Long-term antifibrotic action of interferon-γ treatment in patients with chronic hepatitis B virus infection

Mannheim, Germany

Long-term antifibrotic action of interferon-γ treatment in patients with chronic hepatitis B virus infection

Yi-Jun Wu, Wei-Min Cai, Qi Li, Yan Liu, Hong Shen, Peter R Mertens, Steven Dooley and Hong-Lei Weng

Mannheim, Germany

BACKGROUND: The first priority in treating fibrosis is to eliminate the causes that result in liver injury, e.g., hepatitis B and C virus. However, in many liver diseases the cause is either unknown or untreatable. The present study was designed to investigate the long-term antifibrotic effect of interferongamma (IFN-γ) treatment in patients chronically infected with hepatitis B virus.

METHODS: A total of 42 patients, 30 treated with IFN-γ and 12 controls, were enrolled from an original clinical trial (Clin Gastroenterol Hepatol 2005;3:819.). Three serial liver biopsies that were obtained at the initiation and end of IFN-γ treatment as well as 4 to 6 years after treatment discontinuation were assessed according to the modified Chevallier scoring system.

RESULTS: Twenty-five out of 30 IFN-γ-treated patients were followed up until 4 to 6 years after the treatment was stopped. However, all controls were excluded from follow-up due to death, loss and elevated virus level within 2 years. Twenty-five IFN-γ-treated patients had stable serum liver function and liver fibrosis indices without any further anti-viral or anti-fibrotic treatment. Improved inflammatory and fibrotic scores were found after nine months of IFN-γ treatment according to the modified Chevallier scoring system (inflammation: 11.8±6.5 at the beginning of IFN-γ treatment vs. 9.2±4.1 after 9 months,P＜0.05; fibrosis: 15.0±7.3 at baseline vs. 12.6±6.8 after 9 months,P＜0.05). Among them, 14 patients accepted a third serial liver biopsy 4 to 6 years after treatment discontinuation, and the fibrotic score was increased (14.2±8.3 vs. 11.9±7.6 after 9 months,P＜0.05).

CONCLUSIONS: Nine-month IFN-γ treatment significantly improves the fibrosis score in patients with chronic HBV infection. The majority of patients demonstrate stable serum biochemical indices and quality of life. However, they do not show a long-term benefit according to histological criteria. Given the limited sample size, long-term IFN-γ treatment regimens should be assessed in further clinical trials.

(Hepatobiliary Pancreat Dis Int 2011; 10: 151-157)

hepatic stellate cells; hepatitis B virus; liver fibrosis; inflammation; interferon-gamma

Introduction

The first priority in treating fibrosis is to eliminate the causes that result in liver injury, e.g., hepatitis B and C virus. However, in many liver diseases the cause is either unknown or untreatable. Antiviral drugs, e.g. pegylated interferons and nucleoside analogues such as lamivudine, adefovir, and entecavir, inhibit but do not completely clear HBV or HCV and are only effective in some patients.[1]Thus, additional therapeutic strategies targeting fibrogenesis are urgently required. During the past two decades, the molecular details of liver fibrogenesis have been delineated and now provide options to develop novel therapies, and preclinical studies indeed support the notion that some approaches have the potential to blunt liver fibrogenesis. However, most of these approaches were not successful in clinical trials.[2]

Interferon (IFN)-γ is physiologically produced by natural killer cells and CD4+Th1-lymphocytes andexerts pleiotropic functions, e.g., it modulates immune responses, defends against cancer and acts as an antifibrogenic by inhibiting the collagen synthesis of fibroblasts.[3-5]In primary cultured hepatic stellate cells, the pivotal extracellular matrix (ECM)-derived cells in liver damage, IFN-γ decreases ECM production and fibrogenesis-associated genes by inhibiting transforming growth factor (TGF)-β signalling.[6,7]The latter is the most important pro-fibrotic cytokine in the liver.[8,9]In addition toin vitrodata, IFN-γ has potential anti-fibrotic effects in different animal models.[6,7,10-13]

