)
250 200 150
their acute hepatitis (frequency of the rs12979860 C allele: 67%) and seven developed chronic infection (frequency of the rs12979860 C allele: 64%). Analogous to our cross-sectional analysis, serial measurements revealed slightly higher IL-29 serum levels in carriers of the rs12979860 C allele and a conspicuous difference between resolvers and non-resolvers (Fig. 3A). Individual IL-29 serum levels varied in acute HCV infection but were consistently higher in patients who subsequently spontaneously resolved their infection than in those who progressed to chronic hepatitis (Fig. 3B). Maximum serum levels were reached within the first month after diagnosis in all but two patients with resolved hepatitis C, in whom maximum IL-29 levels were observed at months 2 and 4, respectively. Thus, we could not detect any relationship between early IL-29 induction and outcome of infection. DCs are the primary source of IFN-lambda production; and HCV has already been shown to inhibit IFN-alpha production by DCs [9], although HCV does not infect DCs [32]. To find out if HCV proteins can exert similar inhibition of IFN-lambda production, we triggered IL-29 production in freshly purified DCs from healthy controls with poly I:C and added recombinant HCV proteins core, E2, NS3, NS4, and NS5B, respectively. Fig. 4 illustrates that in vitro stimulation of DCs in the presence of HCV proteins E2 and NS3 resulted in significantly reduced IL-29 production, while HCV-unrelated control proteins HBsAg, HIVgp120, and CMV-p65 as well as HCV proteins core, NS4, and NS5B did not exert any inhibitory effects. Under the same experimental conditions none of the HCV proteins revealed any effect on IFN-alpha production (data not shown). Since HCV proteins E2 and NS3 are known to trigger surface molecules CD81 and TLR2, respectively, we checked if HCV E2- and NS3-mediated inhibition of IL-29 production could be blocked by CD81- or TLR2-specific antibodies. Fig. 5 demonstrates that addition of either neutralising antibody could not prevent inhibition of IL29 production in the presence of HCV E2 and NS3, suggesting that these two receptors were apparently not involved.
100 50 Discussion
0 TT
B IL-29 pg/ml
100 80
CT Genotype Acute Acute
CC
chronic hepatitis C resolved hepatitis C
p = 0.024
60 40 20 0
Diagnosis Maximum >6 months
Fig. 3. IL-29 serum level of patients with acute hepatitis C. Serial serum levels in patients with acute hepatitis C (n = 19) carrying the rs12979860 C allele consistently exhibited higher IL-29 serum levels. (A) This figure demonstrates that there was a conspicuous difference between resolvers and non-resolvers. (B) Individual IL-29 serum levels in 12 patients, who resolved acute hepatitis, and seven patients, who became chronic, varied considerably during the first 6 months of acute HCV infection but IL-29 levels were consistently higher in those patients who subsequently spontaneously resolved their infection than in patients who progressed to chronic infection. Data are presented as means ± SEM.
862
Conventional types I and III IFNs are structurally distinct proteins and activate their own specific receptors on selected cell populations. At the cellular level, however, both types of IFN seem to have similar biological effects, since they activate a highly overlapping set of transcription factors. IFN-lambdas are expressed in virus-infected cells [19] and induce marked antiviral protection in a wide variety of cells [33] especially when cooperating with type I IFNs to induce antiviral gene expression [26]. In HCV infection type III IFNs seem to exert pivotal antiviral functions, since a polymorphism 3 kB upstream of the IL-28B gene appears to be linked to the outcome of infection, although the mode of action is still unknown [27–31]. The present study investigated serum levels of IL-29 and IL-28 in patients with different outcomes of HCV infection and demonstrated that patients with chronic hepatitis C had significantly lower IL-29 serum levels than subjects who had spontaneously cleared a previous HCV infection and healthy controls. Carriers of a rs12979860 C allele consistently tended to have higher IL-29 and IL-28 serum levels than subjects with a T/T genotype in all study groups, while the difference between subjects with a rs12979860 TT versus a CC genotype achieved statistical significance when all patients were studied as a single population. Of note, patients who had resolved
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JOURNAL OF HEPATOLOGY without HCV proteins with HCV proteins
Coincubation with E2
60
50 IL-29 (pg/ml)
IL-29 (pg/ml)
50 p = 0.02
40 30 20
30 20 10
0
0 10
20 30 40 poly I:C (µg/ml)
0
50
Coincubation with Core
60
p = 0.03
40
10
0
10
50
50 IL-29 (pg/ml)
40
n.s.
30 20
40
20 10
0
0 10
20 30 40 poly I:C (µg/ml)
n.s.
30
10
0
50
0
10
Coincubation with NS5B
20 30 40 poly I:C (µg/ml)
50
Coincubation with HBsAg 60
60 50
50
n.s.
IL-29 (pg/ml)
IL-29 (pg/ml)
20 30 40 poly I:C (µg/ml)
Coincubation with NS4
60
50 IL-29 (pg/ml)
Coincubation with NS3
60
40 30 20
30 20
10
10
0
0 0
10
20
30
40
n.s.
