Introduction
Recently, we and others independently identified single nucleotide polymorphisms (SNPs) on chromosome 19 associated with the interleukin-28B gene (
IL28B), encoding IFN-λ3, that were strongly associated with the response to pegylated interferon-α/ribavirin (Peg-IFN/RBV) in chronic hepatitis C (CHC) patients, through a genome-wide association study (GWAS) [
1‐
3]. According to our results, about 80 % of CHC patients with the TT genotype (
rs8099917) showed viral virologic response (VR), including SVR (sustained virologic response) or TVR (transient virologic response), whereas only about 20 % of HCV patients with the TG/GG genotype showed VR [
1]. Thus, by genotyping of
IL28B, we can predict the efficacy of Peg-IFN/RBV before beginning treatment, avoiding unnecessary side effects and the high cost of Peg-IFN/RBV treatment. However, it is still unknown whether genetic variation of
IL28B is a functional phenotype for Peg-IFN/RBV treatment. In addition, genotyping of
IL28B alone failed to predict about 20 % of the response [
1], which would be reasonable because final products of the genes are affected by DNA methylation or chromatin modifications as well as genetic variations [
4].
Type III IFNs, consisting of IFN-λ1, λ2, and λ3 (also known as
IL29,
IL28A and
IL28B, respectively), have recently been characterized [
5,
6]. IFN-λs up-regulate IFN-stimulated genes (ISGs) via Janus kinase/signal transducer and activator of transcription (Jak/STAT) intracellular signaling, inhibiting hepatitis B virus (HBV) or hepatitis C virus (HCV) replication [
7]. Antiviral responses evoked by toll-like receptor (TLR)3 or TLR9 agonists are attenuated in IL28RA
−/− mice [
8], indicating the central role of IFN-λs in antiviral protection. Clinically, early virologic response by Peg-IFN/RBV is associated with a high probability of SVR in HCV patients [
9]. Genetic variations of
IL28B influence spontaneous clearance of HCV [
10], or on-treatment viral kinetics [
11]. These results suggest a mechanistic link between innate immunity and genetic variations of
IL28B.
To recognize viruses and trigger innate antiviral responses, mammals have 2 independent receptors, retinoic acid-induced gene-I (RIG-I)-like receptors (RLRs) and TLRs, distinct families of pattern recognition receptors that sense nucleic acids derived from viruses [
12]. RIG-I is a double-stranded RNA-binding DExD/H box RNA helicase that is essential for initiating the intracellular response to RNA viral infection [
13]. However, NS3/4A, the major serine protease expressed by HCV, disrupts the RIG-I pathway through proteolysis of essential signaling components of IFN regulatory factor 3 (IRF-3) activation [
13,
14], reducing immune response. Alternatively, the TLR-families play an important role in innate immune responses in mammals [
15]. Among them, TLR3 recognizes viral double-stranded RNA, whereas TLR7 recognize single-stranded RNA. Because some TLRs ligands induce IFN-λ in human macrophages [
16], contributing to antiviral defense and HCV is a single-stranded RNA [
8], we hypothesized that IFN-λ3 induced via the TLR pathway might contribute to early antiviral response against HCV, which could lead to accurate prediction of treatment efficacy. Therefore, we investigated IFN-λs production in peripheral blood mononuclear cells (PBMC) in healthy volunteers or CHC patients by ex vivo stimulation with TLR agonists, and analyzed whether this method could predict the responses to Peg-IFN/RBV treatment in clinical practice.
Patients, materials, and methods
Study population
Blood samples were obtained from 12 healthy volunteers and 100 consecutive Japanese outpatients with CHC (genotype 1b and high viral load) who visited our hospital between April 2011 and March 2012. The study protocol was conformed to the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the ethical committee of our institutes (NCGM-G-001023-01). Written informed consent was obtained from all volunteers and patients. All subjects were negative for HBV and human immunodeficiency virus, and did not have hepatocellular carcinoma. IFN treatment was not being given to any patient at the time blood samples were taken. The subjects were all evaluated for SNP near
IL28B (
rs8099917,
rs12979860) using the InvaderPlus assay (Invader Chemistry, Madison, WI, USA) as previously reported [
17].
