Pretreatment up-regulation of hepatic interferon (IFN)-stimulated genes (ISGs) has a stronger association with the treatment-resistant interleukin (IL)28B minor genotype (MI; TG/GG at rs8099917) than with the treatment-sensitive IL28B major genotype (MA; TT at rs8099917). We compared the expression of ISGs in the liver and blood of 146 patients with chronic hepatitis C who received pegylated IFN and ribavirin combination therapy. Gene expression profiles in the liver and blood of 85 patients were analyzed using an Affymetrix GeneChip (Affymetrix, Santa Clara, CA). ISG expression was correlated between the liver and blood of the MA patients, whereas no correlation was observed in the MI patients. This loss of correlation was the result of the impaired infiltration of immune cells into the liver lobules of MI patients, as demonstrated by regional gene expression analysis in liver lobules and portal areas using laser capture microdissection and immunohistochemical staining. Despite having lower levels of immune cells, hepatic ISGs were up-regulated in the liver of MI patients and they were found to be regulated by multiple factors, namely, IL28A/B, IFN-λ4, and wingless-related MMTV integration site 5A (WNT5A). Interestingly, WNT5A induced the expression of ISGs, but also increased hepatitis C virus replication by inducing the expression of the stress granule protein, GTPase-activating protein (SH3 domain)-binding protein 1 (G3BP1), in the Huh-7 cell line. In the liver, the expression of WNT5A and its receptor, frizzled family receptor 5, was significantly correlated with G3BP1. Conclusions: Immune cells were lost and induced the expression of other inflammatory mediators, such as WNT5A, in the liver of IL28B minor genotype patients. This might be related to the high level of hepatic ISG expression in these patients and the treatment-resistant phenotype of the IL28B minor genotype. (Hepatology 2014;59:828–838)
Interferon (IFN) and ribavirin (RBV) combination therapy has been a popular modality for treating patients with chronic hepatitis C (CHC); however, ∼50% of patients usually relapse, particularly those with hepatitis C virus (HCV) genotype 1b and a high viral load. The recently developed direct-acting antiviral drug, telaprevir, combined with pegylated (Peg)-IFN plus RBV, significantly improved sustained virologic response (SVR) rates; however, the SVR rate was not satisfactory (29%-33%) in patients who had no response to previous therapy. Therefore, IFN responsiveness is still an essential clinical determinant for treatment response to triple (Peg-IFN+RBV+DAA) therapy.
A recent landmark genome-wide association study identified a polymorphism in the interleukin (IL)28B, IFN-λ3) gene that was associated with either a sensitive (major genotype; MA) or resistant (minor genotype; MI) treatment response to Peg-IFN and RBV combination therapy and was characterized by either up- (-u) or down-regulation (-d) of interferon-stimulated genes (ISGs).[3-5] However, the underlying mechanism for the association of this polymorphism and treatment response has not been clarified. Previously, we showed that up-regulation of the pretreatment expression of hepatic ISGs was associated with an unfavorable treatment outcome and was closely related to the treatment-resistant IL28B genotype (TG or GG at rs8099917). It could be speculated that the pretreatment activation of ISGs would repress additional induction of ISGs after treatment with exogenous IFN. However, it is unknown how hepatic ISGs are up-regulated in treatment-resistant CHC patients and why patients with high levels of ISG expression cannot eliminate HCV. Therefore, other mechanisms should be involved in the unfavorable treatment outcome of patients with the treatment-resistant IL28B genotype.
In the present study, we performed gene expression profiling in the liver and blood and compared the expression of ISGs between them. Furthermore, ISG expression in liver lobules and portal areas was analyzed separately using a laser capture microdissection (LCM) method. Finally, we identified an immune factor that is up-regulated in patients with the treatment-resistant IL28B genotype and mediates favorable signaling for HCV replication.
The underlying mechanism for the association of the IL28B genotype with treatment responses to IFN-based therapy for HCV has not yet been clarified. We and others have shown that pretreatment up-regulation of hepatic ISGs was associated with an unfavorable treatment outcome[7, 12, 13] and was closely related to treatment-resistant MI IL28B, compared with treatment-sensitive MA IL28B.
By comparing ISG expression in liver and blood, we found that their expression was correlated in MA patients, but not in MI patients. LCM analysis of ISG expression in CLLs and CPAs showed the loss of the correlation between CLLs and CPAs in MI patients (Fig. 2A). This might be the result of the impaired migration of immune cells into liver lobules that was demonstrated by decreased expression of immune cell-surface markers in CLLs by LCM (Supporting Fig. 5A) and IHC staining (Fig. 2B). Lymphocyte accumulation in the portal area (portal-tract-associated lymphoid tissue; PALT) might be involved in extravasation of lymphocytes from vessels in the portal area, but others demonstrated that DCs appeared in the sinusoidal wall and passed through the space of Disse to PALT, where the draining lymphatic duct is located. There should be an active movement of immune cells between liver lobules and PALT, as reflected by the correlation of ISG expression in CLLs and CPAs in the MA patients of this study.