Clinically, IFN-γ has been used to treat fibrotic patients with chronic HCV and HBV infection. However the results are controversial. One report based on a large, double-blind, placebo-controlled multicenter trial found no improvement of fibrosis in the liver tissue of 402 patients with chronic HCV infection after 48 weeks of IFN-γ treatment.[14]Another smaller clinical trial based on 89 chronic HBV-infected patients showed significant improvement of fibrosis in the liver.[15]In addition, longterm effects of IFN-γ treatment in fibrotic diseases have not been reported so far. In the present study we followed up 42 patients chronically infected with HBV from an original study[15]over 4 to 6 years (30 IFN-γ-treated and 12 control patients). We assessed the histology and serum biochemical indices of these patients.

Methods

Patient selection and study design

The patients were selected from a clinical trial performed from 1998 to 2003.[15]Details about recruitment of the patients and study design have been described previously.[15]Briefly, inclusion criteria were positivity for HBsAg in serum for at least 6 months, ages varied from 18 to 65 years, and liver biopsy showing hepatic fibrosis stages 2 to 4 according to the Scheuer criteria. All patients obtained diammone-glycyrrhizinate (Foscarnet Sodium; Chiaking Pharmaceutical, Lianyungang, China) and potassium-magnesium aspartate (Panangin; Shangdongquancheng Pharmaceutical, Quancheng, China). Patients in the IFN-γ group received 50 g (106units) recombinant human interferon-gamma 1b (IFN-γ; Clonbiotech, Shanghai, China) intramuscularly daily for the first 3 months and on alternate days for the following 6 months. Patients with hepatitis C or D virus infection, psychosis, pregnancy, lactation, serious heart and/or renal failure were excluded, as were patients with the following therapies within 3 months: IFN-γ or lamivudine, medications prescribed to reduce fibrogenesis such as colchicin, herbal recipe 861 and extracts from Semen percise. Also excluded were patients with decompensated liver disease, e.g. serum albumin ＜3.5 g/dl, serum total bilirubin ＞85.5 μmol/L, serum alanine aminotransferase (ALT) ＞400 U/L and prothrombin activity 60%, history of hepatic encephalopathy, ascites and/or white blood cell counts of less than 4×109/L and/or platelet counts of less than 50×109/L. Immunotolerant or HBeAg seroconverted patients were excluded in this study.

After completion of the original clinical trial, the patients were seen during the first three months, and every 6 to 12 months thereafter, for a mean (SD) followup period of 5.3±0.6 years (median 5.6 years; range 4-5.8 years). Severe liver-related clinical complications were prospectively defined and included the following: death, development of hepatocellular carcinoma, need for orthotopic liver transplantation (OLT), variceal hemorrhage, hepatic encephalopathy and ascites. The time of follow-up was calculated from the end of the original clinical trial until death, need for OLT or the last recorded visit before October 2007. At each visit, the patients received a complete physical examination, and blood was taken for routine liver function tests and examination of hepatic fibrosis indices (hyaluronic acid, laminin, collagen type IV and procollagen type III), hepatitis B serology (HBsAg, anti-HBs, HBeAg, anti-HBe, HBcAg and HBV DNA copy number) as well as whole blood cell and platelet counts.

The 42 patients from the original clinical trial were enrolled in the follow-up investigation. Among them, 12 patients who were only treated with diammone glycyrrhizinate/potassium magnesium aspartate (foscarnet) served as a control group while 30 who received IFN-γ as add-on treatment for nine months as a IFN-γ group.

The study protocol involving human samples was approved by the local Ethics Committee and every patient gave written informed consent. The study protocol adhered to the ethical guidelines ofthe 1975 Declaration of Helsinki.

Histological study

Serial liver biopsies were performed at three time points, i.e., immediately before and after 9-month IFN-γ treatment and between 4 and 6 years after the ending of treatment. Percutaneous puncture was guided by ultrasound. A 15-gauge biopsy needle (100×1.9 mm, Angiomed, Germany) was used and sample length was greater than 2 cm in all patients. After fixation in 4% formaldehyde, paraffin sections of the sample were stained with hematoxylin/eosin, Masson's trichrome, and Sirius red and observed for reticular fibers.

