Meifang Han

Acute liver failure: mechanisms of immune-mediated liver injury

Acute liver failure (ALF) is a syndrome of diverse aetiology, including hepatic encephalopathy, renal, cardiac and pulmonary failures, which result in a rapid loss of hepatic function. The mechanisms of liver injury contributing to ALF can be summarized into two categories: direct damage and immune‐mediated liver injury. This review summarizes current concepts of immune‐mediated liver injury from both clinical studies and animal models. We highlight immune responses of ALF from the liver injury perspective, which combines a variety of molecular and cellular mechanisms, particularly, the contribution of cytokines and the innate immune system. Hepatic and circulating inflammatory cytokines play a significant role in the pathophysiology of ALF including hepatocyte necrosis, extrahepatic complications and hepatocyte regeneration. Overproduction of cytokines, if unchecked, is hazardous to the host and may cause severe outcomes. Measuring pro‐inflammatory cytokines in ALF may be of value for predictors of outcome. Innate and adaptive immune systems both involved in ALF contribute to immune‐mediated liver injury. The innate immune response is activated much more rapidly compared with adaptive immunity, particularly in acute liver injury where the host has little time to trigger an effective adaptive immune response. From this point of view, the innate immune system may make a more profound contribution than the adaptive immune system. Furthermore, immune responses crosstalk with other physiological or pathophysiological factors, for example, coagulation factors which in turn determine the outcome of ALF and these are discussed.

Switching from entecavir to PegIFN alfa-2a in patients with HBeAg-positive chronic hepatitis B: A randomised open-label trial (OSST trial)

BACKGROUND & AIMS Durable post-treatment response is uncommon in chronic hepatitis B (CHB) patients on nucleos(t)ide analogue therapy. Response, response predictors and safety were assessed in patients who switched from long-term entecavir (ETV) to peginterferon alfa-2a. METHODS Hepatitis B e antigen (HBeAg)-positive CHB patients who had received ETV for 9-36 months, with HBeAg <100 PEIU/ml and HBV DNA ⩽1000 copies/ml, were randomised 1:1 to receive peginterferon alfa-2a 180 μg/week or ETV 0.5mg/day for 48 weeks. The primary endpoint was HBeAg seroconversion at week 48 (ClinicalTrials.gov: NCT00940485). RESULTS 200 patients were randomised; 197 received ⩾1 study drug dose. Five patients who were anti-HBe-positive at baseline were excluded from the modified intention-to-treat population (peginterferon alfa-2a, n = 94; ETV, n = 98). Patients who switched to peginterferon alfa-2a achieved higher week 48 HBeAg seroconversion rates vs. those who continued ETV (14.9% vs. 6.1%; p = 0.0467). Only patients receiving peginterferon alfa-2a achieved HBsAg loss (8.5%). Among peginterferon alfa-2a-treated patients with HBeAg loss and HBsAg <1500 IU/ml at randomisation, 33.3% and 22.2% achieved HBeAg seroconversion and HBsAg loss, respectively. Early on-treatment HBsAg decline predicted response at week 48; highest rates were observed in patients with week 12 HBsAg <200 IU/ml (HBeAg seroconversion, 66.7%; HBsAg loss, 77.8%). Alanine aminotransferase elevations were not associated with viral rebound (n = 38). Peginterferon alfa-2a was well-tolerated. CONCLUSIONS For patients who achieve virological suppression with ETV, switching to a finite course of peginterferon alfa-2a significantly increases rates of HBeAg seroconversion and HBsAg loss. A response-guided approach may identify patients with the greatest chance of success.

