Xiaogang Xiang

IL-22 and non-ELR-CXC chemokine expression in chronic hepatitis B virus-infected liver

Hepatitis B virus infection is still a major global health problem, despite decades of research. Interleukin (IL)‐22 induces acute phase reactants and chemokines, favors anti‐microbial defence and protects tissues from damage. IL‐22 is important in chronic skin inflammation, but its role in chronic hepatitis B (CHB) is unclear. This study explores the association between intra‐hepatic IL‐22 expression, its relevant associated cytokines and the severity of liver inflammation/fibrosis in CHB patients. IL‐22, IL‐17, IL‐10, IL‐6, non‐ELR‐CXC chemokines (CXCL‐9, CXCL‐10, CXCL‐11), fibroblast growth factors and Kupffer cell (KC) numbers were measured in patients with CHB (n=65), acute hepatitis B (AHB; n=4), chronic hepatitis C (CHC; n=14) and non‐viral hepatitis (n=23), using immunohistochemistry. Expression of IL‐22, IL‐17, IL‐10, IL‐6, non‐ELR‐CXC chemokines and number of KCs in liver tissues were substantially higher in AHB patients than others. In CHB patients, the expression of IL‐22, IL‐6, CXCL‐9 and CXCL‐10 were significantly higher with alanine aminotransferase (ALT) levels ⩽twice the upper limit of normal (ULN), compared with those with ALT levels >twice the ULN, whereas IL‐10 and IL‐17 showed a reverse pattern. IL‐22 was inversely (P<0.01), but IL‐17 was positively (P<0.05), correlated with the histological activity index) in these patients, and a significant negative correlation between the fibrosis stage and IL‐22 or non‐ELR‐CXC chemokines was observed. Furthermore, immunofluorescent labeling demonstrated a close spatial association of IL‐22, CXCL‐9, ‐10 or ‐11 in the CHB liver. We speculate that IL‐22 and non‐ELR‐CXC chemokines synergistically may provide protection in liver inflammation/fibrosis during CHB infection.

Protective effect of Th22 cells and intrahepatic IL-22 in drug induced hepatocellular injury.

BACKGROUND & AIMS Th22 cells regulate host immunity against pathogenic invasion, including protecting host against chronic hepatitis B; however, the relationship between drug induced liver injury (DILI) and Th22/Th17 cells is still unclear. We investigated the role of Th22 cells in DILI development. METHODS The frequencies of peripheral Th22/Th17/Th1 cells and intrahepatic IL-22/IL-17 production from DILI, non-DILI liver diseases, and healthy controls were examined. Plasma IL-22/IL-17 and the related cytokines were determined in DILI patients at week 0 (defined as the occurrence of liver injury within 7days), 4 and 24. Multivariable stepwise logistic regression was applied to explore the associations between various factors and recovery of DILI. RESULTS The frequencies of Th22/Th17 cells were significantly higher in DILI onset patients than HC. Significant increase of Th22 cells and the related cytokines levels was observed in DILI with hepatocellular injury type. There was a positive correlation between intrahepatic IL-22 level and liver regeneration. Plasma IL-22 level was higher in DILI patients with improved liver function than unimproved function. Multivariable analysis showed that the odds ratio (OR) of plasma IL-22 at 4weeks was 1.054 [95% confidence interval (CI), 1.012, 1.124]. CONCLUSIONS Increased peripheral and intrahepatic IL-22-secreting cells are detected in DILI. Th22 and its related cytokines might be hepato-protective, which might provide new perspective for understanding the immunopathogenesis of DILI. Plasma IL-22 might be a reliable indicator to evaluate the prognosis of DILI and provide a novel therapeutic target for DILI treatment.

A Novel Strategy To Develop a Robust Infectious Hepatitis C Virus Cell Culture System Directly from a Clinical Isolate

ABSTRACT Hepatitis C virus (HCV) infection is a leading cause of chronic liver diseases. Progress in the HCV field was greatly enhanced by constructing infectious cDNA clone of JFH-1. Since then, JFH-1-based intra- and intergenotypic recombinants have been developed, and this permitted the study of vaccines and antiviral inhibitors for all genotypes. Recently, highly efficient HCV culture systems have been established by using consensus sequence-based clones. We developed a novel strategy to construct infectious HCV cDNA clone by combining functional screening of sequences directly from a genotype 2a clinical isolate (PR63) and cell culture adaptation. Using JFH-1 cDNA as the starting backbone, we sequentially replaced the JFH-1 fragments with a sequence from the pools of PR63 sequences. Through engineering adaptive mutations that improve HCV infectivity, we finally established a full-length cell culture-derived infectious clone of PR63, named PR63cc, that could efficiently produce virus particles in Huh7-derived cells, with peak titers of 1.6 × 105 focus-forming units/ml. The PR63cc could be neutralized by an anti-E2 antibody and inhibited by antiviral agents but appeared more resistant to an NS5A inhibitor than JFH-1. In summary, we developed a new approach to construct an infectious HCV cDNA clone that can produce viruses efficiently in cell culture. This approach could be applied to other viral isolates, with potential implications for individualized treatments of HCV patients.