40
50
0
poly I:C (µg/ml)
10
20
30
40
50
60
poly I:C (µg/ml)
Fig. 4. HCV proteins NS3 and E2 inhibit IL-29 production in DCs. IL-29 production was induced in freshly purified DCs from healthy individuals (50,000/well) by in vitro stimulation with poly I:C (10–50 lg/ml) in the presence of HCV proteins core, NS3, NS4, NS5B, and E2 (5 lg/ml each). The figure illustrates that in vitro stimulation of DCs in the presence of HCV E2 and NS3 resulted in significantly reduced IL-29 production, while HCV proteins core, NS4, and NS5B as well as the HCV-unrelated control protein HBsAg did not exert inhibitory effects. Further control proteins such as HIV-gp120 and CMV-p65 also did not affect IL-29 production (data not shown).
their infection had consistently higher IL-29 and IL-28 serum levels even if the data were corrected for the different rs12979860 T/ C genotypes. These findings suggest that low IFN-lambda levels may predispose patients to chronic HCV infection and that the rs12979860 C allele may contribute to viral clearance because it is linked with increased IFN-lambda production. This hypothesis is further supported by our serial measurements in patients
with acute hepatitis C infection, which confirmed the effect of the rs12979860 C allele on IL-29 serum levels and also provided evidence that high IL-29 peak serum levels during the acute phase are associated with a self-limited course of HCV infection. Nevertheless, our data have to be interpreted with some caution, because patients with acute symptomatic hepatitis C, who have a greater chance of spontaneous viral elimination, are likely
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Research Article A IL-29 (pg/ml)
30
p = 0.003
20
10
0 poly I:C
B
50
poly I:C + E2
poly I:C poly I:C + E2 + E2 +anti-CD81 + control antibody
p = 0.004
IL-29 (pg/ml)
40 30 20 10 0 poly I:C
poly I:C + NS3
poly I:C poly I:C + NS3 +NS3 + anti-TLR2 + control antibody
Fig. 5. Inhibition of IL-29 production by HCV proteins NS3 and E2 is not mediated via TLR2 or CD81. HCV proteins E2 and NS3 trigger surface molecules CD81 and TLR2, which are also expressed on normal DCs. Thus, we checked if HCV E2- and NS3-mediated inhibition of IL-29 production could be blocked by a neutralising CD81 or TLR2 antibody. DCs (50,000/well) were pre-incubated with 5 lg/ml of either antibody or isotype control antibody, before DCs were stimulated with poly I:C (40 lg/ml) in the presence of HCV E2 (A) and NS3 (B), respectively. The figure illustrates that neither neutralising anti-CD81 nor antiTLR2 prevented inhibition of IL-29 production by HCV proteins E2 and NS3. Data are shown as means ± SEM of 10 (A) and four (B) independent experiments.
to be overrepresented in our group with acute HCV infection explaining the unexpectedly high rate of spontaneous resolution. IL-28B has the greatest relative antiviral activity among the IFN-lambda family [34], and unfortunately we could not differentiate between IL-28A and IL-28B due to the cross-reactivity of our capture antibody. However, IL-29 is probably still the major biologically relevant IFN-lambda, because its high abundance over IL-28A/B in serum is quite likely to compensate for the twofold higher intrinsic activity of IL-28B. Finally, down-regulation of IFN-lambda may be a feature in multiple chronic viral infections. For instance, low IFN-lambda serum levels have also been reported in patients with chronic hantavirus infection [35]. Currently it remains unclear why the rs12979860 T/C polymorphism affects IFN-lambda levels. Of note, the IL-28B gene is located on the minus DNA strand, whereas IL-28A and IL-29 genes are encoded on the plus DNA strand on chromosome 19. Thus, the rs12979860 T/C polymorphism is upstream of the promoter region for the IL-28B gene as well as the IL-28A and IL-29 genes and in principle can affect all three IFN-lambda genes. Thus, the rs12979860 T/C polymorphism may be a surrogate marker of altered promoter activity in this particular region. Recent studies have identified multiple strategies employed by HCV to attenuate type I IFN responses [3], also involving an 864
impaired ability of HCV-exposed DCs to produce IFN-alpha upon in vitro stimulation [11,32,36]. Inhibition of type I IFN production was exerted by both infectious and non-infectious HCV and was not abrogated by neutralising antibodies. Thus, HCV infection and replication in DCs was not required to inhibit in vitro type I IFN production, in line with the fact that HCV cannot be propagated in DCs in vitro [32]. Furthermore, type I and type III IFNs were similarly suppressed in epithelial cells and macrophages harbouring the NS1 and NS2 proteins of respiratory syncytial virus [37]. Therefore, we reasoned that IFN-lambda production during chronic hepatitis C might be impaired in an analogous fashion due to interactions of HCV viral antigens with DCs. Therefore, we studied IL-29 production in DCs after exposure to HCV antigens and found that exposure of DCs to HCV E2 and NS3 resulted in reduced IL-29 secretion in response to TLR3 and RIG-I stimulation with poly I:C. This finding supports the idea that circulating HCV components might attenuate IFN-lambda production in a similar fashion as has been described for type I IFNs. HCV proteins E2 and NS3 can bind to multiple host proteins to alter cellular functions. For instance, NS3 triggers TLR2 activation [38], and E2 inhibits natural killer cells via binding to CD81 [39,40]. Since TLR2 and CD81 are also expressed on DCs, we studied whether reduced IL-29 production in the presence of HCV proteins E2 and NS3 was mediated via CD81 or TLR2. However, neutralising antibodies to CD81 and TLR2 failed to prevent inhibition of IL-29 production arguing against involvement of either receptor. Taken together, our study demonstrated high IFN-lambda serum levels to be prevalent in carriers of the rs12979860 C allele and to be associated with a favourable outcome of HCV infection confirming important antiviral properties of type III IFNs. Furthermore, our in vitro findings suggest that HCV has evolved mechanisms to attenuate production of type III IFNs by DCs in a similar fashion as production of type I IFNs. Downregulated IFN-lambda may have further importance for the overall antiviral immune response in HCV infection, since in addition to the potent antiviral activity, IL-29 is an immunomodulator downregulating IL-13, so that the TH1/TH2 balance is shifted towards greater efficiency of adaptive antiviral immune responses [41,42].
