Objectives: Hepatitis B virus, which mainly affects normal liver function, leads to severe acute and chronic hepatitis, resulting in cirrhosis and hepato­cellular carcinoma, but can be safely treated after liver transplant. Evaluation of determinative biomarkers may facilitate more effective treatment of post­transplant rejection. Therefore, we investigated interferon regulatory factor 1 expression in hepatitis B virus-infected liver transplant patients with and without previous rejection compared with controls.

Materials and Methods: Hepatitis B virus-infected liver recipients were divided into those with (20 patients) and without a rejection (26 patients), confirmed by pathologic analyses in those who had a rejection. In addition, a healthy control group composed of 13 individuals was included. Expression levels of interferon regulatory factor 1 were evaluated during 3 follow-ups after transplant using an in-house comparative SYBR green real-time polymerase chain reaction method. Statistical analyses were performed with SPSS software (SPSS: An IBM Company, version 16.0, IBM Corporation, Armonk, NY, USA).

Results: Modifications of interferon regulatory factor 1 gene expression levels in patient groups with and without rejection were not significant between days 1, 4, and 7 after liver transplant. Interferon regulatory factor 1 mRNA expression levels were down-regulated in patients without rejection versus patients with rejection, although not significantly at day 1 (P = .234) and day 4 (P = .302) but significantly at day 7 (P = .004) after liver transplant.

Conclusions: Down-regulation of interferon regulatory factor 1 gene expression in hepatitis B virus patients without rejection emphasized counteraction between hepatitis B virus replication and interferon regulatory factor 1 production. On the other hand, interferon regulatory factor 1 gene overexpression in patients with rejection may result in inflammatory reactions and ischemic-reperfusion injury. Therefore, a better understanding of the association between interferon regulatory factor 1 and hepatitis B virus pathogenesis in a larger population with longer follow-up is needed.

Key words : IRF, HBV, Rejection

Introduction

Hepatitis B virus (HBV) mainly affects normal liver function after proliferation in hepatocytes. There are approximately 400 million people with chronic HBV infection worldwide. Hepatitis B virus infection leads to acute and chronic hepatitis, which causes end-stage liver disease such as cirrhosis and hepatocellular carcinoma.^1-7 Liver transplant is the final therapeutic approach for these patients; however; acceptance of a transplanted liver is not tolerable or safe over the longer term. In general, immunologic responses in recipients to a liver graft divide patients to acceptance or rejection.

Hepatitis B viral nucleic acid (DNA/RNA) has been recognized by pattern recognition receptors, which are an important part of innate immune response in mammalian cells. Cytosolic pattern recognition receptors and membrane-bound toll-like receptors (TLRs) are 2 types of these receptors.8 The activation of the innate immune system leads to induction of cytokines and chemokines and also initiation of adaptive immune responses. Interferon (IFN) type I or IFN-α/β and IFN type II or IFN-γ are important cytokines in the innate immune response to HBV.^4,9

Studies on hepatoma cell lines and transgenic mouse models have found that IFN types I and II reduce HBV replication. Expression of some adaptor proteins in pattern recognition receptor signaling pathways in hepatoma cell lines decreased steady-state levels of viral mRNAs and restricted HBV replication after affecting the nuclear factor κB signaling pathway. It was reported that intravenous injection of activating ligands of TLR signaling pathways, such as TLR-3-5, TLR-7, and TLR-9, in transgenic mouse can promote IFN-α/β production and effectively prevent HBV replication. Hepatitis B virus replication can also be suppressed by anti-HBV CD8-positive T cells induced by IFN-γ. Reduction of HBV replication can be influenced by interleukin 12 (IL-12) and IL-18, which are induced by IFN-γ and IFNα/β.¹⁰

Interferon regulatory factor 1 (IRF-1) is the first recognized member of the IRF family.^11-13 Interferon regulatory factors belong to the family of regulatory transcription factors that modulate expression of IFN and IFN-related genes. Interferon regulatory factors have crucial roles in the interaction with viral infections, regulation of the immune system, and induction of apoptosis, as well as show antitumor effects. Nine members of the IRF family have been recognized in mammals, with each having a DNA binding domain identifying an IFN-stimulated response element in IRF-inducible genes.^8,14,15 Interferon regulatory factor 1 is a highly conserved and ubiquitous nuclear transcription factor that is expressed in all cell types. This regulatory molecule has a reciprocal relation with the IFN-signaling pathway, counteracting with IFN molecules. Other stimuli, such as double-stranded RNA, cytokines, and hormones, also activate IRF-1 expression, and IRF-1 induces expression of proinflammatory cytokines (tumor necrosis factor α, inducible nitric oxide synthase, IL-12, IL-27), chemokines (CXCL10, CCL5), and major histocompatibility complex class I and II molecules.^16-20

Up-regulation of IRF-1 has been found in animal models of acute rejection in liver,²¹ kidney,²² heart,^23-26 and islet cells.²⁷ Interferon regulatory factor 1 has also played an antiviral role in the innate immune response. Overexpression of this regulatory factor in some viral infections such as vesicular stomatitis virus, encephalomyocarditis virus, Newcastle disease virus,²⁸ HBV,¹⁰ and hepatitis C virus²⁹ was confirmed earlier. Therefore, in this study, we evaluated the expression levels of IRF-1 mRNA in HBV-infected liver transplant patients with and without graft rejection.