Biopsy samples were independently assessed by two histopathologists who were blinded to the samplingsequence and other clinical details according to the Scheuer[16]and the modified Chevallier fibrosis and Knodell inflammation scoring systems as reported previously.[15]A consensus was achieved by collective discussion.

Serum hepatic fibrosis indices, liver function and HBV serology assay

The indices of serum hepatic fibrosis were hyaluronic acid, laminin, collagen type IV and procollagen type III, which were determined by radioimmune assays from Shanghai Navy Medical Institute. Serum values for ALT, AST, albumin and bilirubin were determined by standard clinical assays.

HBV copy numbers were determined by PCR as described,[17]providing a linear range between 1×103and 5×107copies (PG Biotech, Shenzhen, China).

Statistical analysis

The data were expressed as means for rank data and included standard deviation for quantitative data. Analysis for statistical significance was made according to the Wilcoxon's rank-sum test.Pvalues ＜0.05 were considered statistically significant.

Results

Patient characteristics and follow-up

The 30 patients in the IFN-γ group and 12 patients in the control group from the original clinical trial[15]were enrolled in this follow-up study. Baseline clinical characteristics revealed no significant differences inage, gender, serum ALT and AST, serum bilirubin and albumin, HBV serology and histological changes (Table 1).

Table 1. Patient characteristics at initiation of IFN-γ treatment

After the clinical trial, the 42 patients were followed up. In the IFN-γ group, 3 patients died within 4 to 6 years after treatment (2 died of hepatocellular carcinoma and 1 of acute myocardial infarction). Two patients were subjected to lamivudine treatment 4 years after treatment due to the presence of HBV DNA in serum samples and increasing ALT values. These 5 patients were excluded from analysis of serum biochemistry and fibrosis indices. None of the remaining 25 patients received anti-fibrotic and/or anti-viral treatment until the end of follow-up. In the control group, 4 patients died of hepatocellular carcinoma within 3 years after the clinical trial. Six patients accepted lamivudine treatment because of elevated ALT levels and HBV DNA serology. Two patients were lost after a 2-year follow-up. All 12 of these patients were excluded from the follow-up.

IFN-γ treatment ameliorates disease severity in HBV patients

A previous study demonstrated significant histological improvement in the degree of inflammation and fibrosis in 54 IFN-γ-treated patients compared with 29 control patients.[15]The current investigation investigated patients from the original study.

The Scheuer and modified Chevallier scoring systems were used to enroll fibrotic patients before treatment. The advantages and disadvantage of the two systems were described previously.[15]The present study used the modified Chevallier scoring system to analyse histological changes in serial liver biopsies.

Improved inflammatory and fibrotic scores were found after 9-month IFN-γ treatment according to the modified Chevallier scoring system (inflammation: 11.8± 6.5 at the beginning of IFN-γ treatment vs. 9.2±4.1 after 9-month,P＜0.05; fibrosis: 15.0±7.3 at baseline vs. 12.6±6.8 after 9-month,P＜0.05). Ten of 20 patients (50%) improved and only 3/30 (10%) had a worsened hepatic fibrosis score after IFN-γ treatment. However, the controlpatients showed increased inflammation in the liver (Fig. 1 and Table 2).

Table 2. Histological changes before and after nine-month treatment in the IFN-γ and control groups according to the modified Chevallier scoring system

No histological benefit 4 to 6 years after IFN-γ discontinuation

Fourteen patients (46.7%) in the IFN-γ group accepted a third liver biopsy 4 to 6 years after treatment, whereas none of the patients in the control group accepted a third liver biopsy 4 years later. Five patients accepted further anti-viral treatment within 2 years and were excluded from further investigation.