Financial support This work has been supported by a Grant from the Deutsche Forschungsgemeinschaft (DFG; SP 483/4-1 and SFB/TRR57), the Wilhelm Sander Stiftung (Project 2009.045.1), and the H.W. and J. Hector Foundation (M42). Conflict of interest The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript. References [1] Lauer GM, Walker BD. Hepatitis C virus infection. N Engl J Med 2001;345:41–52. [2] Pachiadakis I, Pollara G, Chain BM, Naoumov NV. Is hepatitis C virus infection of dendritic cells a mechanism facilitating viral persistence? Lancet Infect Dis 2005;5:296–304.
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JOURNAL OF HEPATOLOGY [3] Rehermann B. Hepatitis C virus versus innate and adaptive immune responses: a tale of coevolution and coexistence. J Clin Invest 2009;119:1746–1964. [4] Longman RS, Talal AH, Jacobson IM, Albert ML, Rice CH. Presence of functional dendritic cells in patients chronically infected with hepatitis C virus. Blood 2004;103:1026–1029. [5] Longman RS, Talal AH, Jacobson IM, Rice CM, Albert ML. Normal functional capacity in circulating myeloid and plasmacytoid dendritic cells in patients with chronic hepatitis C. JID 2005;192:497–502. [6] Piccioli D, Tavarini S, Nuti S, Colombatto P, Brunetto M, Bonino F, et al. Comparable functions of plasmacytoid and monocyte-derived dendritic cells in chronic hepatitis C patients and healthy donors. Hepatology 2005;42:61–67. [7] Barnes E, Salio M, Cerundolo V, Francesco L, Pardoll D, Klenerman P, et al. Monocyte derived dendritic cells retain their functional capacity in patients following infection with hepatitis C virus. J Viral Hepat 2008;15:219–228. [8] Bain C, Fatmi A, Zoulim F, Zaraki JP, Trepo C, Inchauspe G. Impaired allostimulatory function of dendritic cells in chronic hepatitis C. Gastroenterology 2001;120:512–524. [9] Auffermann-Gretzinger S, Keeffe EB, Levy S. Impaired dendritic cell maturation in patients with chronic, but not resolved, hepatitis C virus infection. Blood 2001;97:3171–3176. [10] Kanto T, Inoue M, Miyatake H, Sato A, Sakakibara M, Yakushijin T, et al. Reduced numbers and impaired ability of myeloid and plasmacytoid dendritic cells to polarize T helper cells in chronic hepatitis C virus infection. J Infect Dis 2004;190:1919–1926. [11] Dolganiuc A, Chang S, Kodys K, Mandrekar P, Bakis G, Cormier M, et al. Hepatitis C virus (HCV) core protein-induced, monocyte-mediated mechanisms of reduced IFN-alpha and plasmacytoid dendritic cell loss in chronic HCV infection. J Immunol 2006;177:6758–6768. [12] Stark GR, Kerr IM, Williams BR, Silverman RH, Schreiber RD. How cells respond to interferons. Annu Rev Biochem 1998;67:227–264. [13] Kotenko SV, Gallagher G, Baurin VV, Lewis-Antes A, Shen M, Shan NK, et al. IFN-lambdas mediate antiviral protection through a distinct class II cytokine receptor complex. Nat Immunol 2003;4:69–77. [14] Sheppard P, Kindsvogel W, Xu W, Henderson K, Schlutsmeyer S, Whitmore TE, et al. IL-28, IL-29 and their class II cytokine receptor IL-28R. Nat Immunol 2003;4:63–68. [15] Diebold SS, Montoya M, Unger H, Alexopoulou L, Roy P, Haswell LE, et al. Viral infection switches non-plasmacytoid dendritic cells into high interferon producers. Nature 2003;424:324–328. [16] Pollara G, Jones M, Handley ME, Rajpopat M, Kwan A, Coffin RS, et al. Herpes simplex virus type-1-induced activation of myeloid dendritic cells: the roles of virus cell interaction and paracrine type I IFN secretion. J Immunol 2004;173:4108–4119. [17] Coccia EM, Severa M, Giacomini E, Monneron D, Remoli ME, Julkunen I, et al. Viral infection and Toll-like receptor agonists induce a differential expression of type I and lambda interferons in human plasmacytoid and monocytederived dendritic cells. Eur J Immunol 2004;34:796–805. [18] Onoguchi K, Yoneyama M, Takemura A, Akari A, Taniguchi T, Namiki H, et al. Viral infections activate types I and III interferon genes through a common mechanism. J Biol Chem 2007;282:7576–7581. [19] Ank N, Iversen MB, Bartholdy C, Staeheli P, Hartmann R, Jensen UB, et al. An important role for type III interferon (IFN-lambda/IL-28) in TLR-induced antiviral activity. J Immunol 2008;180:2474–2585. [20] Osterlund P, Veckman V, Sirén J, Klucher KM, Hiscott J, Matikainen S, et al. Gene expression and antiviral activity of alpha/beta interferons and interleukin-29 in virus-infected human myeloid dendritic cells. J Virol 2005;79:9608–9617. [21] Osterlund PI, Pietilä TE, Veckman V, Kotenko SC, Julkunen I. IFN regulatory factor family members differentially regulate the expression of type III IFN (IFN-lambda) genes. J Immunol 2007;179:3434–3442. [22] Brand S, Beigel F, Olzak T, Zitzmann K, Eichhorst ST, Otte JM, et al. IL-28A and IL-29 mediate antiproliferative and antiviral signals in intestinal epithelial
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Interferon-lambda serum levels in hepatitis C Bettina Langhans1,⇑, Bernd Kupfer2, Ingrid Braunschweiger1, Simone Arndt1, Wibke Schulte1, Hans Dieter Nischalke1, Jacob Nattermann1, Johannes Oldenburg3, Tilman Sauerbruch1, Ulrich Spengler1 1 Department of Internal Medicine I, University of Bonn, Bonn, Germany; 2Institute of Virology, University of Bonn, Bonn, Germany; 3Institute for Experimental Hematology and Transfusion Medicine, University of Bonn, Bonn, GermanyReceived 11 January 2010; received in revised form 29 July 2010;