Hepatitis B Virus-induced hFGL2 Transcription Is Dependent on c-Ets-2 and MAPK Signal Pathway

Fibrinogen-like protein 2 (FGL2)/fibroleukin plays a pivotal role in the pathogenesis of experimental and human fulminant and chronic viral hepatitis. To define the transcription factor(s) and upstream signal transduction pathways involved in the transcription of human FGL2 (hFGL2) in response to hepatitis B (HB) virus, hepatitis B core (HBc), hepatitis B virus S protein (HBs), or hepatitis B virus X protein (HBx) protein, expression plasmids were cotransfected with an hFGL2 promoter luciferase reporter construct into Chinese hamster ovary and HepG2 cells, respectively. HBc and HBx proteins, but not HBs protein, enhanced hFGL2 transcription in both cell lines. A strong regulatory region from -712 to -568 (relative to the transcriptional starting site) was shown to be responsible for hFGL2 gene transcription in response to both HBc and HBx proteins. c-Ets-2 was shown to be translocated to the nucleus in association with hFGL2 expression in response to both HBc and HBx proteins. Short hairpin RNA (shRNA) interference of c-Ets-2 expression inhibited hFGL2 gene transcription by 64.8 and 60.0% in response to HBc and HBx, respectively. c-Ets-2 protein was highly expressed in peripheral blood mononuclear cells from patients with severe chronic hepatitis B (CHB) in contrast to patients with mild CHB. Increased phosphorylation of ERK and JNK was detected in peripheral blood mononuclear cells from patients with severe CHB. ERK inhibitor PD098059 or ERK shRNA abolished the nuclear c-Ets-2 DNA binding activity and hFGL2 induction in response to HBc, whereas JNK inhibitor SP600125 or JNK shRNA abolished the nuclear c-Ets-2 DNA binding activity and hFGL2 induction in response to HBx. In conclusion, HBc and HBx proteins enhance transcription of hFGL2 through c-Ets-2 dependent on MAPK signal pathways.

Negative regulation of virus-triggered IFN-β signaling pathway by alternative splicing of TBK1

Induction of Type I IFNs is a central event in antiviral responses and must be tightly controlled. The protein kinase TBK1 is critically involved in virus-triggered type I IFN signaling. In this study, we identify an alternatively spliced isoform of TBK1, termed TBK1s, which lacks exons 3–6. Upon Sendai virus (SeV) infection, TBK1s is induced in both human and mouse cells and binds to RIG-1, disrupting the interaction between RIG-I and VISA. Consistent with that result, overexpression of TBK1s inhibits IRF3 nuclear translocation and leads to a shutdown of SeV-triggered IFN-β production. Taken together, our data indicate that TBK1s plays an inhibitory role in virus-triggered IFN-β signaling pathways.

Sustained immune control in HBeAg-positive patients who switched from entecavir therapy to pegylated interferon-α2a: 1 year follow-up of the OSST study.

BACKGROUND In the OSST study, hepatitis B e antigen (HBeAg)-positive chronic hepatitis B patients who switched from long-term entecavir (ETV) therapy to pegylated interferon-α2a (PEG-IFN-α2a; 40 kDa) achieved higher rates of HBeAg seroconversion and hepatitis B surface antigen (HBsAg) loss than those who continued ETV. Herein we report the sustainability of serological responses during 1 year of untreated follow-up in patients who switched from ETV to PEG-IFN-α2a therapy. METHODS A total of 62 patients who completed 48 weeks of PEG-IFN-α2a therapy were followed-up for 48 weeks off treatment. Primary end points were HBeAg seroconversion and maintenance of HBeAg seroconversion at 48 weeks post-treatment. Secondary end points included HBsAg loss, HBV DNA <1,000 copies/ml and alanine aminotransferase normalization (<1× upper limit of normal). RESULTS The HBeAg seroconversion rate increased from 17.7% (11/62) at the end of treatment to 38.7% (24/62) 1 year post-treatment. Sustained HBeAg seroconversion was achieved by 63.6% (7/11) patients with end-of-treatment responses, while late HBeAg seroconversion was achieved by 33.3% (17/51) of patients who did not have end-of-treatment responses. Sustained HBsAg loss was documented in 6 of 7 patients, and sustained HBV DNA suppression was achieved in 60% (27/45) of patients with an end-of-treatment response. CONCLUSIONS In patients who do not achieve HBeAg seroconversion during long-term ETV therapy, switching to finite treatment with PEG-IFN-α2a produces HBeAg seroconversion in a substantial proportion of patients at end of treatment and during 1 year of follow-up. Moreover, HBeAg seroconversion and HBsAg loss are sustained in most patients during 1 year of untreated follow-up.