The 14 patients who accepted the third liver biopsy felt well and did not display any adverse symptoms and signs. Inflammation improvement was observed in liver tissue compared to the ending of treatment (from 8.8± 4.5 to 8.1±4.9) though there was no significant difference. However, the hepatic fibrosis score increased as assessed by the modified Chevallier scoring system (from 11.9± 7.6 to 14.2±8.3,P＜0.05, Table 3). Only 3 patients (21.4%) showed an improved hepatic fibrosis score by the third biopsy, 3 (21.4%) had no change in score, and 8 (57.1%) had worsened score 4 to 6 years after IFN-γ treatment in contrast to immediately after IFN-γ treatment (Fig. 1). The results suggest that 9-month IFN-γ treatment does not ameliorate liver fibrosis in patients chronically infected with HBV in a long period.

Effect of IFN-γ on compensated cirrhotic patients

In the IFN-γ group, 9 patients (30%) with compensated cirrhosis (stage 4) were enrolled in this study. Improved inflammatory and fibrotic scores were found after 9-month IFN-γ treatment assessed by the modified Knodell and Chevallier scoring system (inflammation: 8.9±4.7 after 9-month vs. 11.2±6.2 at baseline,P＜0.05; fibrosis: 16.9±5.3 after 9-month vs. 19.7±8.4 at baseline,P＜0.05) (Table 4). Eight of the 9 patients (88.9%) improved and 1/9 patients (11.1%) showed no change in the hepatic fibrosis score after IFN-γ treatment (Fig. 1).

Fig. 1. Detailed fibrosis and inflammation scores at different time points in 30 IFN-γ-treated and 12 control patients with chronic HBV infection. A: Inflammatory scores in IFN-γ-treated and control patients at different time points. B: Fibrotic scores in IFN-γtreated and control patients at different time point. *, HBeAg seroconversion 4 or 6 years after IFN-γ treatment stopped; #, died due to hepatocellular carcinoma; **, died due to myocardial infarction; △, obtained lamivudine treatment. These patients were excluded from further analysis of serum biochemical and fibrosis indices.

Table 3. Histological changes in IFN-γ group (n=14) after ninemonth treatment and 4 to 6 years after treatment stopped according to the modified Chevallier scoring system

Table 4. Histological changes in 9 patients with compensated cirrhosis after nine-month IFN-γ treatment according to the modified Chevallier scoring system

Three patients died during the follow-up. These were patients with compensated cirrhosis. In the remaining 6 patients, 3 accepted a third liver biopsy, which was taken between 4 and 6 years after IFN-γ treatment. Nonsignificant inflammation improvement (from 8.7±1.5 to 7±0) was observed in liver biopsies. However, the hepatic fibrosis score increased as assessed by the modified Chevallier scoring system (from 16.5±4.8 to 21.5±8.4,P＜0.05) 4 to 6 years after IFN-γ treatment (Table 5). One patient (patient 1 in Fig. 2) had improved and 2 (patients 2 and 3 in Fig. 2) had worsened hepatic fibrosis scores 4 to 6 years after IFN-γ treatment.

Fig. 2. Histological changes in 3 representative patients with compensated cirrhosis before and after IFN-γ treatment and 4 to 6 years after treatment stopped. Sirius red staining.

Table 5. Histological changes in 3 patients with compensated cirrhosis in IFN-γ groups after 4 to 6 years after treatment stop according to the modified Chevallier scoring system

Table 6. Serum ALT, AST and fibrotic indices at different time points

Serum hepatic fibrosis indices, liver function tests, and HBV serological assay

The original study revealed that the serum hepatic fibrosis indices, hyaluronic acid, collagen type IV, laminin and pro-collagen type III decreased significantly after 9-month of IFN-γ treatment in the IFN-γ group compared with the control group.[15]Unfortunately, we lost all control patients within 2 years. We could only follow up changes in serum fibrosis indices in the IFN-γ group.

Four to 6 years after treatment, the serum levels of these indices were still lower than those before IFN-γ treatment. There were similar results in serum ALT levels (Table 6).

