Breaking Tolerance with CLDC-HBsAg in HBV Transgenic Mice

The objective of this research was to study the CYP2E1 gene expression in carbon tetrachloride (CCl4)-induced acute liver injury in hepatitis B virus (HBV) transgenic mice. Twenty-four HBV(-) and 24 HBV(+) transgenic mice aged 8 to 10 weeks were selected for the present study. Intraperitoneal injection of 1.0 μL/g of CCl4 (1:4 dissolved in olive oil) to mice was performed to induce acute liver injury model. Eight normal clean-grade C57BL/6 mice were taken as the control group. The control group received saline intraperitoneally. The mice in each group were killed 3, 6, 12, 24, 48, and 72 h after injection. The liver tissue samples of mice were collected. The liver histological changes at different time points in each group were observed under light microscope. The quantitative PCR methods were utilized to measure the relative mRNA levels of CYP2E1 gene in liver tissues. Immunohistochemistry and Western blot techniques were used to observe tissue expression levels of CYP2E1 in each group. Compared with that of the control group, mRNA and protein expression levels of CYP2E1 significantly increased both in the HBV(-) group and in the HBV(+) group after the CCl4 induced the acute liver injury, and it reached the peak at 72h after the CCl4 injection. Compared with the HBV(-) group, the mice in the HBV(+) group had severe liver damage and significantly increased CYP2E1 gene and protein expression levels. In the CCl4-induced acute liver injury of HBV transgenic mice, the CYP2E1 gene expression significantly increased. The results provided evidence for the HBV-induced liver damage and liver cancer pathogenesis.

Persistent hepatitis B virus (HBV) infection is characterized by a weak adaptive immune response, which is considered to be due to an imbalance of T helper cell types 1 and 2 (Th1/Th2). Suppressors of cytokine signaling (SOCS) family members, particularly SOCS1 and SOCS3, have been demonstrated to be important in the regulation of T cell differentiation. Previous studies by our group showed that the expressed and purified fusion protein of cytoplasmic transduction peptide (CTP) and HBV core antigen 18‑27 (HBcAg18‑27)‑tapasin was able to enter the cytoplasm of bone marrow‑derived dendritic cells (BMDCs), promoting the maturation of BMDCs and efficiently enhancing T cell immune responses in vitro. In the present study, HBcAg‑specific immune responses induced by CTP‑HBcAg18‑27‑tapasin in HBV were assessed in transgenic mice, and SOCS1 and SOCS3 were identified as negative regulators of this response. The Th1/Th2 cytokine ratio was analyzed by ELISA. The expression of T cell‑specific T‑box transcription factor (T‑bet) and GATA‑binding protein 3 (GATA‑3), SOCS1 and SOCS3 were detected by real‑time quantitative polymerase chain reaction and western blot analysis. The results demonstrated that CTP‑HBcAg18‑27‑tapasin significantly increased the Th1/Th2 cytokine ratio in HBV transgenic mice. CTP‑HBcAg18‑27‑tapasin immunization more efficiently suppressed the expression of serum hepatitis B surface antigen (HBsAg), HBV DNA as well as liver HBsAg and HBcAg in HBV transgenic mice. Furthermore, CTP‑HBcAg18‑27‑tapasin promotes T‑bet but reduces GATA‑3 expression. In addition, the expression of SOCS1 and SOCS3 was significantly downregulated in the CTP‑HBcAg18‑27‑tapasin group compared with the control groups. In conclusion, the present study demonstrated that CTP‑HBcAg18‑27‑tapasin enhanced the Th1/Th2 cytokine ratio and antiviral immunity by suppressing SOCS1/3 in HBV transgenic mice.