Definition of treatment responses
Non-virologic response (NVR) was defined as less than a 2-log-unit decline in the serum level of HCV RNA from the pre-treatment baseline value within the first 12 weeks, and detectable viremia 24 weeks after initiation of treatment. VR was defined as achieving SVR or TVR. SVR was defined as undetectable HCV RNA in the serum 6 months after the end of treatment, whereas TVR was defined as reappearance of HCV RNA in the serum during or after completion of treatment.
Preparation of PBMC and selection of plasmacytoid or myeloid dendritic cells (DCs)
Whole blood anti-coagulated with EDTA was obtained from healthy volunteers and CHC patients. PBMC were isolated by Ficoll-Hypaque (Mediatech, Herndon, VA, USA) density gradient centrifugation. BDCA-1, 3, 4+DCs were negatively or positively selected by BDCA-1+DC isolation kit, BDCA-3 MicroBead kit and BDCA-4/Neuropilin-1 MicroBead kit, respectively (Miltenyi Biotec, Auburn, CA, USA) according to the manufacturer’s instructions.
Ex vivo induction of IFN-λ1, IFN-λ2, and IFN-λ3
After pre-treatment with or without 100 U/ml of IFN-α (Hayashibara Co. Ltd., Okayama, Japan) in 200 μl of Roswell Park Memorial Institutes (RPMI) medium supplemented with 10 % fetal bovine serum for 16 h, 100,000 of mononuclear cells were stimulated with 30 μg/ml of poly I:C (TLR3 agonist; Imgenex, San Diego, CA, USA), or 5 μg/ml of imiquimod (R-837; TLR7 agonist, Imgenex) as previously reported [
16]. For chemiluminescence enzyme immunoassays (CLEIA), 200,000 cells were subjected to the same stimulation protocol.
RNA isolation and cDNA synthesis
After stimulation with TRL-agonists for 4 h, the PBMC were lysed with ISOGEN-II (Nippon Gene, Tokyo, Japan). In some experiments, PBMC were harvested at each indicated time point. The lysate was supplemented with chloroform, incubated for 15 min on ice, and centrifuged at 22,000 g for 15 min. The aqueous layer was removed and precipitated with isopropanol. The RNA was pelleted by centrifugation, washed with ethanol, and dissolved in 20 μl of water. Reverse transcription was performed using the SuperScript III first-strand synthesis system (Invitrogen, Carlsbad, CA, USA).
Real-Time quantitative polymerase chain reaction (PCR)
Quantitative real-time PCR was performed to estimate IFN-λ1, IFN-λ2, and IFN-λ3 mRNA expression based on SYBR green fluorescence (Roche Diagnostics Japan), using TaqMan Universal PCR master mix (Roche Diagnostics Japan), according to the manufacturer’s protocol. Relative gene expression was calculated as a fold induction. Data were analyzed using the 2-∆∆C(t) method with Sequence Detector version 1.7 software (Applied Biosystems, Carlsbad, CA, USA) and were normalized using human hypoxanthine phosphoribosyltransferase (HPRT). A standard curve was prepared by serial 10-fold dilutions of human cDNA. The curve was linear over 7 log units with a 0.998 correlation coefficient. Quantitative mRNA expression was determined by triplicate real-time PCR.
Chemiluminescence enzyme immunoassays
We recently developed a CLEIA system for IFN-λ3 that showed a wide detection range of 0.1–10,000 pg/ml with little or no cross-reactivity to IFN-λ1 or IFN-λ2 [
18]. In addition, this CLEIA system can correctly detect IFN-λ3 from different
IL28B genotypes.
Acoustic radiation force impulse (ARFI) elastography
For non-invasive evaluation of liver fibrosis, ARFI elastography was performed using a Siemens Acuson S2000™ ultrasound system (Mochida Siemens Medical System Co, Ltd, Tokyo, Japan) as previously reported [
19]. We performed 5 measurements for each patient, and a median value was calculated. Liver stiffness was expressed as the shear wave velocity (m/s) and has been reported to be well correlated with histological liver fibrosis [
19].
Statistical analyses
Continuous variables between groups were compared using the Mann–Whitney U test, and categorical data were compared using the Chi square test or Fisher’s exact test. Correlations between continuous variables were searched using the Pearson correlation test. Values of p < 0.05 were considered significant.
