Y.-C. Fan

Implication of Th17 and Th1 Cells in Patients with Chronic Active Hepatitis B

ObjectiveThe pathogenesis of hepatitis B virus (HBV)-associated chronic liver disease is still not fully understood. The immune imbalance of cytokine profile exerts a profound influence on the resolution of HBV infections and HBV clearance. This present study aimed to evaluate the immune status of the peripheral T helper (Th) 17 and Th1 cells in the active patients with chronic HBV infection.Materials and MethodsThirty patients with chronic active hepatitis B were included in our present study. The frequency of peripheral Th 17 cells (CD3+CD8−IL-17+ T cells), Th1 cells (CD3+CD8−IFN-γ+ T cells), and Tc1 cells (CD3+CD8+IFN-γ+ T cells) in chronic hepatitis B (CHB) were analyzed by flow cytometry. The protein and mRNA levels of interleukin-17 (IL-17) and interferon-gamma (IFN-γ) were measured by enzyme-linked immunosorbent assay and quantitative real-time polymerase chain reaction (PCR).ResultsThe percentage of Th17 cells in peripheral blood of CHB patients (1.53 ± 0.52%) was significantly increased than that in normal controls (0.92 ± 0.20%; P < 0.05). In contrast, the percentage of Th1 and Tc1 cells of CHB patients was significantly decreased as compared with that of control group. The frequency of Th17 cells had a negative correlation with Th1 cells, and a positive correlation with serum alanine aminotransferase in CHB patients.ConclusionThe elevated peripheral Th17 cells were obtained in the patient with chronic active hepatitis B, suggesting its potential role in the immune activation of chronic HBV infection.

DNA methylation patterns of peroxisome proliferator-activated receptor gamma gene associated with liver fibrosis and inflammation in chronic hepatitis B

Peroxisome proliferator‐activated receptor gamma (PPAR gamma) is a nuclear receptor that regulates gene expression of inflammatory mediators in liver injury. Hepatitis B virus (HBV) suppresses the PPAR gamma‐mediated transactivation in liver cancerous cell lines. However, the role of PPAR gamma in patients with chronic HBV infection has not fully demonstrated. Our present study was firstly to determine the clinical relevance of peripheral PPAR gamma mRNA levels in chronic hepatitis B (CHB) patients, and then, the DNA methylation of PPAR gamma promoter was investigated. Peripheral blood mononuclear cells (PBMCs) were isolated from 91 CHB patients and 18 healthy controls. The mRNA level of PPAR gamma was determined by quantitative real‐time PCR; meanwhile, the CpG island methylation was assessed by methylation‐specific PCR. CHB patients showed significantly lower mRNA level of PPAR gamma than healthy controls (P = 0.005). The mRNA level was decreased in HBV‐DNA‐positive group than HBV‐DNA‐negative group (P = 0.041). Interaction analysis demonstrated that the DNA methylation pattern was responsible for the suppression of peripheral PPAR gamma transcription in CHB patients (P = 0.003). Furthermore, the hypermethylation of PPAR gamma gene promoter was significantly associated with liver inflammation and fibrosis in CHB. In conclusion, DNA methylation patterns were responsible for the decreased mRNA level of peripheral PPAR gamma in CHB patients. Liver inflammation and fibrosis were found to be associated with hypermethylation of PPAR gamma promoter.

IL-17A but not IL-22 suppresses the replication of hepatitis B virus mediated by over-expression of MxA and OAS mRNA in the HepG2.2.15 cell line.