KCTD9 contributes to liver injury through NK cell activation during hepatitis B virus-induced acute-on-chronic liver failure

We explored the expression of a newly identified potassium channel tetramerisation domain containing 9 (KCTD9) protein in 113 blood and 81 liver samples, from patients with mild chronic hepatitis B (CHB) or HBV-induced acute-on-chronic liver failure (HBV-ACLF). KCTD9 was highly expressed in peripheral and hepatic NK cells from HBV-ACLF patients compared with mild CHB patients, and this correlated positively with the severity of liver injury. The role of KCTD9 was further investigated in NK92 cells in vitro. KCTD9 overexpressed NK92 cells exhibited a marked increase in CD69 expression, cytotoxicity, IFN-γ secretion and a significant decrease in NKG2A receptor expression. Inhibition of KCTD9 by shRNA resulted in reduced cytotoxic function. These results suggest the involvement of KCTD9 in NK cell activation and provide additional insight into a potential therapeutic target for molecular manipulation for HBV-ACLF patients.

The Nucleocapsid Protein of SARS-CoV Induces Transcription of hfgl2 Prothrombinase Gene Dependent on C/EBP Alpha

Abstract Fibrin deposition was universal in the lungs of SARS patients and fgl2 prothrombinase gene, a novel procoagulant, was demonstrated to express highly in a clinically relevant SARS model. To investigate whether and which structural protein of SARS-CoV induced transcription of hfgl2 prothrombinase gene, three eukaryotic expression plasmids expressing nucleocapsid protein (N), membrane protein (M) and spike protein 2 (S2) of SARS-CoV were co-transfected with hfgl2 promoter luciferase-reporter plasmids and β-galactosidase plasmid in CHO cells, respectively. M, N and S2 protein of SARS-CoV were detected by western blotting and immunohistochemistry analysis. Further assays demonstrated that expression of hfgl2 gene was related with N protein, but not with M or S2 protein in THP-1 cells and Vero cells. N protein significantly induced functional procoagulant activity in comparison with control group. Luciferase assay showed that N protein of SARS-CoV could activate the transcription of hfgl2 promoter compared with the pcDNA3.1 empty vector. Site-directed mutagenesis and EMSA assay further demonstrated that transcription factor C/EBP alpha band with its cognate cis-element in hfgl2 promoter. The results showed that N protein of SARS-CoV induced hfgl2 gene transcription dependent on the transcription factor C/EBP alpha, which maybe contribute to the development of thrombosis in SARS.

Resistant mutations and quasispecies complexity of hepatitis B virus during telbivudine treatment.

Ultra-deep pyrosequencing (UDPS) was used to analyse the dynamics of quasispecies and resistant mutations during telbivudine (LDT) treatment of hepatitis B patients. Twenty-six HBeAg-positive chronic hepatitis B patients were treated with LDT for a period of 104 weeks and were characterized as 16 responders, six partial responders and four viral breakthrough patients based on hepatitis B virus (HBV) DNA levels. The plasma samples were subjected to UDPS of the reverse transcriptase (RT) region of HBV. Mutations rtM204I, rtL80I and rtL80V were detected in at least three of the four viral breakthrough patients, indicating the significant roles of the mutations in resistance to LDT. The degree of complexity of viral quasispecies remained in a steady state in the absence of selection pressure, but increased after the LDT treatment. The complexity in the responder group at week 12 was significantly higher than that in the group comprising partial responders and viral breakthrough patients. In vitro replication efficiency analyses showed that the RT mutations had different impacts on HBV replication, with a tendency of rtM204I>rtL80V>rtL80I. Furthermore, double mutations rtL80I/M204I and rtL80V/M204V had replication efficiency similar to that of rtL80I and rtL80V, respectively. Consistent with previous studies, mutation rtM204I was found to be highly resistant to LDT. However, in contrast with their sensitivity to lamivudine, rtL80I and rtL80V were moderately resistant to LDT. Our results indicated that rtL80I and rtL80V may not only serve as replication complementary mutations to rtM204I, but also directly contribute to the LDT resistance.

