Increased apoptosis in HepG2.2.15 cells with hepatitis B virus expression by synergistic induction of interferon‐γ and tumour necrosis factor‐α

Background: Interferon‐γ (IFN‐γ) and tumour necrosis factor‐α (TNF‐α) were thought to be important immune mediators in host defence against hepatitis B virus (HBV) infection. Aims: To examine the synergistic effect of IFN‐γ and TNF‐α on HBV‐expressing HepG2.2.15 cells and its potential mechanisms. Methods: Cell viability was quantitatively measured by 3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐diphenyl tetrazolium bromide assay. Cell morphology was captured using light microscopy. The typical DNA ladder test was performed using agarose gel electrophoresis. HBsAg and HBeAg titre changes were quantified by the enzyme‐linked immunosorbent assay method. Gene expression was analysed using cDNA macroarrays. Results: Interferon‐γ (1000 U/ml) alone or combined with TNF‐α (5 ng/ml) treatment resulted in apoptosis in HepG2.2.15 cells, but no significant apoptosis in the parent non‐virus expressing HepG2 cells. IFN‐γ‐ and TNF‐α‐mediated apoptosis was reduced by lamivudine treatment in HepG2.2.15 cells. IFN‐γ combined with TNF‐α reduced the titre of hepatitis B surface antigen and hepatitis B e antigen in the HepG2.2.15 cell line. For apoptosis‐related gene changes, IFN regulatory factor 1 (IRF‐1) (12.2‐fold), c‐myc (V00568 4.7‐fold, L00058 2.4‐fold) and caspase 7 (2.3‐fold) genes were upregulated in the combination treatment group. Conclusion: Interferon‐γ and TNF‐α play a role in the cell death of HBV‐expressing HepG2.2.15 cells. Expression of HBV leads to IFN‐γ‐ and TNF‐α‐mediated apoptosis in the cells. Increased IRF‐1, c‐myc and caspase 7 gene expression may be responsible for the synergistic induction of apoptosis by IFN‐γ and TNF‐α.     

Disparity of basal and therapeutically activated interferon signalling in constraining hepatitis E virus infection

Hepatitis E virus (HEV) represents one of the foremost causes of acute hepatitis globally. Although there is no proven medication for hepatitis E, pegylated interferon‐α (IFN‐α) has been used as off‐label drug for treating HEV. However, the efficacy and molecular mechanisms of how IFN signalling interacts with HEV remain undefined. As IFN‐α has been approved for treating chronic hepatitis C (HCV) for decades and the role of interferon signalling has been well studied in HCV infection, this study aimed to comprehensively investigate virus–host interactions in HEV infection with focusing on the IFN signalling, in comparison with HCV infection. A comprehensive screen of human cytokines and chemokines revealed that IFN‐α was the sole humoral factor inhibiting HEV replication. IFN‐α treatment exerted a rapid and potent antiviral activity against HCV, whereas it had moderate and delayed anti‐HEV effects in vitro and in patients. Surprisingly, blocking the basal IFN pathway by inhibiting JAK1 to phosphorylate STAT1 has resulted in drastic facilitation of HEV, but not HCV infection. Gene silencing of the key components of JAK‐STAT cascade of the IFN signalling, including JAK1, STAT1 and interferon regulatory factor 9 (IRF9), stimulated HEV infection. In conclusion, compared to HCV, HEV is less sensitive to IFN treatment. In contrast, the basal IFN cascade could effectively restrict HEV infection. This bears significant implications in management of HEV patients and future therapeutic development.     