Amelioration of liver injury by continuously targeted intervention against TNFRp55 in rats with acute-on-chronic liver failure.

Background Acute-on-chronic liver failure (ACLF) is an acute deterioration of established liver disease. Blocking the TNF (tumor necrosis factor)/TNFR (tumor necrosis factor receptor) 1 pathway may reduce hepatocyte apoptosis/necrosis, and subsequently decrease mortality during development of ACLF. We demonstrated that a long-acting TNF antagonist (soluble TNF receptor: IgG Fc [sTNFR:IgG-Fc]) prevented/reduced development of acute liver failure by blocking the TNF/TNFR1 (TNFRp55) pathway. However, it is still unclear if sTNFR:IgG-Fc can inhibit hepatocyte damage during development of ACLF. Methodology Chronic liver disease (liver fibrosis/cirrhosis) was induced in Wistar rats by repeatedly challenging with human serum albumin (HSA), and confirmed by histopathology. ACLF was induced with D-galactosamine (D-GalN)/lipopolysaccharide (LPS) i.p. in the rats with chronic liver disease. Serum and liver were collected for biochemical, pathological and molecular biological examinations. Principal Findings Reduced mortality was observed in sTNFR:IgG-Fc treated ACLF rats, consistent with reduced interleukin (IL)-6 levels in serum and liver, as well as reduced hepatic caspase-3 activity, compared to that of mock treated group. Reduced hepatic damage was confirmed with histopathology in the sTNFR:IgG-Fc treated group, which is consistent with reduced Bcl-2 and Bax, at mRNA and protein levels, but increased hepatocyte proliferation (PCNA). This is also supported by the findings that caspase-3 production was up-regulated significantly in ACLF group compared to the mock treated group. Moreover, up-regulated caspase-3 was inhibited following sTNFR:IgG-Fc treatment. Finally, there was up-regulation of hepatic IL-22R in sTNFR:IgG-Fc treated ACLF rats. Conclusions sTNFR:IgG-Fc improved survival rate during development of ACLF via ameliorating liver injury with a potential therapeutic value.

Viral sequence evolution in Chinese genotype 1b chronic hepatitis C patients experiencing unsuccessful interferon treatment.

The efficiencies of IFN-α based therapy in chronic genotype 1b HCV patients are still unsatisfied to date. The mechanisms underlining treatment failure remain unclear and controversial. To investigate HCV sequence evolution in unsuccessfully treated genotype 1b patients before, during and after the therapy, full-length open-reading-frame of HCV genomes at week 0, week 48 and year 5 in one breakthrough and one nonresponse patients were amplified by reverse transcription (RT)-nested-PCR and sequenced. Mutations were scored and analyzed according to their locations in the HCV genome. HCV sequences in the breakthrough patient displayed significantly more mutations during the one-year therapy than that in the nonresponse patient, with p7 and NS2 encoding regions having the highest mutation rates. Most of the mutations selected during the therapy phase in the breakthrough patient were maintained and few new mutations arose in the four-year post-therapy phase, suggesting these mutations might not compromise viral fitness. Altogether our data suggest that mutations occurred during the therapy phase in the breakthrough patient are likely driven by the action of interferon and ribavirin, and these mutations may have important effects on the responses to interferon based therapy in genotype 1b HCV patients.

Circulating cell death biomarker: good candidates of prognostic indicator for patients with hepatitis B virus related acute-on-chronic liver failure