BackgroundWhile the immune pathogenesis caused by hepatitis B virus (HBV) infection has been studied extensively, little is known about direct pathogenic effects of HBV surface proteins. Here, we have investigated pathological cellular effects of HBV surface protein expression in the liver of transgenic mice with different genetic background.MethodsThe impact of HBV surface protein expression on the liver was studied in two mouse strains, BALB/c and C57BL/6. Histology and hydroxyproline assays were performed to investigate liver morphology and fibrosis. Gene expression and signaling were analyzed by microarray, qPCR and Western blotting.ResultsExpression of HBV surface proteins in the liver of transgenic mice induced activation of protein kinase-like endoplasmic reticulum kinase (PERK) and eukaryotic initiation factor 2α (eIF2α) phosphorylation. Phosphorylation of eIF2α resulted in activation of the ER stress markers glucose regulated protein (GRP) 78 and pro-apoptotic C/EBP homologous protein (CHOP) in transgenic mice on BALB/c genetic background leading to stronger liver injury and fibrosis in comparison with transgenic mice on C57BL/6 background. Hepatic stellate cells represented the main collagen-producing liver cells in HBV transgenic mice. The key regulators of hepatocyte proliferation, transcription factors c-Jun and STAT3 were activated in HBV transgenic mice. Tumour incidence in transgenic mice was strain- and sex-dependent.ConclusionsExtent of liver injury, fibrosis, and tumour development induced by hepatic HBV surface protein expression considerably depends on host genetic background.

Therapeutic potential of dendritic cell-based immunization against HBV in transgenic mice

Using hepatitis B virus (HBV) transgenic mice as recipients of virus-specific cytotoxic T lymphocytes (CTLs), we recently showed that polymorphonuclear neutrophils (PMNs) and the matrix-degrading metalloproteinases (MMPs) they produce are necessary for the intrahepatic recruitment of antigen nonspecific mononuclear cells that amplify the liver damage initiated by the CTLs. We now report that the high-mobility group box 1 protein (HMGB1) is also involved in this process. Transfer of CTLs in HBV transgenic mice induces the translocation of HMGB1 from the nucleus to the cytoplasm of hepatocytes surrounding CTL-containing necroinflammatory liver foci, without significant net synthesis of HMGB1. Treatment of CTL-injected HBV transgenic mice with either recombinant Box-A or glycyrrhizin, two functional inhibitors of extracellular HMGB1, significantly decreases the intrahepatic recruitment of PMNs and all other inflammatory cells, in the face of intact homing of virus-specific CTLs into the liver. The inhibition of PMN chemoattraction explains the mode of action of glycyrrhizin, which has long been used in Japan for the treatment of hepatitis, and suggests that new and more potent inhibitors of HMGB1 may be useful for the treatment of patients chronically infected with HBV.

For complete or functional cure of hepatitis B virus (HBV) infection, application of immunotherapy is now being attempted. Recently, we reported that a 6-mer hepatitis B virus (HBV)-derived peptide, Poly6, exerts a strong anticancer effect in tumor-implanted mice through inducible nitric oxide synthase (iNOS)-producing DCs (Tip-DCs) in a type 1 interferon (IFN-I)-dependent manner, suggesting its potential as a vaccine adjuvant. In this study, we explored the potential of Poly6 in combination with HBsAg as a therapeutic vaccine against hepatitis B virus infection. We investigated the immunotherapeutic potential of Poly6 combined with HBsAg vaccination against hepatitis B virus infection in C57BL/6 mice or an HBV transgenic mouse model. In C57BL/6 mice, Poly6 enhanced DC maturation and DC migration capacity in an IFN-I-dependent manner. Moreover, the addition of Poly6 to alum in combination with HBsAg also led to enhanced HBsAg-specific cell-mediated immune (CMI) responses, suggesting its potential as an adjuvant of HBsAg-based vaccines. In HBV transgenic mice, vaccination with Poly6 combined with HBsAg exerted a strong anti-HBV effect via induction of HBV-specific humoral and cell-mediated immune responses. In addition, it also induced HBV-specific effector memory T cells (TEM). Our data indicated that vaccination with Poly6 in combination with HBsAg exerts an anti-HBV effect in HBV transgenic mice, which is mainly mediated by HBV-specific CMI and humoral immune responses via IFN-I-dependent DC activation, suggesting the feasibility of Poly6 as an adjuvant for an HBV therapeutic vaccine.