干扰素α抗病毒活性的实验研究

目的比较不同亚型干扰素α（IFNα2b、IFNα2a、IFNαIb）对JAK-信号转导活化转录因子（STAT）通道中重要信号传导分子STAT1、STAT2、干扰素α受体（IFNAR）、蛋白激酶（PKR）、核糖核酸酶L（RNaseL）表达的影响，研究其抗病毒活性的差别，并评价IFNα抗病毒活性的关键性信号传导分子。方法1000U／ml的不同亚型IFNα分别作用于HepG2细胞后，用逆转录聚合酶链反应（RTPCR）方法检测HepG2细胞内STAT1、STAT2、IFNAR、PKR、RNase L mRNA表达水平。用western blot方法检测细胞内STAT1及IFNAR蛋白表达水平。结果RT-PCR的实验结果：（1）IFNα1b处理组IFNAR、STAT1、STAT2 mRNA的表达水平较IFNα2b组高，两组比较差异无统计学意义。IFNα1b、IFNα2b处理组IFNAR、STAT1、STAT2 mRNA水平明显较IFNα2a组高，差异均有统计学意义，，值分别为5．26、15．6、4．67，P值均〈0．05。（2）IFNα1b、IFNα2b、IFNα2a处理后的PKR表达水平相似，3组比较差异无统计学意义。（3）RNaseL表达量极少，无法比较不同处理组间RNase L mRNA表达水平的差异性。Western blot实验结果：（1）IFNα1b处理组IFNAR、STAT1蛋白水平较IFNα2b处理组高，两者比较差异无统计学意义，IFNα1b、IFNα2b处理组IFNAR、STAT1蛋白水平较IFNα2a处理组明显升高，差异有统计学意义，F值分别为17．7、20．1，F值均〈0．05。结论IFNα2b、IFNα2a、IFNα1b均可促进JAK—STAT信号通道中STAT1、STAT2、IFNAR mRNA及蛋白表达，IFNα1b和IFNα2b作用较强，IFNα2a作用较弱。初步表明，IFNα1b、IFNα2b的抗病毒活性较IFNα2a强，IFNAR、STAT1、STAT2可作为评价IFNα抗病毒活性的关键性指标，而PKR、RNaseL能否作为评价指标有待进一步的实验证实。     

Interferon-α suppressed granulocyte colony stimulating factor production is reversed by CL097, a TLR7/8 agonist

Background and Aim: Neutropenia, a major side‐effect of interferon‐α (IFN‐α) therapy can be effectively treated by the recombinant form of granulocyte colony stimulating factor (G‐CSF), an important growth factor for neutrophils. We hypothesized that IFN‐α might suppress G‐CSF production by peripheral blood mononuclear cells (PBMCs), contributing to the development of neutropenia, and that a toll‐like receptor (TLR) agonist might overcome this suppression. Methods: Fifty‐five patients who were receiving IFN‐α/ribavirin combination therapy for chronic hepatitis C virus (HCV) infection were recruited. Absolute neutrophil counts (ANC), monocyte counts and treatment outcome data were recorded. G‐CSF levels in the supernatants of PBMCs isolated from the patients and healthy controls were assessed by enzyme‐linked immunosorbent assay following 18 h of culture in the absence or presence of IFN‐ α or the TLR7/8 agonist, CL097. Results: Therapeutic IFN‐α caused a significant reduction in neutrophil counts in all patients, with 15 patients requiring therapeutic G‐CSF. The reduction in ANC over the course of IFN‐α treatment was paralleled by a decrease in the ability of PBMCs to produce G‐CSF. In vitro G‐CSF production by PBMCs was suppressed in the presence of IFN‐α; however, co‐incubation with a TLR7/8 agonist significantly enhanced G‐CSF secretion by cells obtained both from HCV patients and healthy controls. Conclusions: Suppressed G‐CSF production in the presence of IFN‐α may contribute to IFN‐α‐induced neutropenia. However, a TLR7/8 agonist elicits G‐CSF secretion even in the presence of IFN‐α, suggesting a possible therapeutic role for TLR agonists in treatment of IFN‐α‐induced neutropenia.     

Amino acid substitutions of hepatitis C virus core protein are not associated with intracellular antiviral response to interferon‐α in vitro

Background: Studies on patients with hepatitis C virus (HCV) of genotype 1b have suggested that amino acids (aa) 70 and/or 91 of the HCV core protein affect the outcome of interferon (IFN)‐α and ribavirin (RBV) therapy, although there are no clear supporting data in vitro. Aims: This study was designed to determine the differences among the antiviral activities of HCV core proteins with various substitutions at aa70 and/or aa91. Methods: The retroviral vectors expressing the HCV core proteins with substitutions of arginine/leucine, arginine/methionine, glutamine/leucine or glutamine/methionine at aa70/aa91 were transiently transfected or stably transducted into an immortalized hepatocyte line (PH5CH8), hepatoma cell lines and an HCV‐RNA replicating cell line (sOR) to evaluate antiviral responses to IFN‐α or IFN‐α/RBV. Sequence analysis was performed using genome‐length HCV‐RNA replicating cells (OR6 and AH1) to evaluate HCV core mutations during IFN‐α treatment. Results: The promoter activity levels of IFN‐stimulated genes in the transiently transfected cells or the mRNA levels of 2′‐5′‐oligoadenylate synthetase in the stably transducted PH5CH8 cells were not associated with the HCV core aa70 and/or aa91 substitutions during IFN‐α treatment. Antiviral responses to IFN‐α or IFN‐α/RBV treatment were enhanced in sOR cells stably transducted with the HCV core, although there were no differences in antiviral responses among the cells expressing different core types. Sequence analysis showed no aa mutations after IFN‐α treatment. Conclusions: Antiviral activities were enhanced by HCV core transduction, but they were not associated with the HCV core aa70 and/or aa91 substitutions by in vitro analysis.     