Materials and Methods

Patients
Forty-six HBV-infected patients who underwent liver transplant at Namazi Hospital (Shiraz, Iran) were recruited between 2012 and 2014. Three EDTA-treated blood samples were collected from each patient 1, 4, and 7 days after transplant. The buffy coat and plasma of each EDTA-treated blood sample were isolated and stored at -80°C until analyses. Hepatitis B virus-infected patients were divided into those with rejection (20 patients) and those without rejection (26 patients), with rejection confirmed by pathologic analyses. In addition, a healthy control group composed of 13 individuals was included in our study. This study was approved by the Ethical Committee of Shiraz University of Medical Sciences and conducted according to the ethical guidelines of the 1975 Declaration of Helsinki. Recipients received livers from deceased donors based on ABO blood group compatibility. In our transplant center, HLA matching is not routinely done for all liver transplant recipients. Pretransplant simultaneous coinfection with human immuno­deficiency virus and hepatitis C virus were ruled out in all studied patients. For prevention of the recurrence of active HBV infection, an anti-HBV conditioning regimen, which included oral tenofovir (300 mg/d) or oral lamivudine (50-100 mg/d), was administered to all studied chronic HBV-infected recipients after transplant.

Hepatitis B virus diagnosis
Hepatitis B virus infection was confirmed in HBV-infected study patients before transplant. The presence of HBV immunologic markers was indicated in plasma samples of all patients using hepatitis B surface antigen, hepatitis B e antigen, hepatitis B e antibody, and hepatitis B core antibody methods before transplant. The HBV viral load was determined in plasma samples of all HBV-confirmed patients using quantitative real-time polymerase chain reaction.

Laboratory tests
Liver function tests, including alanine transaminase, aspartate transaminase, alkaline phosphatase, total bilirubin, direct bilirubin, albumin, and creatinine levels, were collected for each patient in both groups before transplant and at 1, 4, and 7 days after transplant.

RNA extraction and cDNA synthesis
Total RNA was extracted from each patient’s buffy coat blood samples using RNX Plus (CinnaGen, Tehran, Iran), and cDNA was synthesized using PrimeScript RT Reagent Kit (Takara Bio, Otsa, Shiga, Japan) according to instructions from the manufacturer. The amount, concentration, and purity of total RNA were measured with the use of NanoDrop (Thermo Fisher Scientific Inc., Rockford, IL, USA) at 260/280 nm.

Real-time polymerase chain reaction
The expression levels of IRF-1 in HBV patients with and without rejection were determined using an in-house SYBR green real-time PCR protocol by Step One Plus Real-Time Instrument (ABI, Step One Plus, Foster City, CA, USA). For elimination of test errors, glyceraldehyde-3-phosphate dehydrogenase gene was used as an internal control. The primer sequences were designed for amplification of IRF-1 and glyceraldehyde-3-phosphate dehydrogenase transcripts (NM_002198.2; NM_001289745.1) (Table 1). The PCR mix consisted of SYBR Premix Ex Taq II Bulk (SYBR green mix, RNase H, and dye from Takara Bio), forward and reverse primers (5 pmol), and template cDNA (Table 1). Melting point curves of target and internal control genes were analyzed to confirm the specificity of PCR reactions.

Molecular and antigenic analyses of cytomegalovirus infection
Cytomegalovirus genome was extracted from plasma samples of HBV patients both with and without rejection using Invisorb Spin Virus RNA Mini Kit (Invitek, Birkenfeld, Germany) according to the manufacturer’s instruction. Cytomegalovirus genomic DNA load was analyzed using Gensig Quantitative Real-Time PCR kit (Primer Design, Advanced kit, York House, UK) according to the manufacturer’s instruction as previously described.³⁰ Sensitivity of the real-time PCR assay for detection of cytomegalovirus infection was as few as 10 copies of the viral genome per milliliter of sample.

