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Hepatic Metallothionein Expression in Chronic Hepatitis C Virus Infection Is IFNL3 Genotype-Dependent

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Hepatic Metallothionein Expression in Chronic Hepatitis C Virus Infection Is IFNL3 Genotype-Dependent Genes and Immunity (2014) 15, 88–94 & 2014 Macmillan Publishers Limited All rights reserved 1466-4879/14 www.nature.com/gene ORIGINAL ARTICLE Hepatic metallothionein expression in chronic hepatitis C virus infection is IFNL3 genotype-dependent KS O’Connor1, G Parnell1, E Patrick2, G Ahlenstiel3, V Suppiah1,3, D van der Poorten3, SA Read3,4, R Leung1,3, MW Douglas3,4, JYH Yang2, GJ Stewart1, C Liddle3, J George3 and DR Booth1 The IFNL3 genotype predicts the clearance of hepatitis C virus (HCV), spontaneously and with interferon (IFN)-based therapy. The responder genotype is associated with lower expression of interferon stimulated genes (ISGs) in liver biopsies from chronic hepatitis C patients. However, ISGs represent many interacting molecular pathways, and we hypothesised that the IFNL3 genotype may produce a characteristic pattern of ISG expression explaining the effect of genotype on viral clearance. For the first time, we identified an association between a cluster of ISGs, the metallothioneins (MTs) and IFNL3 genotype. Importantly, MTs were significantly upregulated (in contrast to most other ISGs) in HCV-infected liver biopsies of rs8099917 responders. An association between lower fibrosis scores and higher MT levels was demonstrated underlying clinical relevance of this association. As expected, overall ISGs were significantly downregulated in biopsies from subjects with the IFNL3 rs8099917 responder genotype (P ¼ 2.38 Â 10 À 7). Peripheral blood analysis revealed paradoxical and not previously described findings with upregulation of ISGs seen in the responder genotype (P ¼ 1.00 Â 10 À 4). The higher MT expression in responders may contribute to their improved viral clearance and MT-inducing agents may be useful adjuncts to therapy for HCV. Upregulation of immune cell ISGs in responders may also contribute to the IFNL3 genotype effect. Genes and Immunity (2014) 15, 88–94; doi:10.1038/gene.2013.66; published online 16 January 2014 Keywords: IFNL3; metallothioneins; hepatitis C virus; interferon-stimulated genes INTRODUCTION Type I and type III IFNs upregulate the same set of ISGs but 15,16 Approximately 3% of the world’s population has been infected signal through different receptor complexes. However, the with hepatitis C virus (HCV).1 With spontaneous clearance rates of level and timing of upregulation of immediate, early and late ISGs 9,15 only 20–30%, a substantial proportion of these individuals are at appears to be affected by the type of IFN. Although signalling risk of becoming chronic carriers of the virus and developing long- through the same receptor, the various Type 1 IFNs can have very term sequelae, including cirrhosis and hepatocellular carcinoma.2 different outcomes on ISG response in vivo and in vitro. This has The aim of HCV therapy is to achieve a sustained virological been attributed to the different avidity and kinetics of their binding response (SVR), defined as undetectable 6-month post-treatment to the receptor, interacting with different receptor densities and 17 HCV RNA in plasma. A significant number of individuals will fail to other microenvironmental factors. Factors controlling ISG achieve an SVR or develop major side effects from therapy. patterns in response to Type III IFNs have yet to be established, Genome-wide association studies have identified single- as well as how these patterns are affected by context. nucleotide polymorphisms in the vicinity of IFNL3 that are We sought to determine a hepatic ISG signature in HCV-infected predictive of response to pegylated interferon (IFN) alpha and liver biopsies and peripheral blood that is associated with IFNL3 ribavirin therapy in patients with HCV genotype 13–5 and haplotypes. We used the comparison of two methods, gene genotype 4.6 expression microarray and RNA high-throughput sequencing IFNL3 encodes IFN-lambda-3, a member of the type III IFN family (RNA-Seq), to improve specificity in detecting the most signifi- (INFL1, IFNL2 and IFNL3) that are functionally closely related to cantly associated ISGs and confirmed these findings by quantita- Type I IFNs.7,8 Both Type I (for example, IFNA) and Type III IFNs tive PCR (qPCR). induce interferon stimulated genes (ISGs) in HCV-infected cells.9 Before genome-wide association studies, studies had demonstrated that individuals with a non-SVR (NSVR) have RESULTS higher pre-treatment hepatic ISG expression.10,11 Notably, it has Association of IFNL3 genotype and ISG expression in whole blood subsequently been reported that there is a strong association and in liver biopsies between the IFNL3 unfavourable genotype and higher pre- In blood 182 ISGs were detected and in infected liver biopsies 338 treatment hepatic ISG levels.12–14 There is even evidence ISGs were detected. suggesting that hepatic ISG expression may be a better Expression microarray analysis of 32 whole-blood samples predictor of SVR than IFNL3 genotype.12,13 (Cohort 1) revealed a higher