Investigations on survival of patients with hepatitis B virus related acute-on-chronic liver failure (HBV-ACLF) are sparse and urgently needed. The current study aimed to evaluate the prognostic value of circulating cell death biomarkers (M30-anigen, M65-antigen and HMGB1) for HBV ACLF. In this prospective study (2/2013–8/2014), 94 patients including 54 HBV-ACLF and 40 chronic hepatitis B (CHB) patients were recruited. 40 healthy controls (HC) were also recruited. HBV-ACLF were followed up for 3 months for short-term mortality. All three biomarkers were significantly elevated in HBV-ACLF compared with CHB or HC. M30- and M65-antigens could significantly discriminate between non-survivors and survivors in HBV-ACLF. However, HMGB1 showed no prognostic value. By Cox regression analysis, M30- and M65-antigens and MELD were identified as independent predictors for short-term mortality. A novel prognostic model, MELD-CD (MELD-cell death) was established based on the multivariate results. The adjusted Harrell’s C-index of MELD-CD was 0.86 (P < 0.001) and was significantly higher (P < 0.001 for all) than the currently used models, MELD (C-index, 0.71, P < 0.001), MELD-NA (0.67, P < 0.001), CTPs (0.61, P < 0.05). Dynamic analyses further confirmed the prognostic utility of M30- and M65-antigen. Future studies are warranted to validate the results.

Impairment of the retinoic acid-inducible gene-I-IFN-β signaling pathway in chronic hepatitis B virus infection

Chronic hepatitis B (CHB) virus infection is caused by compromised host immunity, but the precise underlying mechanism remains unclear. Retinoic acid-inducible gene I (RIG-I) triggers antiviral immunity by inducing interferon-β (IFN-β) production following viral infection. To investigate the role of the RIG-I-IFN-β signaling pathway in monocyte-derived dendritic cells (moDCs) during CHB infection, moDCs were generated by stimulating CD14+ monocytes in vitro. MoDCs from patients with CHB, acute hepatitis B (AHB) and healthy controls (HCs) were challenged with vesicular stomatitis virus (VSV) and the levels of RIG-I, IFN-β promoter stimulator 1 (IPS-1) and IFN-β in the stimulated moDCs were determined. Following 16 h of VSV stimulation, RIG-I expression was reduced by 50% in moDCs from CHB patients and by 70% in moDCs from AHB patients relative to HC moDCs, concomitant with a 20% decrease in IFN-β expression in CHB patients relative to AHB patients and HCs. Additionally, a significant correlation between the RIG-I/IPS-1 ratio and alanine aminotransferase (ALT) level was observed. To further investigate the function of RIG-I in chronic hepatitis B virus (HBV) infection, HepG2 or HepG2.2.15 (HBV-transformed) cell lines were challenged with VSV following RIG-1 transfection. IFN-β induction was suppressed in HepG2.2.15 cells, but was restored following RIG-I transfection. Taken together, these data indicate that compromised moDC function in CHB patients is attributable to an impaired RIG-I-IFN-β signaling pathway, which results in compromised host viral clearance and HBV persistence in a susceptible population.

Persistently elevated circulating Th22 reversely correlates with prognosis in HBV-related acute-on-chronic liver failure.

Hepatitis B virus‐related acute‐on‐chronic liver failure (HBV‐ACLF) is an acute deterioration of liver function on chronic liver disease with immune disorder. Th22 cells and IL‐22 were correlated with inflammatory and autoimmune diseases. However, Th22 cells and IL‐22 in the pathogenesis of HBV‐ACLF remains to be elucidated. It was investigated the correlation between Th22 and prognosis in HBV‐ACLF.

Identification of acetyltransferase genes (HAT1 and KAT8) regulating HBV replication by RNAi screening

BackgroundThe initiation of hepatitis B virus (HBV) replication involves the formation of covalently closed circular DNA (cccDNA) and its transcription into pregenomic RNA (pgRNA) in hepatocyte nuclei. The regulatory mechanism of HBV replication by acetyltransferase is thus far not well understood, but human acetyltransferase has been reported as being involved in the regulation of HBV replication.ResultsDepletion of KAT8 or HAT1 via RNA interference (RNAi) markedly down-regulated HBV-DNA and pgRNA levels in HepG2.2.15 cells, with KAT8 knockdown reducing both HBsAg and HBeAg more than HAT1 knockdown. Consistent with these observations, HBV replication regulators hepatocyte nuclear factor-4-α (HNF4α) and peroxisome proliferator-activated receptor gamma coactivator- (PPARGC-) 1-α were decreased following knockdown of HAT1 or KAT8.ConclusionsThese data suggest that KAT8 or HAT1 regulate HBV replication and may be potential drug targets of anti-HBV therapy.

Algorithm of Golgi protein 73 and liver stiffness accurately diagnoses significant fibrosis in chronic HBV infection.

Serum Golgi protein 73 (GP73) is a potential biomarker for fibrosis assessment. We aimed to develop an algorithm based on GP73 and liver stiffness (LS) for further improvement of accuracy for significant fibrosis in patients with antiviral‐naïve chronic hepatitis B virus (HBV) infection.