Upregulation of NKG2C+ natural killer cells, TLR-2 expression on monocytes and downregulation of regulatory T-cells influence PEG-IFN treatment efficacy in entecavir-suppressed patients with CHB.

BACKGROUND There is increasing evidence that host immune responses influence antiviral efficacy in chronic hepatitis B (CHB). The aim of this study was to characterize the immunological features responsible for improved treatment responses with pegylated interferon (PEG-IFN)-α2a in entecavir (ETV)-suppressed patients with CHB. METHODS Peripheral natural killer (NK) cells, Toll-like receptors (TLRs), T-cell subsets, regulatory T-cells (Tregs) and programmed death 1 (PD-1) were evaluated dynamically in 77 patients undergoing a clinical trial (OSST trial, NCT00940485) by flow cytometry. Response was defined as hepatitis B e antigen (HBeAg) seroconversion, hepatitis B surface antigen (HBsAg) loss and HBsAg seroconversion (either as singular events or in combination at week 48). RESULTS Compared with ETV responders or PEG-IFN-α non-responders, PEG-IFN-α responders exhibited a significant decline in HBsAg during treatment (P=0.033 and P<0.001, respectively) and a significant decline in Treg proportions from week 12 to week 24 (P=0.036 and P=0.004, respectively). Moreover, PEG-IFN-α responders showed a significantly higher increase in the NKG2C(+) NK cell proportions from baseline to week 12 (P=0.0073) and of TLR2(+) monocytes at week 12 than PEG-IFN-α non-responders (P=0.039). CONCLUSIONS Successful response to PEG-IFN-α correlates with an early significant restoration of impaired immune responses. Although antiviral treatment response can be achieved by both IFN and ETV, the underlying immunological features vary which may explain the generally observed difference in off-treatment durability of response between the two treatments, as well as effects on HBsAg.

IFN-α-mediated Base Excision Repair Pathway Correlates with Antiviral Response Against Hepatitis B Virus Infection

Previous studies identified APOBEC deaminases as enzymes targeting hepatitis B virus (HBV) DNA in the nucleus thus affecting its persistence. Interferon (IFN)-α treated chimpanzees and hepatitis C patients showed elevated APOBEC expression. We thus hypothesized that the responses to IFN-α treatment of chronic hepatitis B (CHB) patients is influenced by IFN-induced base excision repair (BER). CHB-treatment naïve patients, patients treated with PEGylated IFN-α, and patients with sequential treatment of Entecavior and PEGylated IFN-α were recruited. Blood and liver biopsy samples were collected before treatment and at treatment endpoint. BER genes were assessed by quantitative RT-PCR. BER gene expression levels and IFN treatment responses were correlated in patient liver biopsies. APOBEC3A, -B, -C, -D/E, and-G mRNA levels were up-regulated in IFN-treated patients. APOBEC3A expression was significantly higher in IFN-responders than in non-responders. BER genes NEIL3 was down-regulated in IFN-treated patients. APOBEC3 and BER gene expression at treatment endpoints partially correlated with the corresponding absolute DNA level or degree of HBsAg and HBV DNA decline. Our study suggests that the expression of APOBEC3A positively correlates with IFN-treatment responses in CHB patients, while NEIL3 shows negative correlation. These genes may involve to IFN mediated viral suppression and serve as biomarkers for CHB disease management.