At the beginning of the study, 21 patients (70%) in the IFN-γ group were positive for HBeAg. We identified only one patient who converted from HBeAg-positive to anti-HBe-positive and from HBV DNA-positive to HBV DNA-negative in a 9-month period of IFN-γ treatment. The mean values of HBV DNA before and after IFN-γ treatment were 5.99±1.21×105and 5.78±1.84×105copies/ml (P＞0.05). Unexpectedly, the HBV DNA value decreased to 1.89±0.32×104copies/ml (P＜0.01, compared with the value before treatment) 4-6 years after treatment. Five of 18 patients (27.8%, 3 HBeAg-positive patients died) converted from HBeAg-positive to anti-HBe-positive and from HBV DNA-positive to HBV DNA-negative during the follow-up for 4 to 6 years. In the control group, none of the patients showed seroconversion from HBeAgpositive to anti-HBe-positive and from HBV DNA-positive to HBV DNA-negative after treatment. On the contrary, 5 patients (41.7%) accepted anti-virus treatment because of significantly elevated HBV-DNA levels.

Discussion

IFN-γ is a potential anti-fibrotic cytokinein vitroand in animal models.[6,7,10-13]However it is controversial whether IFN-γ treatment benefits fibrotic patients with chronic liver disease. In the early 1990s, the anti-viral effect of IFN-γ was tested by comparing it with IFN-γ in a small cohort of patients chronically infected with HCV.[18]IFN-γ did not show any anti-viral effect in these patients. However, this cytokine reduced the degree of liver fibrosis in some HCV patients. A subsequent pilot study focusing on the antifibrotic effect of IFN-γ was performed in 20 chronic HCV patients.[19]Six patients (30%) showed a more than 1% reduction of absolute fibrotic score in liver tissue. These data led to a large, double-blind, placebocontrolled multicenter trial, in which 502 patients with chronic HCV infection were enrolled.[14]Unfortunately, serial liver biopsies based on 420 patients revealed noreduction in the Ishak fibrotic score between IFN-γ treated and control patients. However, subgroup analysis showed that HCV-infected patients with ＞60% induction of serum interferon-inducible T-cell by 24 weeks of IFN-γ treatment had a clearly better outcome of liver histology. This subgroup of HCV-infected patients might be an IFN-γ-responsive population.

In contrast to the study of chronic HCV patients, we performed a randomized, open-label multicenter trial in 315 patients with chronic HBV infection.[15]All enrolled patients were treated with diammone glycyrrhizinate/ potassium magnesium aspartate (foscarnet). Among them, 194 patients received IFN-γ as an add-on treatment for 9 months. Eighty-three patients accepted liver biopsies pre- and post-treatment, including 54 patients treated with IFN-γ. Histology and serum indices showed a better outcome in patients receiving IFN-γ treatment according to the modified Chevallier scoring system.[15]

The above conflicting trials suggested that an IFN-γ response might occur in some fibrotic patients. For instance, although the large clinical trial of Pockros and colleagues did not find improvement of liver fibrosis in patients with chronic HCV infection, they reported that most patients (84%) were cirrhotic at baseline, representing a difficult-to-treat group.[14]In addition to the phase of disease, etiology is a crucial factor for liver fibrogenesis and disease treatment.[8]One pivotal anti-fibrotic mechanism of IFN-γ in damaged liver is the inhibition of TGF-β signalling.[20]Albeit TGF-β is commonly recognized as the most important pro-fibrotic cytokine,[8,9]and its role in different chronic liver diseases is etiology-dependent.[5,21]A recent study investigated the correlation between phospho-Smad2, a major active marker of TGF-β downstream signalling, and liver fibrotic stage in different chronic liver diseases, including chronic hepatitis B and C.[22]TGF-β/phospho-Smad2 showed a remarkable correlation with liver fibrotic stage in chronic HBV patients, but not in patients with chronic HCV infection.[22]The study suggested an etiologydependent mechanism in liver fibrogenesis. The finding may partly explain why IFN-γ is more effective in HBV-infected patients than in those infected with HCV.