Discussion
The present study demonstrated that the amount of endogenous IFN-λ3 in PBMC induced by the TLR7 agonist determines the outcome of Peg-IFN/RBV therapy, though its induction was basically dependent on the
IL28B genotype. Since this method can evaluate the final gene products, the genetic factor or epigenetic factor is not necessary for consideration, which could provide more accurate prediction of the response to Peg-IFN/RBV therapy than
IL28B genotyping. In addition, an annoying informed consent about gene handling is not necessary, and the cost is lower than genomic analyses. IFN-based treatment with novel drugs such as protease inhibitors or polymerase inhibitors could be still predictive by this method because genetic variations in
IL28B are strongly associated with response to telaprevir/Peg-IFN/RBV treatment [
24].
IFN-λs were shown to inhibit the replication of a number of viruses in vitro, including HBV or HCV [
5‐
7], by up-regulation of ISGs. Several GWAS studies suggested that IFN-λ3 played a key role in the response to HCV [
1‐
3,
10,
11]. Meanwhile, type III IFNs, produced by hepatocytes, in response to HCV infection, predominantly lead functional ISGs induction in comparison to type I IFNs [
25]. The combination of
IL28B genotype between recipients and donors determines the outcome of Peg-IFN/RBV therapy for recurrent hepatitis C after liver transplantation [
26]. Therefore, a favorable response to anti-HCV treatment might be dependent on the amount of endogenous IFN-λ3 produced by both lymphocytes and hepatocytes.
Unsuccessful induction of IFN-λs by IFN-α or TLR-agonists alone (Fig.
1a) may explain controversial results of IFN-λ3 expression in either PBMC or intrahepatic lymphocytes from pre-treatment patients [
1,
3,
23,
27]. IFN-α up-regulates the expression of TLR, TRIF, and MyD88, common adaptor molecules associated with TLR signaling [
16]. UV-inactivated viruses, as well as infectious viruses, can induce IFN-λ, suggesting viruses can be sensed through a non-infectious route such as endocytosis of infectious or non-infectious viral particles [
28]. TLR7 appears to play an important role in the induction of antiviral responses against single-stranded RNA viruses [
29‐
31], and imidazoquinoline activates immune cells via the TLR7-MyD88-dependent signaling pathway [
32]. Synthetic TLR7 agonists have recently been characterized with respect to their ability to induce cytokines [
33]. Furthermore, in our study, IFN-λ3 induced by R-837 clearly differentiated the response to Peg-IFN/RBV treatment. Therefore, sequential stimulation with IFN-α following a TLR7 agonist (R-837) may mimic IFN therapy in CHC patients in terms of IFN-λs induction, and our sensitive CLEIA system may contribute to clear differentiation among the responses to Peg-IFN/RBV treatment in this study. It has recently been reported that expression levels and function of TLR7 were impaired in HCV-infected human hepatoma cells [
34]. However, in the present study, a TLR7 agonist was able to induce IFN-λs in PBMC from HCV patients, although the response was slightly impaired in HCV patients compared with that in healthy volunteers. Therefore, impairment of expression or function of TLR7 in HCV patients may not be a critical factor for whole innate immunity.
IFN-λs display high sequence homology. In particular, IFN-λ2 and IFN-λ3 are virtually identical with 96 % amino acid homology [
6]. However, the GWAS revealed that only SNPs near the
IL28B, not
IL28A or
IL29, showed strong associations with response to Peg-IFN/RBV treatment [
1‐
3]. In the current study, we found that the expression level of IFN-λ3 in the PBMCs was better correlated with genetic variations in
IL28B or response to Peg-IFN/RBV treatment. Furthermore, the antiviral effect of recombinant IFN-λ3 was more potent than those of IFN-λ1 or IFN-λ2 [
35]. Collectively, these findings may support our GWAS data. Alternatively, there are significant differences in IFN-λ2 as well as IFN-λ3 between genotypes or treatment response. The specific primers and probe sets for the IFN-lambda family that we previously developed achieved approximately 10
7-fold specificity in each IFN-λ [
18]. However, Osterlund et al. [
36] reported that both IFN-λ2 and IFN-λ3 were regulated by a similar pathway of IRF7 resembling those of IFN-α gene expression. Therefore, it is possible that the upregulation of IFN-λ3 could affect that of IFN-λ2 because the homology of regulatory sequence between IFN-λ2 and IFN-λ3 is pretty high. On the other hand, IFN-λ1 and λ2, λ3 show rather low homology (81 %), which could explain the different responses among them.