Detection of active cytomegalovirus infection was performed for both HBV patients with and without rejection using an antigenemia method with CMV Brite Turbo kit (IQ Products, Groningen, The Netherlands) according to the manufacturer’s instruction as previously described.³⁰

Statistical analyses
For evaluation of the IRF-1 gene expression levels, we conducted intragroup and intergroup analyses of HBV liver transplant patients with and without rejection and the control group between 3 time points using the 2-ΔΔCT (Livak) method. Statistical analyses were performed with SPSS software (SPSS: An IBM Company, version 16.0, IBM Corporation, Armonk, NY, USA), and P < .05 was considered significant.

Results

The HBV group without rejection was composed of 26 patients, with 7 female patients (27%) and 19 male patients (73%), who received liver transplants without having acute rejection. Patients in this group ranged from 26 to 74 years old (mean of 51.62 ± 10.6 y). The HBV group with rejection was composed 20 patients, with 3 female patients (15%) and 17 male patients (85%). Patients in this group ranged from 27 to 69 years old (mean of 50.95 ± 10.61 y). The most frequent ABO blood group was O positive in both patient groups. Patient demographics and laboratory liver tests for both groups are presented in Table 2.

Cytomegalovirus coinfections
The presence of each Cytomegalovirus genomic DNA and pp65 antigenemia were negative in all studied liver transplant patients from both groups.

Interferon regulatory factor 1 gene expression in liver transplant patients with hepatitis B virus infection with and without rejection
Expression levels of the IRF-1 gene were compared between and among groups at 1, 4, and 7 days after liver transplant. Expression levels of the IRF-1 gene in HBV patients without rejection were up-regulated at day 4 and down-regulated at day 7 after transplant (Figure 1A). Modifications in the expression levels of the IRF-1 gene in the group without rejection were not significant (P = .54). The results showed that IRF-1 gene expression levels were up-regulated at days 4 and 7 after transplant in HBV patients with rejection (Figure 1B). However, modifications in the expression levels of the IRF-1 gene in the rejection group during the follow-up times were not significant (P = .338).

Comparison between interferon regulatory factor 1 gene expression in liver transplant patients with hepatitis B virus infection with and without rejection and in controls
Interferon regulatory factor 1 gene expression levels were compared among HBV patients with and without rejection and the control group at 3 follow-ups after transplant (Figure 2). Expression levels of the IRF-1 gene was down-regulated but not significantly in HBV patients with rejection compared with HBV patients without rejection at day 1 (P = .372) and day 4 (P = .280) but significantly at day 7 (P = .022) after transplant (Figure 2A). Interferon regulatory factor 1 gene expression levels were down-regulated in HBV patients without rejection compared with controls but not significantly at days 1 (P = .213), 4 (P = .571), and 7 (P = .213) after transplant (Figure 2B). Interferon regulatory factor 1 gene expression levels were up-regulated but not significantly in HBV patients with rejection compared with controls at days 1 (P = .888), 4 (P = .472), and 7 (P = .150) after liver transplant (Figure 2C).

The IRF-1 gene expression level was compared between HBV patients with and without rejection at 3 follows-ups after transplant (Figure 3). Expression levels of IRF-1 mRNA were down-regulated but not significantly in HBV patients without versus HBV patients with rejection at day 1 (P = .234) and 4 (P = .302), with significance shown at day 7 (P = .004) after liver transplant.

Discussion

Hepatitis B virus infection can cause a variety of clinical outcomes, from acute self-limited to fulminant hepatitis. Chronic hepatitis may also lead to end-stage liver disease such as cirrhosis and hepatocellular carcinoma, with need for liver transplant as the final effective therapeutic approach.^1-7 Some recipients experience rejection episodes after allograft surgery. Evaluation of some immunologic biomarkers may facilitate effective management of posttransplant rejection. Evaluation of the actions between a transplant recipient’s immune system and the effects of immuno­suppressive conditioning regimens is possible by analyzing changes in the gene expression of graft-related biomarkers.²¹ Therefore, in this study, we evaluated expression levels of the IRF-1 gene in HBV patients with and without rejection and in a control group at 1, 4, and 7 days after liver transplant. In this study, IRF-1 production was increased in HBV patients without rejection, although not significantly, at day 4 after transplant. This increase perhaps results from transplant surgery or negligible ischemic-reperfusion injury. Interferon regulatory factor 1 production was also decreased but not significantly in HBV patients without rejection at day 7 after transplant, perhaps indicating safe allograft reception and return of the inflammatory expression level to baseline (Figure 1A). On the other hand, expression levels of IRF-1 were up-regulated in HBV patients with rejection at days 4 and 7 posttransplant but not significantly, perhaps indicating results of a promoting rejection process (Figure 1B).