Interleukin-17A (IL-17A) and interleukin-22 (IL-22), mainly secreted by interleukin-17-producing T help cells (Th17), are pleiotropic cytokines that regulate the biological responses of several target cells, including hepatocytes. Th17 frequency was reported to negatively correlate with plasma hepatitis B virus (HBV) DNA load in patients with HBV infection. Several studies have indicated that cytokines, such as IL-6 and IL-4, are involved in the noncytopathic suppression of HBV replication. We therefore hypothesized that IL-17A and IL-22 might have a potent suppressive effect on HBV replication. In our present study, we analyzed the suppressive effect of IL-17A and IL-22 on HBV replication in the hepatocellular carcinoma cell line HepG2.2.15. IL-17A did not inhibit the proliferation of HepG2.2.15 cells. It decreased the levels of HBV s antigen (HBsAg) and HBV e antigen (HBeAg) in culture medium and the levels of intracellular HBV DNA. By contrast, blockage of IL-17 receptor (IL-17R) increased the levels of HBsAg and extracellular HBV DNA in culture medium and the levels of intracellular HBV DNA. The expression of antiviral proteins, including myxovirus resistance A (MxA) and oligoadenylate synthetase (OAS), was enhanced by IL-17A. IL-22 and anti-human IL-22 receptor (IL-22R) antibody did not change any indexes. We demonstrated that IL-17A effectively suppressed HBV replication in a noncytopathic manner and the over-expression of MxA and OAS mRNA was involved in the suppression of HBV replication by IL-17A.

Elevated expression of tumour necrosis factor‐α‐induced protein 8 (TNFAIP8)‐like 2 mRNA in peripheral blood mononuclear cells is associated with disease progression of acute‐on‐chronic hepatitis B liver failure

Aberrant immunity response contributes to the pathogenesis of acute‐on‐chronic hepatitis B liver failure. Tumour necrosis factor‐α‐induced protein‐8 like‐2 (TIPE2) is a recently identified molecular to maintain immune homoeostasis, but its role in acute‐on‐chronic hepatitis B liver failure (ACHBLF) is unknown. We detected TIPE2 mRNA levels in peripheral blood mononuclear cells (PBMCs) from 56 patients with ACHBLF, 60 chronic hepatitis B patients, 24 healthy controls and analysed its role in disease severity and prognosis. TIPE2 mRNA expression in patients with ACHBLF was higher than that of patients with chronic infection or healthy controls. In patients with ACHBLF, TIPE2 mRNA level was positively correlated with serum total bilirubin, international normalized ratio and model for end‐stage liver disease scores. Furthermore, the level of TIPE2 mRNA was significantly higher in nonsurvivors than survivors in patients with ACHBLF. The mRNA level of TIPE2 gradually decreased week by week in survivors accompanied by recovery from patients with ACHBLF, while its expression sustained at high levels in nonsurvivors. TIPE2 mRNA level after lipopolysaccharide (LPS) stimulation ex vivo in patients with ACHBLF was higher compared with controls and patients with chronic infection. Meanwhile, cytokines ex vivo secreted were measured as a marker of immune activation. After LPS stimulation, interleukin (IL)‐6 and tumour necrosis factor (TNF)‐α mRNA expression were reduced in patients with ACHBLF, and a significantly negative correlation was found between TIPE2 and TNF‐α mRNA levels. In conclusion, our results identified the potential role of TIPE2 in predicting disease progression and prognosis in patients with ACHBLF by negative regulating of cell‐mediated immunity.

The tumour necrosis factor-α-238A allele increases the risk of chronic HBV infection in European populations

Summary.  Tumour necrosis factor‐α (TNF‐α) plays a pivotal role in viral clearance and host immune response to hepatitis B virus (HBV) infection, of which the production capacity in individuals is demonstrated to be influenced by a single nucleotide polymorphism within the promoter region of TNF‐α genes. However, there have been conflicting results reported in previous studies on TNF‐α‐238 and TNF‐α‐863 gene promoter polymorphisms in chronic HBV infection. To derive a more precise estimation of their relationship, we searched Pubmed (January, 1966–August, 2010) and China Biological Medicine Database (January, 1978–August, 2010) and carried out a meta‐analysis involving nineteen studies that included 5245 chronic HBV infection cases and 3181 controls describing G238A genotypes, and eleven studies totalling 3576 cases and 2044 controls describing C863A genotypes. The overall meta‐analysis did not suggest significant associations of TNF‐α‐238 and TNF‐α‐863 gene promoter polymorphisms with chronic HBV infection. However, in subgroup analysis by ethnicity, it indicated that TNF‐α‐238A allele carriers (GA + AA) in European populations had an increased risk of developing chronic HBV infection (OR = 2.22, 95% CI: 1.07–4.58, P = 0.032; OR = 4.46, 95% CI: 1.75–11.38, P = 0.002, respectively), when compared with spontaneous recovered and healthy populations, respectively. However, no significant associations were found in Asian populations in all genetic models. So, we draw the conclusion that the TNF‐α‐238A allele may increase the risk of chronic HBV infection in European populations.