accepted 4 August 2010
See Editorial, pages 844–847
Background & Aims: Dendritic cells (DCs) trigger adaptive immune responses and are an important source of antiviral cytokines. In hepatitis C virus (HCV) infection DC function is markedly impaired. Thus far, studies have focused on types I and II interferon (IFN). We studied IFN-lambda1 (IL-29) and IFN-lambda2/3 (IL-28A/ B) serum levels in patients with different outcomes of HCV infection. Methods: IFN-lambdas were measured by ELISAs detecting IL-29 or IL-28A and IL-28B, respectively. Results were stratified with respect to the recently discovered rs12979860 T/C polymorphism upstream of the IL-28B gene. Results: In general IL-29 serum levels exceeded IL-28A/B at least twofold, with IL-29 and IL-28A/B levels being significantly higher in carriers of the rs12979860 C allele than in TT homozygous individuals (p <0.02). IL-29 levels were substantially lower in patients with chronic hepatitis C than in healthy controls (p = 0.005) and patients with spontaneously resolved hepatitis (p = 0.001). Patients with acute hepatitis C showed IL-29 levels intermediate between chronic hepatitis C and normal controls; and IL-29 serum levels were higher in patients who spontaneously resolved hepatitis C than in those who became chronic. In vitro HCV proteins NS3 and E2 directly inhibited IL-29 production in poly I:C-stimulated purified DCs. Conclusions: Our data suggest that HCV proteins modify IFNlambda production in DCs. Carriers of the rs12979860 C allele associated with resolution of HCV infection exhibited increased IFN-lambda levels. Moreover, high IFN-lambda levels predisposed to spontaneous resolution of HCV infection. Thus, IFN-lambdas seem to play an important role in the control of hepatitis C. Ó 2010 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.
Keywords: Hepatitis C virus (HCV); IFN-lambdas (IL-29, IL-28A/B); IL-28B rs12979860 T/C polymorphism; Dendritic cells (DCs). Received 11 January 2010; received in revised form 29 July 2010; accepted 4 August 2010; available online 23 October 2010 q DOI of original article: 10.1016/j.jhep.2010.10.008. ⇑ Corresponding author. Address: Department of Internal Medicine I, University of Bonn, Sigmund-Freud-Straße 25, 53105 Bonn, Germany. Tel.: +49 228 287 51416; fax: +49 228 287 51419. E-mail address: [email protected] (B. Langhans).
Introduction Hepatitis C virus (HCV) infection is a major cause of chronic liver disease worldwide [1]. Chronic infection is characterized by weak and functionally impaired cellular immune responses against HCV proteins. Recent evidence suggests a pivotal role of dendritic cell (DC) dysfunction for the outcome of HCV infection [2,3]. DCs are important for the initiation of innate and adaptive immune responses, because they can secrete interferons (IFNs) and trigger activation of T-lymphocytes. Although some studies described normal DCs function [4–7], others demonstrated poor allostimulatory DC activity in chronic hepatitis C [8]. DCs maintain an immature phenotype and do not respond to maturation stimuli [9]. Importantly, DCs of HCV-infected patients produce reduced levels of IFN-alpha and tumor necrosis factor alpha (TNF-alpha) so that antiviral cytokines may not reach sufficient levels for viral elimination [10,11]. IFNs consist of several protein classes, among which type I IFNs, such as IFN-alpha and IFN-beta [12] play a pivotal role in the first line defense against viruses. Recently, a novel IFN class, designated type III IFNs, has been discovered comprising the closely related proteins IL-29 (IFN-lambda1), IL-28A (IFN-lambda2), and IL-28B (IFN-lambda3) [13,14]. IFN-lambdas interact with shared unique cell surface receptors composed of the specific ligand-binding chain IFN-lR (also known as IL-28R) and the accessory receptor chain IL-10R2, which differ from the type I IFN receptors. IFNlambda receptors are preferentially expressed on hepatocytes and B cells, but not on other haematopoietic cells [14]. IFN-lambdas are mainly produced by DCs in response to viral proteins or toll-like receptor (TLR) agonists [15–19]. IFN-lambdas trigger the same signalling pathways as type I IFNs ultimately resulting in phosphorylation and nuclear translocation of IFNstimulated regulatory factor 3 (IRF-3) to induce expression of IFN-responsive genes [18,20,21]. Thus, IFN-lambdas can inhibit the replication of various viruses [13,14,22] including hepatitis B virus and HCV [23–26]. In particular, IFN-lambdas may play a pivotal role in the antiviral response against HCV, because polymorphisms in close proximity to the IL-28B gene have recently been linked to the outcome of HCV infection during spontaneous and treatment induced elimination of HCV [27–31]. However, the