Differential expression of innate immune response genes in clinical phases of chronic hepatitis B infection

We investigated innate immune gene expression in clinical phases of chronic hepatitis B infection, including immune tolerant (IT), immune active (IA), inactive carrier (IC) and hepatitis B e antigen (HBeAg)‐negative phases, as well as healthy controls. Expression levels of interferon types I, II and III, their receptor subunits, IRFs, TLRs and other IFN‐induced genes in peripheral blood mononuclear cells were compared. Forty HBsAg‐positive treatment‐naïve subjects without co‐infection with HIV, HCV or HDV were enrolled. To complement the viral load, the expression levels of 37 innate immune genes were measured by qPCR. The highest response of the innate immune system was observed in the IT and HBeAg‐negative phases, and the IC phase had the lowest response; 31 of the 37 studied genes reached their maximum mRNA expression levels in the IT and HBeAg‐negative phases, and the minimum expression levels of 23 genes were found in the IC phase. The highest mRNA expression levels of IFNs, IFN receptor subunits, IRFs and TLRs genes in all clinical phases were IFN‐λ2 and 3, IFN‐γR2, IRF7 and TLR7, and the lowest levels of mRNA expression were observed for IFN‐α, IFN‐λR1, IRF8 and TLR2. We conclude that innate immune response genes are expressed differentially among chronic HBV phases, and this difference may help to develop new precise and noninvasive methods to determine the progression of disease in chronic HBV patients.     

Characterization of microRNA expression profiles associated with hepatitis B virus replication and clearance in vivo and in vitro

Background and Aim: Alpha interferon (IFN‐α) is an approved treatment for chronic hepatitis B (CHB). MicroRNA (miRNA) are currently known as a part of IFN‐mediated antiviral defense. We aimed at characterizing the miRNA expression associated with hepatitis B virus (HBV) replication and IFN‐mediated HBV clearance. Methods: We investigated the expression patterns of cellular miRNA induced by HBV replication and/or IFN‐α treatment in HepG2 cells, and also analyzed the miRNA response in peripheral blood mononuclear cells in CHB patients on IFN‐α treatment. The differentially expressed miRNA were verified using quantitative real‐time polymerase chain reaction and an miRNA expression pattern was classified based on the final virological response. Results: A total of 223 miRNA were differentially expressed (> 1.5 folds) between the HepG2.2.15 and HepG2 cells, including 24 highly differentially expressed miRNA (> 5 folds). With 12 h of IFN‐α treatment, 23 totally differentially expressed miRNA were identified in HepG2 cells; whereas only five miRNA were identified in HepG2.2.15 cells. Similar amounts of the miRNA were regulated in patients with HBeAg or non‐HBeAg seroconversion; whereas levels of eight miRNA were significantly differentially expressed between the two groups. Conclusions: HBV replication alters miRNA expression profiles and impairs IFN‐inducible miRNA response in HepG2 cells. The miRNA expression pattern of peripheral blood mononuclear cells in CHB patients with IFN therapy can be associated with their therapeutic outcome.     

The inhibition of TNF‐α‐induced leucocyte apoptosis by melatonin involves membrane receptor MT1/MT2 interaction