Tumour necrosis factor-α-857T allele reduces the risk of hepatitis B virus infection in an Asian population.

Summary.  Tumour necrosis factor‐α (TNF‐α) plays a pivotal role in hepatitis B virus (HBV) clearance and host immune response determining the chronicity of HBV infection. However, studies of the association between TNF‐α‐857 polymorphism and chronic HBV infection have reported conflicting results. So a meta‐analysis was carried out to draw a more precise conclusion. Pubmed (January, 1966–March, 2011) and the China Biological Medicine Database (January, 1978–March, 2011) were searched using the keywords TNF‐α gene polymorphism in combination with HBV infection without language restriction. Fourteen studies including 4929 chronic HBV infection cases and 2702 controls describing the C857T genotype were included in the meta‐analysis. All fourteen studies focussed on an Asian population. The overall meta‐analysis suggested that TNF‐α‐857T allele reduced the risk of chronic HBV infection in the Asian population (OR = 0.82, 95% CI: 0.71–0.95, P = 0.008) when compared with a spontaneously recovered population. In the sensitivity analyses of the groups obeying Hardy–Weinberg equilibrium (HWE), without the largest study population and without the smallest study population, a similar association was revealed (OR = 0.81, 95% CI: 0.68–0.98, P = 0.043; OR = 0.77, 95% CI: 0.68–0.87, P = 0.0001; OR = 0.81, 95% CI: 0.70–0.95, P = 0.009, respectively). However, when compared with a healthy population, no significant association was found in the Asian population in all groups. So, we draw the conclusion that the TNF‐α‐857T allele reduces the risk of chronic HBV infection in this Asian population.

Aberrant GSTP1 promoter methylation predicts short-term prognosis in acute-on-chronic hepatitis B liver failure

Glutathione‐S‐transferase P1 (GSTP1) methylation has been demonstrated to be associated with oxidative stress induced liver damage in acute‐on‐chronic hepatitis B liver failure (ACHBLF).

Exportin 4 gene expression and DNA promoter methylation status in chronic hepatitis B virus infection.

Exportin 4 (XPO4) is a novel identified candidate tumour‐suppressor gene involved in the pathogenesis of hepatocellular carcinoma (HCC). This study was aimed to determine the clinical features of XPO4 mRNA expression and promoter methylation status in peripheral blood mononuclear cells (PBMCs) of patients with chronic hepatitis B virus (HBV) infection. PBMCs were isolated from 44 HCC, 38 liver cirrhosis (LC), 34 chronic hepatitis B (CHB) patients and 17 healthy controls (HCs). The mRNA level and promoter methylation status of XPO4 were determined by quantitative real‐time RT‐PCR and methylation‐specific PCR, respectively. XPO4 mRNA level of HCC patients was significantly lower compared with LC and CHB patients as well as HCs (all P < 0.01, respectively), and significant differences of the XPO4 mRNA level were found in LC and CHB group than in HCs (LC vs HCs, P < 0.01; CHB vs HCs, P < 0.05). Methylation rate of XPO4 promoter was significantly increased in patients with HCC than in patients with CHB and HCs (both P < 0.05). DNA methylation pattern was responsible for the suppression of XPO4 transcription in the progression of HBV infection (P = 0.000). Furthermore, AFP level was significantly higher in HCC patients with XPO4 methylation than in those without methylation ((8702 ± 15635) μm vs (1052 ± 5370) μm, P < 0.05). In conclusion, transcription of XPO4 gene was gradually decreased and methylation rate of XPO4 promoter was increased with the progression of HBV infection. Methylation status of XPO4 in PBMCs tended to be a noninvasive biomarker to predict HCC and the progression of HBV infection.