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Research Article mechanisms, how polymorphisms in the region of the IFN-lambda genes affect antiviral host responses have remained elusive. To better understand the role of IFN-lambdas in HCV infection we performed a cross-sectional analysis of IFN-lambda serum levels in HCV infected patients with different outcomes of HCV infection and studied the in vitro effects of HCV viral proteins on IFN-lambda production by DCs. Materials and methods Patients Sera had been collected from the blood of 19, 60, and 29 patients with acute, chronic persistent, and previous self-limited hepatitis C infection, respectively. Furthermore, 26 HCV-seronegative un-infected subjects served as a reference population. All persons in the study had Caucasian ethnicity. Sera were immediately collected, aliquoted, and frozen at 20 °C. HCV viremia was quantified by Roche Amplicor HCV Monitor or TaqMan HCV (Roche Diagnostics, Grenzach, Germany). Serum from patients with acute hepatitis C was collected at the earliest possible date after discovery of HCV-RNA by PCR. The study protocol was approved by the local ethics committee and conformed to the ethical guidelines of the 1975 Declaration of Helsinki, and informed consent had been obtained from each individual included in the study. IL-28B genotyping Several highly correlated single nucleotide polymorphisms (SNPs) in close proximity to the IL-28B gene are linked to the outcome of treatment-induced and spontaneous viral clearance [27–31], with the closest correlation being described for the rs12979860 T/C polymorphism. Thus, we determined rs12979860 genotypes in our patients analogous to the study of Thomas et al. [29] and Ge et al. [30]. Genotyping was performed using the commercially available LightSNiP rs12979860 hu IL28B assay (TIB MOLBIOL, Berlin, Germany) on a Light Cycler instrument according to the manufacturer’s instructions. Analysis of IL-29 and IL-28 serum levels IL-29 was determined with the human IL-29 ELISA Ready-SET-Go (NatuTec, Frankfurt, Germany). IL-28 was detected by ELISA using R&D IL-28 DuoSet antibodies (R&D Systems Wiesbaden, Germany). The capture antibody in this kit (clone 248512) is directed against IL-28A but also significantly cross-reacts with IL-28B (96% amino acid identity). For this reason combined IL-28A and IL-28B immunoreactivity measured with this ELISA is referred to as IL-28 throughout this manuscript. ELISAs were carried out according to the manufacturer’s instruc-
tions. Briefly, serum samples were used undiluted and analysed in duplicates. After 2 h incubation in anti-cytokine-coated plates, avidin-coupled horseradish peroxidase was added and the plates were developed with tetramethylbenzidine (TMB; Roth, Karlsruhe, Germany). Plates were measured in an ELISA-Reader (Orgentec, Mainz, Germany) at 450 nm. The IL-29 ELISA Ready-SET-Go assay has a detection limit of 8 pg/ml and has been validated in the range between 8.0 and 1000 pg/ml with 7.0% and 9.6% intraand inter-assay variations, respectively. The detection limit was 15.0 pg/ml in the IL-28 DuoSet ELISA with a validated detection range between 15.0 and 2000 pg/ ml. Intra- and inter-assay variations were 3.0% and 29.2%, respectively. To further validate the IL-28 DuoSet ELISA regarding analysis of serum samples, spiking experiments were performed. For this purpose increasing amounts of IL-28A (62.5–500 pg/ml; taken from the kit standard) were added to representative serum samples from both patients with chronic hepatitis C (n = 3) and healthy controls (n = 3). Sera were analysed by ELISA as described above. Reagents Synthetic polyinosinic-polycytidylic acid (poly I:C; ALEXIS Biochemicals; Lörrach, Germany) was used to stimulate IFN production in DCs via triggering TLR3 and RIG-I. Recombinant HCV proteins (core, NS3, NS4, and NS5B; <4.0 pg/lg LPS contamination) corresponding to HCV genotype 1 were purchased from Mikrogen GmbH (Martinsried, Germany). HCV E2 protein was kindly provided by Chiron Corporation (Emeryville, California, USA). Viral surface proteins (HBsurface Ag; kindly provided from Rhein Biotec GmbH, Düsseldorf, Germany; recombinant HIV-1 IIIB gp120 from NIH AIDS Research & Reference Reagent Program, MD, USA) and cytomegalovirus (CMV) glycoprotein p65 (AUSTRAL Biologicals, San Ramon, California, USA) were used as controls to check antigen-specificity. RPMI 1640 medium, supplemented with 10% human AB serum, glutamine (200 mg/ml; all PAA Laboratories GmbH) was used for cell culture. In vitro modulation of IL-29 production by HCV-antigens DCs were separated from freshly isolated peripheral blood mononuclear cells (PBMC) of healthy blood donors using the human blood dendritic cell isolation kit II (Miltenyi Biotec, Bergisch Gladbach, Germany). This immunomagnetic separation kit enables the concurrent isolation of pDCs and mDCs from human blood at physiological ratios. Isolation of DCs was performed in a two-step procedure first using a ‘‘Non-Dendritic Cell Depletion Cocktail’’ followed by a ‘‘Dendritic Cell Enrichment Cocktail’’ according to the manufacturer’s instructions. Then, 50,000 DCs were plated into 96-well plates and stimulated with poly I:C (10–50 lg/ml) at 37 °C in 5% CO2. To test modulation of IL-29 production by HCV proteins, poly I:C-stimulated DCs were co-stimulated with recombinant HCV core, NS3, NS4, and NS5B and E2 protein (5 lg/ml). Specificity was checked with a CMV control protein (5 lg/ml). Supernatants were collected after 20 h and IL-29 measured by ELISA.
Table 1. Demographic and virological data of the study groups.
Acute hepatitis C
Chronic hepatitis C
Self-limited hepatitis C
Healthy Controls
Numbers
19
60
29
26
Sex (% males) a Age (years)
53 33 (25-63)
60 49 (25-79)
96 35 (23 - 76)
42 30 (22-52)
HCV load (IU/ml) a
7.046.068
503.924
<10
n.d.