The pro‐apoptotic signalling cascades induced by tumour necrosis factor‐alpha (TNF‐α) have been intensively studied in multiple cellular systems. So far, it is known that TNF‐α can simultaneously activate survival and apoptotic cell death responses. The balance between these signals determines the ultimate response of the cell to TNF‐α. Moreover, emerging evidence suggests that melatonin may be involved in the protection of different cell types against apoptosis. Thus, the objective of this study was to evaluate the effect of melatonin on TNF‐α‐induced apoptosis in human leucocytes. Cells were treated with TNF‐α alone or in the presence of cycloheximide (CHX), which promotes caspase‐8 activation by eliminating the endogenous caspase‐8 inhibitor, c‐FLIP. Treatment with TNF‐α/CHX led to apoptotic cell death, as ascertained by annexin V/propidium iodide (PI) staining. Likewise, in the presence of CHX, TNF‐α stimulation produced cFLIP down‐regulation and subsequent caspase‐8 activation, thus directly triggering caspase‐3 activation and causing Bid truncation and subsequent caspase‐9 activation. Conversely, pre‐incubation of cells with melatonin inhibited TNF‐α‐/CHX‐evoked leucocyte apoptosis. Similarly, pretreatment of leucocytes with melatonin increased cFLIP protein levels, thereby preventing TNF‐α‐/CHX‐mediated caspase processing. Blockade of melatonin membrane receptor MT1/MT2 or extracellular signal‐regulated kinase (ERK) pathway with luzindole or PD98059, respectively, abolished the inhibitory effects of melatonin on leucocyte apoptosis evoked by TNF‐α/CHX. In conclusion, the model proposed by these findings is that the MT1/MT2 receptors, which are under the positive control of melatonin, trigger an ERK‐dependent signalling cascade that interferes with the anti‐apoptotic protein cFLIP modulating the cell life/death balance of human leucocytes.     

Inhibition of nuclear factor κB and phosphatidylinositol 3‐kinase/Akt is essential for massive hepatocyte apoptosis induced by tumor necrosis factor α in mice

Background/aims: Tumor necrosis factor (TNF)‐α itself does not induce liver injury in normal mice or hepatocytes. Rather, this event, especially in vitro, is explained by the fact that the TNF‐α/TNF receptor system not only triggers downstream signals leading to apoptosis but also induces an antiapoptotic pathway through the activation of nuclear factor (NF)‐κB. The aim of this study was to determine whether inhibition of antiapoptotic pathways influences the susceptibility of mice to TNF‐α. Here, we focused on the roles of NF‐κB and phosphatidylinositol 3‐kinase (PI3K)‐regulated serine/threonine kinase Akt. Methods: TNF‐α was administered to BALB/c mice after treatment with an adenovirus expressing a mutant form IκBα (Ad5IκB), the PI3K inhibitor wortmannin, or both. Liver injury was assessed biochemically and histologically. The expression of Bcl‐2 family members and caspase activity were examined. Results: In the mice livers, treatment with Ad5IκB or the wortmannin suppressed the activation of NF‐κB or Akt, respectively. Suppression of either NF‐κB or Akt showed a slight increase in transaminase levels and focal liver cell death after TNF‐α administration. However, in mice treated with both Ad5IκB and wortmannin, TNF‐α administration resulted in massive hepatocyte apoptosis and hemorrhagic liver destruction in mice. The combination of Ad5IκB, wortmannin, and TNF‐α markedly increased the activation of caspase‐3 and ‐9, and activated caspase‐8 to a lesser degree, suggesting that TNF‐α‐induced hepatocyte apoptosis is dependent on type II cell death signaling pathway, probably through the mitochondria. Inhibition of the NF‐κB and PI3K/Akt pathways had no effect on expression of Bcl‐2 families. Conclusion: The inducible activation of NF‐κB and constitutive activation of Akt regulate hepatocyte survival against TNF‐α, which occurs independent of Bcl‐2 families.     

Caspase activation during hepatocyte apoptosis induced by tumor necrosis factor‐α in galactosamine‐sensitized mice