Increased peripheral RORα and RORγt mRNA expression is associated with acute-on-chronic hepatitis B liver failure

Summary.  T helper cells17 (Th17) have accurate but inconclusive roles in the pathogenesis of acute‐on‐chronic hepatitis B liver failure (ACHBLF). Retinoic acid‐related orphan receptor γ t(RORγt) and RORα are two lineage‐specific nuclear receptors directly mediating Th17 differentiation. This study was aimed to evaluate the gene expression of RORα and RORγt and their potential role in ACHBLF. Forty patients with liver failure, 30 with chronic hepatitis B (CHB) and 20 healthy controls were studied. The mRNA levels of RORα and RORγt in peripheral mononuclear cells were determined by quantitative real‐time polymerase chain reaction. The frequency of peripheral Th17 cells was determined using flow cytometry. The serum levels of interleukin‐6(IL‐6), transforming growth factor –β (TGF‐β), interleukin‐17(IL‐17), interleukin‐23(IL‐23) and interferon‐γ (IFN‐γ) were measured by enzyme‐linked immunosorbent assay. The frequency of peripheral Th17 cells in patients with liver failure was significantly increased compared to patients with CHB and controls. The peripheral mRNA levels of RORα and RORγt in hepatitis B‐associated acute‐on‐chronic liver failure were significantly higher than in patients with CHB and controls as were the serum levels of IL‐6 and TGF‐β. The serum level of IFN‐γ in patients with acute‐on‐chronic liver failure from HBV was significantly higher than patients with CHB but lower than controls. In patients with acute‐on‐chronic liver failure associated with HBV, RORγt, IL‐6 and IL‐23 were positively correlated with the frequency of Th17 cells, while RORα, TGF‐β and IFN‐γ had no correlation with the latter. The mRNA level of RORγt was positively correlated with model of end‐stage liver disease (MELD) score, but there was no correlation of RORα and MELD score. RORγt plays an important role in the pathogenesis of acute‐on‐chronic HBV‐associated liver failure and might be considered to be a candidate factor consistent with the severity of disease.

Up-regulation of A20 gene expression in peripheral blood mononuclear cells is associated with acute-on-chronic hepatitis B liver failure

Aberrant immunity contributes to the pathogenesis of acute‐on‐chronic hepatitis B liver failure (ACHBLF), and A20 is a newly identified negative regulatory molecule of the immune response. However, no data have been reported for the role of A20 in ACHBLF. This study aimed to investigate A20 mRNA expression in ACHBLF and to determine the potential of A20 as a biomarker for the prognosis of ACHBLF. Quantitative real‐time polymerase chain reaction (qPCR) was used to measure the mRNA expression of A20 in peripheral blood mononuclear cells (PBMCs) from 137 ACHBLF patients, 105 chronic hepatitis B (CHB) and 35 healthy controls (HCs). A secondary cohort with 37 ACHBLF patients was set up as validation data set. The plasma levels of interleukin (IL)‐1β, IL‐6 and IL‐10 were determined using enzyme‐linked immunosorbent assay (ELISA). Receiver‐operating characteristic (ROC) curves were used to determine the predictive value of A20 for the prognosis of ACHBLF patients. A20 mRNA expression in ACHBLF was significantly higher compared with CHB and HCs. In ACHBLF patients, A20 mRNA was closely associated with total bilirubin, albumin, international normalized ratio, prothrombin time activity and model for end‐stage liver disease. Furthermore, A20 mRNA was significantly correlated with IL‐6 and IL‐10. An optimal cut‐off value of 12.32 for A20 mRNA had significant power in discriminating survival or death in ACHBLF patients. In conclusion, our results suggest that the up‐regulation of the A20 gene might contribute to the severity of ACHBLF and A20 mRNA level might be a potential predictor for the prognosis of ACHBLF.