HCV genotypes
(681 - 61.038.462) 69, 10, 17, 4
(3.648 - 5.306.449) 86, 0, 14, 0
n.a.
n.a.
(%; GT1, GT2, GT3, GT4) rs12979860 genotypes
16, 37, 47
11, 44, 45
7, 39, 54
5, 36, 59
(%; TT, CT, CC) a
Median (range). n.d. = not done. n.a. = not applicable.
860
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JOURNAL OF HEPATOLOGY To identify putatively involved HCV binding receptors, we used azide-free antibodies and isotype controls (5 lg/ml) to block interactions via CD81 (clone JS-81; BD Pharmingen) and TLR2 (clone TL2.1; eBioscience) (5 lg/ml each), respectively. Statistics All calculations were performed on a personal computer with the Prism software package 4.0 (Graph Pad Software, San Diego California, USA). Differences between experiments were compared by t-test and Wilcoxon test as appropriate. A p-value <0.05 was considered to indicate significant differences.
A
p = 0.019
160
n.s.
To exclude a potential interference of cytokine-binding proteins regarding quantification of IL-28A/B in serum samples by IL-28 DuoSet ELISA we performed spiking experiments. Of note, the IL-28 DuoSet ELISA showed excellent linearity in serum samples with similar IL-28A recovery rates in spiked serum samples from both patients with chronic hepatitis C (85 ± 4%) and healthy controls (79 ± 11%). Next, we performed a cross-sectional analysis of IL-29 and IL28A/B random serum levels in 60 untreated patients with chronic replicative hepatitis C (frequency of the rs12979860 C allele: 67%), 29 patients who had spontaneously resolved their HCV infection (frequency of the rs12979860 C allele: 73%) and 26
n.s.
120 100
p = 0.009
A
80 60 40 20 0 TT
CT
CC
Genotype
B
p = 0.03
300 p = 0.005
n.s.
100 IL-28A/B (pg/ml)
IL-29 (pg/ml)
140
Results
80
n.s.
60 40 20
p = 0.001
0 TT
CT
CC
200
B 100
0 Healthy control
C
400
Acute
Chronic
Recovered
Hepatitis C
IL-28A/B (pg/ml)
IL-29 (pg/ml)
Genotype 300
200
100
Chronic hepatitis C Resolved hepatitis C
0 Healthy control
Acute
Chronic
Recovered
Hepatitis C
p = 0.054
C p = 0.007
400
100 0 TT
CT Genotype
CC
Fig. 1. IL-29 serum levels of patients with different outcomes of HCV infection. IL-29 serum levels were significantly lower in carriers of a rs12979860 T allele (A). Analysis of serum levels between the study groups shows that IL-29 serum levels were markedly lower in patients with chronic hepatitis C (n = 60) than in individuals who spontaneously resolved from HCV infection (n = 29) and healthy controls (n = 26) (B). Patients with acute hepatitis C (n = 19) showed IL-29 levels intermediate between chronic hepatitis C and normal controls. Differences in IL-29 serum levels between patients with chronic hepatitis C and subjects who had spontaneously resolved their HCV infection were also evident when patients were stratified with respect to rs12979860 genotypes (C). Columns represent means ± SEM.
IL-28A/B (pg/ml)
IL-29 (pg/ml)
p = 0.081
300 200
n.s.
Chronic hepatitis C Resolved hepatitis C
n.s.
300 200 100 0 TT
CT Genotype
CC
Fig. 2. IL-28A/B serum levels of patients with different outcomes of HCV infection. Overall, IL-28A/B serum levels were rather low but still showed increased levels in carriers of the rs12979860 C allele (A). However, differences were not significant when analysis of IL-28A/B serum levels was stratified with respect to study groups (B) or rs12979860 genotypes in patients with resolved and chronic hepatitis C (C). Columns represent means ± SEM.
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Research Article un-infected healthy controls (frequency of the rs12979860 C allele: 67%). The distribution of alleles was in accordance with the Hardy–Weinberg equilibrium in each group. Further demographic and viral data are summarised in Table 1. Overall, IL-29 was detectable in all but two sera from healthy controls and exceeded IL-28A/B at least twofold, so that IL-28 serum levels were above the detection limit in only 27% of subjects. If all study participants were analysed together, IL-29 and IL-28A/B levels were significantly higher in carriers of the rs12979860 C allele than in TT homozygous individuals (Fig. 1A: IL-29; p = 0.019, and Fig. 2A: IL-28A/B; p = 0.009). When allocated to the study group, IL-29 serum levels were markedly lower in patients with chronic hepatitis C than in individuals who spontaneously resolved from HCV infection (p = 0.001) and healthy controls (p = 0.005) (Fig. 1B). Patients with acute hepatitis C showed IL-29 levels intermediate between chronic hepatitis C and normal controls. When IL-29 serum levels in anti-HCV-positive subjects were stratified with respect to rs12979860 alleles, individuals with resolved hepatitis C had consistently higher IL-29 serum levels than patients with chronic hepatitis C (Fig. 1C). Furthermore, there was a non-significant trend for increased IL-29 levels in carriers of the rs12979860 C allele (Fig. 1C). IL-28A/B serum levels were rather low, and differences between patient subgroups were only minor in our analysis (Fig. 2B and C). Thus, further studies were focused exclusively on IL-29. Next, we prospectively studied serial serum levels in 19 patients with acute hepatitis C, among whom 12 patients resolved
A
Acute Acute
chronic hepatitis C resolved hepatitis C p = 0.051
IL-29 (pg/ml)
250 200 150