Abstract: Background/Aims: To clarify the mechanism of hepatocyte apoptosis induced by tumor necrosis factor‐α (TNF‐α), caspase cascade and ceramide formation were investigated in the liver of D‐galactosamine (GalN)‐sensitized mice treated with TNF‐α. Methods: Seven‐week‐old male BALB/c mice were intraperitoneally injected with 20 mg GalN 30 min prior to the intravenous injection of recombinant mouse TNF‐α (0.5 μg/mouse). Cytochrome c release and processing of procaspases in the liver were analyzed by Western blotting. Activities of caspases were measured using chromogenic peptides as substrates. Ceramide content was determined using Escherichia coli diacylglycerol kinase. Results: Apoptosis of hepatocytes was observed in mice treated with both GalN and TNF‐α (GalN/TNF‐α), but not GalN or TNF‐α alone. Activation of caspases‐9 and ‐3, and cytochrome c release were observed only in liver from mice treated with GalN/TNF‐α. In a cell‐free system, processing of procaspases‐9 and ‐3, and cytochrome c release were observed in the postnuclear fraction of liver obtained from GalN/TNF‐α‐treated mice, but not in that from control mice. Processing of procaspase‐3 was inhibited by a caspase‐9 inhibitor, but not by inhibitor for caspase‐8 or ‐2. In a reconstitution assay system, procaspase‐9 processing occurred, when both cytosol and membrane fractions were obtained from the liver of mice treated with GalN/TNF‐α. Ceramide accumulation was observed only in apoptotic liver and preceded cytochrome c release and caspase activation. Conclusion: Cytochrome c release and caspase‐9 activation are required for the activation of executor caspase‐3 in TNF‐α‐induced hepatocyte apoptosis, but caspases‐8 and ‐2 play, if any, a minimal role. Ceramide may be implicated in this apoptotic process.     

High incidence of type 1 diabetes mellitus during or shortly after treatment with pegylated interferon α for chronic hepatitis C virus infection

Background: Development of diabetes mellitus (DM) during or shortly after treatment with interferon α (IFN‐α) in patients with chronic hepatitis C virus (HCV) infection has been reported sporadically. We prospectively screened for DM during and after IFN‐α therapy for chronic HCV infection. Methods: Blood glucose levels of patients with chronic HCV infection were routinely assessed at all outpatient visits during and after treatment with pegylated‐IFN‐α (Peg‐IFN‐α) and ribavirin (Riba). Results: Between December 2002 and October 2005, 189 non‐diabetic patients were treated with Peg‐IFN‐α/Riba, of whom five developed type 1 DM (2.6%), three type 2 DM (1.6%) and one an indeterminate type of DM. Classical symptoms of DM were present in three patients who developed DM shortly after cessation of Peg‐IFN‐α/Riba. In the other patients, symptoms of DM were either indistinguishable from side effects caused by Peg‐IFN‐α/Riba or absent. Conclusion: Our study showed a high incidence of type 1 DM during Peg‐IFN‐α/Riba therapy for chronic HCV infection. Symptoms of DM may be absent or mistaken for Peg‐IFN‐α/Riba‐associated side effects. To diagnose DM without delay, we propose routine assessment of blood glucose at all outpatient visits during and after Peg‐IFN‐α/Riba treatment in chronic HCV patients.     

Therapeutic effects of cytokine modulator Y-40138 in the rat alcoholic liver disease model

Background and Aim: Inflammatory cytokines, such as tumor necrosis factor‐α (TNF‐α) and interferon‐gamma (IFN‐γ), induce liver injury in the rat alcoholic liver disease (ALD) model. Y‐40138 is known to suppress the pro‐inflammatory cytokines and augment the anti‐inflammatory cytokines. We investigated whether or not Y‐40138 may be effective as a novel immunotherapy in the rat ALD model. Methods: Male Wistar rats were fed Lieber‐DeCarli ethanol liquid diet. The effects of Y‐40138 treatment in the ALD models were assessed by analyzing the serum and the liver tissues. Results: The serum levels of alanine aminotransferase (ALT), TNF‐α, and IFN‐γ, and the liver levels of TNF‐α and IFN‐γ were significantly higher in the ethanol‐fed group than in the pair‐fed group. The immunohistochemistry of the liver TNF‐α and 4‐hydroxynonenal (4HNE), and the expressions of TNF‐α and IFN‐γ mRNA were increased, too. The gene expressions of interleukin‐10 (IL‐10) in the ethanol‐fed group were suppressed as compared with the pair‐fed group. The serum triglyceride (TG) and liver TG were increased, and Oil Red O and α‐smooth muscle actin (α‐SMA) staining showed greater expression by ethanol‐fed feeding. After administration of Y‐40138, enzyme linked immunosorbent assay and real‐time polymerase chain reaction of the liver showed that the increased TNF‐α and IFN‐γ were suppressed, and that IL‐10 was augmented. Moreover, ethanol‐induced lipid accumulation in the liver was suppressed by administering Y‐40138. Conclusions: Y‐40138 decreased the inflammation, fibrosis, oxidative stress, and lipid synthesis, and augmented the anti‐inflammatory cytokines of the liver. These results indicate that the multiple cytokine production modulator, Y‐40138, is a promising novel therapy for ALD.