Interferon regulatory factor 1 is a nuclear transcription factor that has a major role in regulating the expression of proinflammatory genes during the inflammatory process, leading to infection and tissue damage. During all phases of allograft transplant, risks of cellular stress and inflammation exist.³¹ Expression of the IRF-1 gene is up-regulated in response to different factors, such as IFN types I and II, cytokines, double-stranded RNA, and hormones.^8,14 During viral infection, the stimulated IFNs activate Janus kinase 1-signal transducer and activator of transcription signaling pathways and induce expression of IRF-1.^8,11-13 From different viral infections, HBV has some strategies to escape from triggering by the innate immune system. Studies of the HepG and PH5CH8 cell lines introduced HBV polymerase as a viral protein that counteracted with the host’s innate immune response after inhibition of pattern recognition receptor signaling. This process suppressed TLR-3-and retinoic acid-inducible gene 1 (RIG-I) signaling pathways by disrupting the interaction between Tank-binding kinase 1/IκB kinase-e and DDX3 DEAD-box RNA helicase. Tank-binding kinase 1 and IκB kinase-e are kinases that phosphorylate and activate IRF-3 and IRF-7, which induce expression of IFN type I-related genes.^1-2,10 In another study, the HBx protein inactivated beta-interferon promoter stimulator 1 in HBV-infected human cell lines and ameliorated degradation of mitochondrial antiviral signaling proteins, which are the adaptor proteins of the RIG-I signaling pathway. Negative effects on production of IFN-β resulted from IFN-β promoter stimulator 1 inhibition and mitochondrial antiviral signaling degradation in these cell lines. Toll-like receptor-activated antiviral response was abolished by HBV virions or viral antigens such as hepatitis B surface antigen and hepatitis B e antigen.^32-34 As a result of suppressing TLR signaling pathway induction of IFN-β, IFN-stimulated genes and transcription factors like IRF-3 and nuclear factor κB were inhibited.^34-36

In HBV-infected hepatoma cell lines and liver biopsies from patients with chronic HBV, the Janus kinase 1-signal transducer and activator of transcription signaling pathway, which induces IFN-α-related genes, was prevented by up-regulation of protein phosphatase 2Ac.13 Up-regulation of 2 inducible IFN-γ genes, IRF-1 and guanylate nucleotide binding protein 2, in both the peripheral leukocytes and liver during acute cellular rejection was confirmed in rat liver transplant using DNA microarray.21 In a mouse model of kidney rejection, induction of IRF-1 expression by IFN-γ had an important role in regulation of major histo­compatibility complex expression and resistance to graft necrosis.²² Studies on heart transplant animal models emphasized the relation between expression levels of IRF-1 and acute cellular rejection. In other animal studies, up-regulation of IFN-γ, IRF-1, and other IFN-related genes and chemokines was found on day 5 after heart transplant rejection.^23-26 Expression of the IRF-1 gene was up-regulated, causing ischemic-reperfusion injury in liver graft cells after activation of the hepatocyte apoptosis pathway and resistance to necrosis.^16,17,19 Transgenic expression of suppressor of cytokine signaling-1 as an inhibitor of IRF-1 resists grafted islets to cell death after treatment with tumor necrosis factor α alone and in combination with IFN-γ.27 A transgenic mouse study suggested that HBV replication was stopped after overexpression of IRF-1, which was induced by increased regional concentration of IFN-α/β or IFN-γ. The anti-HBV clearing role of IFN-γ is related to overexpression of the tripartite motif, which is an IFN-γ-inducible molecule, and up-regulation of IRF-1.^9,21

Similar to other reports, for crosstalk between IFN type I and IRF-1 and for the inhibitory role of HBV proteins in IFN type I production, expression levels of the IRF-1 gene in HBV patients without rejection in our study was less than the other 2 groups at 1, 4, and 7 days posttransplant. The suppressor effect of HBV proteins was more than the activator effect of inflammation on expression of the IRF-1 gene after allograft transplant (Figure 2A and 2B). Unlike HBV patients without rejection, production of IRF-1 was up-regulated in HBV patients with rejection (Figure 2A and 2B). A significant increase in the expression level of IRF-1 at day 7 posttransplant may have induced rejection in HBV patients who had rejection (Figure 3C). Results in our group of HBV patients with infection also paralleled other research findings, which used animal models and cell lines. Despite inhibitory effects of HBV proteins on production of IFN type I, the expression level of the IRF-1 gene was increased. This up-regulation may occur because of the rejection process and elevated levels of inflammatory cytokines and/or ischemic-reperfusion injury after allograft transplant. However, the overexpression of IRF-1 in the rejection group may have an important role in anti-HBV immunity, and the reduced viral load in these patients may have resulted from overexpression of IFN type I.

Conclusions

A significant decrease in the expression levels of the IRF-1 gene in HBV-infected liver transplant patients without rejection compared with HBV patients with rejection emphasized the counteraction between HBV replication and IRF-1 production. Therefore, better discrimination of the role of IRF-1 in HBV pathogenesis in liver transplant patients should be the focus in further basic studies on larger populations with longer follow-up.

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