Background/Aims: Developing engineered dendritic cell (DC)-targeting lentivectors (LVs) have been the target of intense research for their potential to create antigen-directed immunotherapeutics which can be safely administered to patients. In this study, we constructed a DC-directed LV (LVDC-UbHBcAg-LIGHT) as a potential vaccine to induce anti-HBV immune responses. Methods: Specificity of LVDC-UbHBcAg-LIGHT for DCs in vivo was confirmed through live animal imaging studies. The levels of cytokine production in T cells were assessed by flow cytometry. The HBcAg-specific cytotoxic T lymphocyte (CTL) responses and antibody responses induced by direct administration of the LVs were detected by LDH release assay and ELISA respectively. The levels of serum HBsAg and HBV DNA were evaluated by Abbott kits and quantitative PCR respectively. The expression levels of HBsAg and HBcAg in liver tissues of HBV transgenic mice were examined by immunohistochemistry. In addition, molecular mechanism underlying the activation of CD8+ T cells was explored. Results: Live animal imaging studies showed that following subcutaneous administration of LVDC-UbHBcAg-LIGHT, no obvious luminescence signal was detected at the injection site. Immunization with LVDC-UbHBcAg-LIGHT elicited potent T cell responses in HBV transgenic mice evidenced by increased percentages of IFN-γ, TNF-α and GzmB producing CD8+ T cells as well as IFN-γ producing CD4+ T cells, improved HBcAg-specific CTL activities and antibody responses. Additionally, vaccination with LVDC-UbHBcAg-LIGHT efficiently reduced serum HBsAg, HBV DNA levels and the expression of HBsAg and HBcAg in liver tissues of HBV transgenic mice. More importantly, autophagy was induced in the activated CD8+ T cells, and the induced autophagy noticeably promoted the proliferation of T cells and decreased the frequencies of apoptotic CD8+ T cells by selectively degrading ubiquitinated apoptosis and cell cycle-associated protein aggregates. Futhermore, we confirmed the interaction between autophagosomes and ubiquitinated aggregates by confocal microscopy and immunoprecipitation analysis. Conclusions: These results demonstrated that LVDC-UbHBcAg-LIGHT provided a simple method of eliciting effective antiviral immune responses in HBV transgenic mice and might potentially be used as a therapeutic strategy to eradicate HBV with more safety and efficiency. Moreover, our results revealed a direct role of autophagy in promoting the survival and proliferation of activated CD8+ T cells.

Highly representative and relevant cell and mouse models are required for HBV study, including uncovering its lifecycle, investigation of the viral-host interaction, and development and evaluation of the novel antiviral therapy. During the past 40years, both HBV cell culture models and animal models have evolved over several generations, each with significant improvement for specific purposes. In one aspect, HBV cell culture models experienced the original noninfection model including HBV plasmid DNA transfection and HBV genome integrated stable cells such as HepG2.2.15 which constitutively produces HBV virus and HepAD38 cells and its derivatives which drug-regulated HBV production. As for HBV infection models, HepaRG cells once dominated the HBV infection field for over a decade, but its complicated and labor-extensive cell differentiation procedures discouraged primary researchers from stepping in the field. The identification of human NTCP as HBV receptor evoked great enthusiasm of the whole HBV field, and its readily adaptive characteristic makes it popular in many HBV laboratories. Recombinant cccDNA (rc-cccDNA) emerged recently aiming to tackle the very basic question of how to eventually eradicate cccDNA without HBV real virus infection. In the other aspect, HBV transgenic mouse was firstly generated in the 1990s, which was helpful to decipher HBV production in vivo. However, the HBV transgenic mice were naturally immune tolerant to HBV viral products. Subsequently, a series of nonintegrated HBV mouse models were generated through plasmid hydrodynamic tail vein injection and viral vector-mediated delivery approaches, and HBV full life cycle was incomplete as cccDNA was not formed from HBV relaxed circular DNA (rcDNA). Human NTCP transgenic mouse still could not support productive HBV infection, and humanized mouse liver with human hepatocytes which supported whole HBV life cycle still dominates HBV infection in vivo, a value but expensive model until now. Other methods to empower mouse to carry HBV cccDNA were also exploited. In this chapter, we summarized the advantages and disadvantages of each model historically and provided protocols for HBV infection in HepG2-NTCP cells, HBV rc-cccDNA transfection in HepG2 cells, and HBV infection in NRG-Fah-/- liver humanized mouse.