The aim of the current study was to investigate the long-term effect of IFN-γ in fibrotic patients chronically infected with HBV. A subgroup of HBV-infected patients from a previous clinical trial 15 was followed up for at least 4 years to determine the long-term anti-fibrotic effect of IFN-γ. Based on 14 patients who accepted a third liver biopsy, 9-month IFN-γ treatment was not effective in controlling the degree of fibrosis of liver tissue in most of the patients in a long period. However, compared with control patients, the IFN-γ treated patients displayed several aspects of positive response even 4 to 6 years after treatment. First, most of the IFN-γ treated patients (25/30) felt well and survived as healthy people. They did not receive further anti-fibrosis and/or anti-viral treatment. Among them, 14 patients (56%) were willing to accept a third biopsy. In contrast to the IFN-γ-treated patients, the control patients were excluded from a further follow-up for 2 years. Besides 4 deaths and 2 lost patients, the remaining 6 patients had to accept anti-viral treatment because of elevated virus levels. Second, serum hepatic fibrosis indices and liver function remained within the normal range in the IFN-γ-treated patients 4 to 6 years after treatment. On the contrary, the control patients showed increased serum liver function and fibrosis indices soon after treatment. Third, although a deterioration of the fibrosis score was found in 8 patients after treatment, an improvement was found in the inflammation score, as assessed by the modified Knodell system. The results suggest that IFN-γ does not maintain a long-term histological effect in patients with chronic HBV infection; however, IFN-γ treatment improves their quality of life, symptoms, signs and serological indices.

Clinical trials did not show the anti-viral effect of IFN-γ treatment.[23]Immediately after 9-month IFN-γ treatment, seroconversion occurred only in one of the 30 patients. Unexpectedly, serum HBV DNA levels decreased significantly shown by extended follow-up and 5/18 HBeAg-positive patients converted from HBeAg-positive to anti-HBe-positive and from HBV DNA-positive to HBV DNA-negative, indicating an anti-viral effect of IFN-γ because spontaneous and treatment-induced HBeAg seroconversion is infrequent in the immune tolerance phase (less than 5% per year).[24]Such an effect is in line with experimental studies performedin vitroandin vivodemonstrating a significant anti-HBV effect of IFN-γ.[25,26]The anti-fibrotic effect of IFN-γ is mediated by inducing stellate cell apoptosis and cell cycle arrest. Recent studies[27,28]also suggest an additional mechanism of the anti-fibrotic effect of IFN-γ that is mediated by activating natural killer cell killing of stellate cells. However, the precise molecular mechanism acting over time needs to be defined. Because of the limited sample in the current study, further clinical studies are needed to address this question.

Acknowledgements

We are grateful to Drs. Tai-Lin Wang and Jia-Lin Chen for histological evaluation, and Mr. Rong-Hua Liu for technical assistance.

Funding: The study was supported by Else-Kr?ner Fresenius (H.L.W., S.D.), Returned Overseas Chinese Scholars, State Education Ministry (SRF for ROCS, SEM, J20050337491010-G50523), China (H.L.W), Zhejiang Administration of Traditional Chinese Medicine (2009 CA053).

Ethical approval: Not needed.

Contributors: WHL proposed the study. WYJ and WHL wrote the first draft. LY and SH analyzed the data. All authors contributed to the design and interpretation of the study and to further drafts. WHL is the guarantor.

Competing interest: No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

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Received September 16, 2010

Accepted after revision January 17, 2011

Author Affiliations: Department of General Surgery (Wu YJ) and State Key Laboratory for Diagnosis and Treatment of Infectious Diseases (Cai WM), First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou 310003, China; Molecular Hepatology-Alcohol Dependent Diseases, II. Medical Clinic Faculty of Medicine at Mannheim, University of Heidelberg, Mannheim 68167, Germany (Li Q, Liu Y, Shen H, Dooley S and Weng HL); Department of Nephrology and Hypertension, Otto-von-Guericke-University, Magdeburg 64153, Germany (Mertens PR)

Hong-Lei Weng, PhD, Molecular Hepatology-Alcohol Dependent Diseases, II. Medical Clinic Faculty of Medicine at Mannheim, University of Heidelberg, Mannheim 68167, Germany (Tel: 49-621-3832940; Fax: 49-621-3831467; Email: honglei.weng@medma.uniheidelberg.de)

? 2011, Hepatobiliary Pancreat Dis Int. All rights reserved.