ISGs were reportedly up-regulated in hepatocytes of treatment-resistant IL28B genotype patients, but were up-regulated in Kupffer cells of treatment-sensitive genotype patients. Our results confirmed these findings; however, we also showed that expression of various immune cell-surface markers, such as those on DCs, NK cells, macrophages, T cells, B cells, and granulocytes, was lower in MI than in MA patients (Supporting Fig. 5). In addition, we showed that expression of various chemokines was also repressed in MI patients, compared to MA patients (Supporting Fig. 4C-F).
Up-regulation of pretreatment chemokine (C-X-C motif) ligand 10/interferon-gamma-induced protein 10 (CXCL10/IP-10) serum levels is also associated with an unfavorable treatment outcome. CXCL10 expression in the liver was significantly correlated with hepatic ISG expression and was higher in nonresponders than in responders (Supporting Fig. 10). Our results support the usefulness of serum CXCL10 for prediction of treatment outcome. Chemokine (C-X-C motif) receptor 3 (CXCR3) expression, a receptor for CXCL10, was inversely correlated with hepatic ISG expression and was significantly lower in MI than in MA patients (Supporting Fig. 10).
The lower number of immune cells in the liver lobules of MI patients would imply the reduced production of IFN from DCs, macrophages, and so on. Correlation analysis showed that hepatic ISGs were mainly associated with type III IFNs (IL28A/B and IL29), but not type I IFNs (IFN-α or IFN-β), although a significant association with IL29 was only observed in MA patients with up-regulated ISGs. This might be related to the high serum ALT levels in MAu patients (Fig. 3). Closer examination of hepatic ISGs and IL28A/B suggested that factors other than IL28A/B might regulate ISG expression in MI patients. During the preparation of this study, IFN-λ4 was newly identified to be expressed in hepatocytes from treatment-resistant IL28B genotype patients. Interestingly, we found a significant correlation between hepatic ISGs and IFN-λ4 in MI patients (Fig. 3C). Moreover, a closer examination of gene expression profiling in MI patients enabled us to detect up-regulation of the noncanonical WNT ligand, WNT5A. RTD-PCR analysis of 168 patients confirmed up-regulation of WNT5A and its receptor, FZD5, in MI patients. Importantly, WNT5A expression was significantly correlated with hepatic ISG expression in MI patients. A recent report showed that WNT5A induces expression of ISGs, increases sensitivity of keratinocytes to IFN-α, and might be involved in the immune response to influenza virus infection. Therefore, we examined the role of WNT5A in hepatocytes. Interestingly, expression of WNT5A and ISGs was well correlated, and knocking down WNT5A using siRNA reduced expression of ISGs in THLE-5b cells (Supporting Fig. 8). Conversely, transduction of Huh-7 cells with WNT5A using a lentivirus system increased expression of ISGs. Despite the increase in ISG expression, WNT5A did not suppress HCV replication, but rather increased it in Huh-7 cells (Supporting Fig. 8). These results were also confirmed by using Huh-7 cells continuously infected with JFH-1. By knocking down or overexpressing WNT5A in Huh7-JFH1 cells, we showed that HCV-RNA was positively regulated by WNT5A (Fig. 5B,C).
WNT5A and its receptor, FZD5, mediate noncanonical WNT signaling, such as planar cell polarity and the WNT-Ca2+-signaling pathway through G proteins. WNT5A reportedly inhibits B- and T-cell development by counteracting canonical WNT signaling. We found that G3BP1, an SG assembly factor, was up-regulated by WNT5A (Fig. 5C). SGs were reportedly formed by endoplasmic reticulum stress, followed by HCV infection, and localized around lipid droplets with HCV replication complexes. G3BP1 contributes to SG formation and increases HCV replication and infection in Huh-7 cells. Moreover, a recent report demonstrated that G3BP1 is a DVL-associated protein that regulates WNT signaling downstream of the FZD receptor. In this study, repression of WNT5A or FZD5 significantly reduced expression of DVL1-3, G3BP1, Mx1, and IFIT1 as well as HCV infectivity in Huh7-JFH1 cells (Fig. 5 and Supporting Fig. 9).
Importantly, we found a significant correlation between WNT5A and G3BP1 expression in liver tissue samples (Fig. 5E). We also found a significant correlation between FZD5 and G3BP1 expression in liver tissue samples (Fig. 5F). Thus, up-regulated noncanonical WNT5A-FZD5 signaling participates in the induction of ISG expression, but preserves HCV replication and infection in hepatocytes by increasing levels of the SG protein, G3BP1. These findings may explain the pathophysiological state of the treatment-resistant phenotype in MI patients.
In this study, we demonstrated impaired immune cell infiltration of the liver in treatment-resistant IL28B genotype patients, and we also demonstrated that up-regulation of hepatic ISGs in treatment-resistant IL28B genotype patients was mediated by multiple factors, including IL28A/B, IFN-λ4, and WNT5A. We found a significant negative correlation between WNT5A and various chemokines in liver of CHC patients (Supporting Fig. 7). Interestingly, WNT5A directly repressed one of these chemokines, CXCL13, a B-lymphocyte chemoattractant, in HCV-infected hepatocytes. These results indicate that loss of immune cells from the liver may be associated with the induction of other inflammatory factors, such as WNT5A, in MI patients, although we did not identify which cells express WNT5A. Further studies are needed to explore their functional relevance in the pathogenesis of CHC.
The authors thank Mina Nishiyama for her technical assistance.