their acute hepatitis (frequency of the rs12979860 C allele: 67%) and seven developed chronic infection (frequency of the rs12979860 C allele: 64%). Analogous to our cross-sectional analysis, serial measurements revealed slightly higher IL-29 serum levels in carriers of the rs12979860 C allele and a conspicuous difference between resolvers and non-resolvers (Fig. 3A). Individual IL-29 serum levels varied in acute HCV infection but were consistently higher in patients who subsequently spontaneously resolved their infection than in those who progressed to chronic hepatitis (Fig. 3B). Maximum serum levels were reached within the first month after diagnosis in all but two patients with resolved hepatitis C, in whom maximum IL-29 levels were observed at months 2 and 4, respectively. Thus, we could not detect any relationship between early IL-29 induction and outcome of infection. DCs are the primary source of IFN-lambda production; and HCV has already been shown to inhibit IFN-alpha production by DCs [9], although HCV does not infect DCs [32]. To find out if HCV proteins can exert similar inhibition of IFN-lambda production, we triggered IL-29 production in freshly purified DCs from healthy controls with poly I:C and added recombinant HCV proteins core, E2, NS3, NS4, and NS5B, respectively. Fig. 4 illustrates that in vitro stimulation of DCs in the presence of HCV proteins E2 and NS3 resulted in significantly reduced IL-29 production, while HCV-unrelated control proteins HBsAg, HIVgp120, and CMV-p65 as well as HCV proteins core, NS4, and NS5B did not exert any inhibitory effects. Under the same experimental conditions none of the HCV proteins revealed any effect on IFN-alpha production (data not shown). Since HCV proteins E2 and NS3 are known to trigger surface molecules CD81 and TLR2, respectively, we checked if HCV E2- and NS3-mediated inhibition of IL-29 production could be blocked by CD81- or TLR2-specific antibodies. Fig. 5 demonstrates that addition of either neutralising antibody could not prevent inhibition of IL29 production in the presence of HCV E2 and NS3, suggesting that these two receptors were apparently not involved.
100 50 Discussion
0 TT
B IL-29 pg/ml
100 80
CT Genotype Acute Acute
CC
chronic hepatitis C resolved hepatitis C
p = 0.024
60 40 20 0
Diagnosis Maximum >6 months
Fig. 3. IL-29 serum level of patients with acute hepatitis C. Serial serum levels in patients with acute hepatitis C (n = 19) carrying the rs12979860 C allele consistently exhibited higher IL-29 serum levels. (A) This figure demonstrates that there was a conspicuous difference between resolvers and non-resolvers. (B) Individual IL-29 serum levels in 12 patients, who resolved acute hepatitis, and seven patients, who became chronic, varied considerably during the first 6 months of acute HCV infection but IL-29 levels were consistently higher in those patients who subsequently spontaneously resolved their infection than in patients who progressed to chronic infection. Data are presented as means ± SEM.
862
Conventional types I and III IFNs are structurally distinct proteins and activate their own specific receptors on selected cell populations. At the cellular level, however, both types of IFN seem to have similar biological effects, since they activate a highly overlapping set of transcription factors. IFN-lambdas are expressed in virus-infected cells [19] and induce marked antiviral protection in a wide variety of cells [33] especially when cooperating with type I IFNs to induce antiviral gene expression [26]. In HCV infection type III IFNs seem to exert pivotal antiviral functions, since a polymorphism 3 kB upstream of the IL-28B gene appears to be linked to the outcome of infection, although the mode of action is still unknown [27–31]. The present study investigated serum levels of IL-29 and IL-28 in patients with different outcomes of HCV infection and demonstrated that patients with chronic hepatitis C had significantly lower IL-29 serum levels than subjects who had spontaneously cleared a previous HCV infection and healthy controls. Carriers of a rs12979860 C allele consistently tended to have higher IL-29 and IL-28 serum levels than subjects with a T/T genotype in all study groups, while the difference between subjects with a rs12979860 TT versus a CC genotype achieved statistical significance when all patients were studied as a single population. Of note, patients who had resolved
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JOURNAL OF HEPATOLOGY without HCV proteins with HCV proteins
Coincubation with E2
60
50 IL-29 (pg/ml)
IL-29 (pg/ml)
50 p = 0.02
40 30 20
30 20 10
0
0 10
20 30 40 poly I:C (µg/ml)
0
50
Coincubation with Core
60
p = 0.03
40
10
0
10
50
50 IL-29 (pg/ml)
40
n.s.
30 20
40
20 10
0
0 10
20 30 40 poly I:C (µg/ml)
n.s.
30
10
0
50
0
10
Coincubation with NS5B
20 30 40 poly I:C (µg/ml)
50
Coincubation with HBsAg 60
60 50
50
n.s.
IL-29 (pg/ml)
IL-29 (pg/ml)
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Fig. 4. HCV proteins NS3 and E2 inhibit IL-29 production in DCs. IL-29 production was induced in freshly purified DCs from healthy individuals (50,000/well) by in vitro stimulation with poly I:C (10–50 lg/ml) in the presence of HCV proteins core, NS3, NS4, NS5B, and E2 (5 lg/ml each). The figure illustrates that in vitro stimulation of DCs in the presence of HCV E2 and NS3 resulted in significantly reduced IL-29 production, while HCV proteins core, NS4, and NS5B as well as the HCV-unrelated control protein HBsAg did not exert inhibitory effects. Further control proteins such as HIV-gp120 and CMV-p65 also did not affect IL-29 production (data not shown).