expression of ISGs from the 17 1Institute for Immunology and Allergy Research, Westmead Millennium Institute, University of Sydney, Sydney, New South Wales, Australia; 2Department of Mathematics, University of Sydney, Sydney, New South Wales, Australia; 3Storr Liver Unit, Westmead Millennium Institute and Westmead Hospital, University of Sydney, Sydney, New South Wales, Australia and 4Centre for Infectious Diseases and Microbiology, Sydney Emerging infections and Biosecurity Institute, University of Sydney and Westmead Hospital, Sydney, New South Wales, Australia. Correspondence: Dr KS O’Connor, Institute for Immunology and Allergy Research, Westmead Millennium Institute, University of Sydney, Sydney, 2145 New South Wales, Australia. E-mail: [email protected] Received 3 October 2013; revised 11 November 2013; accepted 12 November 2013; published online 16 January 2014 Hepatic MT expression in chronic HCV infection KS O’Connor et al 89 patients who had failed to clear HCV after treatment than 15 who trend towards higher expression in the TG/GG non-responder had cleared virus with pegylated IFN alpha and ribavirin (Table 1; genotype (Figure 1d). In contrast to the liver biopsy samples, P ¼ 1.15 Â 10 À 8 by the Sign test). The HCV-infected blood samples MT1E, MT1G, MT1H or MT1M were not detected by gene (n ¼ 17) exhibited higher ISGs in the favourable rs8099917 expression microarray analysis in peripheral blood. genotype (Table 1; P ¼ 1.00 Â 10 À 4). For rs12979860 a non- significant trend in the same direction was also observed with a greater number of upregulated ISGs seen in the responder Confirmation of metallothionein significance by RNA-Seq and qPCR genotype (CC n ¼ 100) versus the non-responder genotype (TC/TT n ¼ 82). The converse was seen on microarray expression Figure 2 demonstrates t-test values for ISGs from the microarray analysis of the 22 HCV-infected liver biopsies (Cohort 2) examined, cohort plotted against the RNA-Seq subjects (Cohort 3) for rs8099917 TT responders. As expected, the majority of ISGs with highly significant differences demonstrated (Table 1; P ¼ 2.38 À 13 Â 10 À 7). Thus, a greater number of upregulated ISGs were (Po7.8 Â 10 ) plot into the left lower quadrant consistent with detected in the unfavourable genotypes for rs8099917. However, downregulation of ISGs seen in the responder genotype. The right no significant difference was observed for rs12979860 (upregu- upper quadrant represents ISGs upregulated in the responder lated ISGs CC n ¼ 177 and TC/TT n ¼ 161). genotype and a cluster of five MTs are demonstrated (Figure 2). To validate the association between MT expression and rs809917 genotype, we employed qPCR, as a third method, in a Metallothioneins are associated with IFNL3 rs8099917 genotype new cohort of liver biopsy samples (Cohort 4, n ¼ 37) and peripheral blood (Cohort 5, n ¼ 24) samples. MT1F and MT1G Expression microarray analysis of 22 HCV-infected liver biopsies were selected as candidate MTs for qPCR analysis based on earlier (Cohort 2) revealed a statistically significant difference (Po0.05) in findings. Results correlated with RNA-Seq and microarray findings ISG expression between rs8099917 responder (TT) and non- with higher expression of both MT1F and MT1G in liver biopsy responder (TG/GG) genotypes for 22 of the ISGs analysed samples of TT responder genotype (Figure 3) although these (Table 2). Twelve of these showed higher expression and 10 results did not reach statistical significance. There was, however, revealed lower expression in the TT responder genotype. The three most significantly upregulated genes were metallothioneins (MTs), isoforms MT1F, MT1G and MT1H. MT1X and MT1M also featured amongst these 12 genes (Table 2). Given this cluster of Table 2. Correlation of IFNL3 rs8099917 genotype and level of MTs, we extended our analysis to all MTs including those that are expression of ISGs showing significant differences by microarray not in the original ISG list. In liver tissue seven MT1 isoforms were analysis detectable as well as MT2A. All MTs demonstrated higher Fold change TT P-value expression levels in the rs8099917 TT responder genotype and versus TG and GG this was statistically significant in all but two MT genes (Figure1a; Po0.05 for MT1A, MT1H and MT1X and Po0.005 in MT1F and Upregulated rs8099917 MT1G). For rs12979860 a non-significant trend in the same MT1F 1.43 0.0002 direction was observed for all MTs examined with higher MT1G 1.78 0.0005 expression levels seen in the CC responder genotype (data not MT1H 1.69 0.0009 shown). Higher MT levels were also seen for SVRs compared with APOL3 1.086 0.0016 NSVRs, although this only reached statistical significance for MT2A MASTL 1.54 0.0041 MT1X 1.47 0.0047 (Figure 1b; Po0.05). MT1M 1.62 0.0069 MT1A, MT1F, MT1X and MT2A microarray expression data were SLC25A30 1.99 0.0109 available for analysis from peripheral blood microarray data SAA1 1.85 0.0218 (Cohort 1). Comparing HCV-infected subjects (NSVRs) versus ABTB2 1.42 0.0267 those that had cleared virus (SVRs) we detected higher levels SERPINB9 2.70 0.0300 in the infected subjects for all MT1 isoforms and a significantly MAX 1.01 0.0309 higher level for MT2A (Figure 1c; Po0.05).
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