About 20 % of the HCV patients with the TT genotype failed to respond to the treatment [
1]. Indeed, in the current study, 8 of 28 (28.6 %) patients with favorable
IL28B genotype (TT) showed NVR in Peg-IFN/RBV therapy. Interestingly, all these cases who showed NVR despite their favorable
IL28B genotype demonstrated low IFN-λ3 production (Fig.
5a–d). Generally, poor response to HCV treatment is related to a number of factors that are unlikely to be solely due to the
IL28B genotype, such as greater age, male gender, viral factors and liver fibrosis [
37,
38]. Therefore, we have tried to calculate multivariate logistic regression to find the most predictive factors affecting the treatment response including clinical backgrounds and the current data. However, the calculations were impossible because of “complete separation” between the treatment-response groups on ELISA data. Liver fibrosis may be attributed to hyporesponsiveness to Peg-IFN/RBV because surrogate markers of liver fibrosis (low platelet counts and high shear wave velocity) were significantly observed in our NVR cases (Table
2). However, both of surrogate markers showed substantial overlap between VR and NVR (Supplementary Fig. 2). Other factors including genetic deficiency of molecules in the TLR7 signaling pathway [
39] may affect IFN-λs production. Meanwhile, 7 of 18 (38.9 %) patients with the unfavorable
IL28B genotype (TG/GG) showed VR in Peg-IFN/RBV therapy (Table
3) and all of these VR patients showed high IFN-λ3 production. Because recombinant IFN-λ from any
IL28B genetic variant similarly represses HCV RNA [
40], the amount of IFN-λ, whatever the genetic variations of
IL28B are, would be important for antiviral effects. Enhanced transcriptional regulations of
IL28B could partially be attributed to high IFN-λ3 production in patients with TG/GG [
41]. Alternatively, the number of BDCA-4
+DCs was well correlated with IFN-λ3 production when PBMC from healthy volunteers was stimulated with R-837 (Supplementary Fig. 3), which suggest that the number of BDCA-4
+DC strongly affect IFN-λ3 production in our setting. However, the precise mechanisms of regulating IFN-λ3 production or the number of BDCA-4
+DCs in CHC patients should be addressed in the future. Importantly, our methods clearly predict the effectiveness of Peg-IFN/RBV including these exceptional cases.
In the present study, neither the mRNA nor the protein levels of IFN-λ3 were much different among genetic variations in
IL28B (Fig.
4c, d). It is possible that the exceptional cases of IFN-λ3 levels (e.g., low IFN-λ3 levels in the TT genotype and high levels in the TG/GG genotype) affected the statistical differences. Substantial overlap in the mRNA levels of IFN-λ3 was observed between the different treatment response groups whereas the protein levels of IFN-λ3 clearly differentiated (Fig.
5a–d). One possibility would be that our newly developed CLEIA system [
18] is more sensitive than real-time PCR. Moreover, IFN-λs mRNA levels were rapidly induced by R-837, and these effects were limited to several hours after stimulation (Fig.
2c) whereas protein levels of IFN-λ3 were stable from 9 to 24 h after R-837 stimulation (Fig.
2d). In the prospective study, there were no statistical differences in the mRNA levels of IFN-λs. However, the increase of the number of these patients would clarify the differences because the trend of these levels in the prospective study was similar to those in the previously treated group.
In conclusion, our findings suggest that genetic variations in IL28B basically affect IFN-λ3 production; however, the amount of endogenous IFN-λ3 determines the outcome of Peg-IFN/RBV therapy. This study, for the first time, presents compelling evidence that genetic variations in IL28B confer a functional phenotype and potentially explains our GWAS data. In addition, these results may explain discrepant cases related to IL28B genotyping in the response to Peg-IFN/RBV treatment. Thus, ex vivo induction of IFN-λ3 in PBMC by a TLR7-agonist may be a more accurate predictive method for determining the outcome of Peg-IFN/RBV therapy.