their infection had consistently higher IL-29 and IL-28 serum levels even if the data were corrected for the different rs12979860 T/ C genotypes. These findings suggest that low IFN-lambda levels may predispose patients to chronic HCV infection and that the rs12979860 C allele may contribute to viral clearance because it is linked with increased IFN-lambda production. This hypothesis is further supported by our serial measurements in patients
with acute hepatitis C infection, which confirmed the effect of the rs12979860 C allele on IL-29 serum levels and also provided evidence that high IL-29 peak serum levels during the acute phase are associated with a self-limited course of HCV infection. Nevertheless, our data have to be interpreted with some caution, because patients with acute symptomatic hepatitis C, who have a greater chance of spontaneous viral elimination, are likely
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Research Article A IL-29 (pg/ml)
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Fig. 5. Inhibition of IL-29 production by HCV proteins NS3 and E2 is not mediated via TLR2 or CD81. HCV proteins E2 and NS3 trigger surface molecules CD81 and TLR2, which are also expressed on normal DCs. Thus, we checked if HCV E2- and NS3-mediated inhibition of IL-29 production could be blocked by a neutralising CD81 or TLR2 antibody. DCs (50,000/well) were pre-incubated with 5 lg/ml of either antibody or isotype control antibody, before DCs were stimulated with poly I:C (40 lg/ml) in the presence of HCV E2 (A) and NS3 (B), respectively. The figure illustrates that neither neutralising anti-CD81 nor antiTLR2 prevented inhibition of IL-29 production by HCV proteins E2 and NS3. Data are shown as means ± SEM of 10 (A) and four (B) independent experiments.
to be overrepresented in our group with acute HCV infection explaining the unexpectedly high rate of spontaneous resolution. IL-28B has the greatest relative antiviral activity among the IFN-lambda family [34], and unfortunately we could not differentiate between IL-28A and IL-28B due to the cross-reactivity of our capture antibody. However, IL-29 is probably still the major biologically relevant IFN-lambda, because its high abundance over IL-28A/B in serum is quite likely to compensate for the twofold higher intrinsic activity of IL-28B. Finally, down-regulation of IFN-lambda may be a feature in multiple chronic viral infections. For instance, low IFN-lambda serum levels have also been reported in patients with chronic hantavirus infection [35]. Currently it remains unclear why the rs12979860 T/C polymorphism affects IFN-lambda levels. Of note, the IL-28B gene is located on the minus DNA strand, whereas IL-28A and IL-29 genes are encoded on the plus DNA strand on chromosome 19. Thus, the rs12979860 T/C polymorphism is upstream of the promoter region for the IL-28B gene as well as the IL-28A and IL-29 genes and in principle can affect all three IFN-lambda genes. Thus, the rs12979860 T/C polymorphism may be a surrogate marker of altered promoter activity in this particular region. Recent studies have identified multiple strategies employed by HCV to attenuate type I IFN responses [3], also involving an 864
impaired ability of HCV-exposed DCs to produce IFN-alpha upon in vitro stimulation [11,32,36]. Inhibition of type I IFN production was exerted by both infectious and non-infectious HCV and was not abrogated by neutralising antibodies. Thus, HCV infection and replication in DCs was not required to inhibit in vitro type I IFN production, in line with the fact that HCV cannot be propagated in DCs in vitro [32]. Furthermore, type I and type III IFNs were similarly suppressed in epithelial cells and macrophages harbouring the NS1 and NS2 proteins of respiratory syncytial virus [37]. Therefore, we reasoned that IFN-lambda production during chronic hepatitis C might be impaired in an analogous fashion due to interactions of HCV viral antigens with DCs. Therefore, we studied IL-29 production in DCs after exposure to HCV antigens and found that exposure of DCs to HCV E2 and NS3 resulted in reduced IL-29 secretion in response to TLR3 and RIG-I stimulation with poly I:C. This finding supports the idea that circulating HCV components might attenuate IFN-lambda production in a similar fashion as has been described for type I IFNs. HCV proteins E2 and NS3 can bind to multiple host proteins to alter cellular functions. For instance, NS3 triggers TLR2 activation [38], and E2 inhibits natural killer cells via binding to CD81 [39,40]. Since TLR2 and CD81 are also expressed on DCs, we studied whether reduced IL-29 production in the presence of HCV proteins E2 and NS3 was mediated via CD81 or TLR2. However, neutralising antibodies to CD81 and TLR2 failed to prevent inhibition of IL-29 production arguing against involvement of either receptor. Taken together, our study demonstrated high IFN-lambda serum levels to be prevalent in carriers of the rs12979860 C allele and to be associated with a favourable outcome of HCV infection confirming important antiviral properties of type III IFNs. Furthermore, our in vitro findings suggest that HCV has evolved mechanisms to attenuate production of type III IFNs by DCs in a similar fashion as production of type I IFNs. Downregulated IFN-lambda may have further importance for the overall antiviral immune response in HCV infection, since in addition to the potent antiviral activity, IL-29 is an immunomodulator downregulating IL-13, so that the TH1/TH2 balance is shifted towards greater efficiency of adaptive antiviral immune responses [41,42].
Financial support This work has been supported by a Grant from the Deutsche Forschungsgemeinschaft (DFG; SP 483/4-1 and SFB/TRR57), the Wilhelm Sander Stiftung (Project 2009.045.1), and the H.W. and J. Hector Foundation (M42). Conflict of interest The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript. References [1] Lauer GM, Walker BD. Hepatitis C virus infection. N Engl J Med 2001;345:41–52. [2] Pachiadakis I, Pollara G, Chain BM, Naoumov NV. Is hepatitis C virus infection of dendritic cells a mechanism facilitating viral persistence? Lancet Infect Dis 2005;5:296–304.
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