Role of sex steroid hormones on hepatocellular carcinoma (HCC) development in HBV transgenic mice

To establish and identify an animal model of non-alcoholic fatty liver disease in chronic HBV infected mice. Transgenic mice with sustaining HBV production were established by microinjection of ocyte. Then they were randomly assigned into 4 groups (male control, male NAFLD model, female control and female NAFLD model) and treated with high fat diet (2% cholesterol, 10% lard, 88% forage) and common forage, respectively. NAFLD-related physical indexes, liver and kidney function, glucose and lipid metabolism were investigated at the time points of 8 weeks, 16 weeks and 24 weeks. Meanwhile, HBV type, serum levels of HBV DNA and HBeAg, immunohistochemical staining of hepatic HBsAg were detected. The establishment of NAFLD was evaluated by serum levels of total cholesterol (TC), triglycerides, glucose, aspartate aminotransferase (AST), alkaline phosphatase (ALP), alanine aminotransferase (ALT), gamma-glutamyltransferase (GGT), etc. Histological changes were also analyzed by HE, oil red O and Masson's trichrome staining. The status of CHB was assessed on the basis of immunohistochemistry and real-time PCR. The body and liver weights, liver index in HBV transgenic mice were significantly increased in regardless of the gender of HFD feeding, and the levels of serum ALT, AST, ALP, GGT, TBIL, TBA, TC, TG, HDL-C, LDL and FBG were higher in HFD groups as compared with the control mice. Lipid droplets, cytologic ballooning and liver steatosis could be observed in most lobules of HFD groups after 8-week administration, fatty degeneration of hepatocytes, patch necrosis, mild to moderate chronic inflammatory infiltration were also observed in some of HFD feeding, reflecting the emerge of steatohepatitis. At the time point of 24-week perisinusoidal fibrosis and local fibrosis occurred in HFD groups. Immunohistochemical staining and real-time PCR analysis of the liver tissues showed positive signal of HBsAg in all groups of mice, although no significant difference was documented. Our study suggests that animal model of NAFLD can be established in HBV transgenic mice and provide a nice animal model for further studies on NAFLD with chronic hepatitis B infection.

The role of hepatocyte nuclear factor 1alpha (HNF1 alpha) in the regulation of hepatitis B virus (HBV) transcription and replication in vivo was investigated using a HNF1 alpha-null HBV transgenic mouse model. HBV transcription was not measurably affected by the absence of the HNF1 alpha transcription factor. However, intracellular viral replication intermediates were increased two- to fourfold in mice lacking functional HNF1 alpha protein. The increase in encapsidated cytoplasmic replication intermediates in HNF1 alpha-null HBV transgenic mice was associated with the appearance of nonencapsidated nuclear covalently closed circular (CCC) viral genomic DNA. Viral CCC DNA was not readily detected in HNF1 alpha-expressing HBV transgenic mice. This indicates the synthesis of nuclear HBV CCC DNA, the proposed viral transcriptional template found in natural infection, is regulated either by subtle alterations in the levels of viral transcripts or by changes in the physiological state of the hepatocyte in this in vivo model of HBV replication.

A hepatitis B virus (HBV) transgenic mice model was used to establish the fatty liver superimposed model by feeding the methionine choline-deficient (MCD) diet for 8 weeks, with or without the gavage of 2 mg/kg zeaxanthin dipalmitate (ZD) three times per week. Both wild-type and HBV transgenic mice, with MCD diet, gained typical non-obese non-alcoholic steatohepatitis (NASH) and HBV symptoms. Coadministration with ZD exhibited evident therapeutic effects through alleviating those pathological events. Moreover, long-term vehicle-ZD treatment was found to be safe. Thus, ZD is a promising and safe hepato-protective agent against hepatic injury induced by superimposed HBV and NASH in non-obese mice.

The objective of this work is to examine the possible modulation of carcinogen metabolism (activation by cytochrome P450s and detoxification by conjugation via glutathione S-transferases [GST]) in relation to hepatitis B virus (HBV)-associated liver injury. In HBV transgenic mouse lineage 107.5, the hepatitis B surface antigen (HBsAg) is expressed at noncytopathic concentrations but after injection of an HBsAg-specific, major histocompatibility complex (MHC) class I restricted cytotoxic T-lymphocyte (CTL) clone, the mice develop a severe acute necroinflammatory liver disease that reaches maximum severity within 3 days and gradually subsides during the next 2 to 3 weeks. In this model, using immunohistochemical analysis, we observed an increase of P450s (CYP1A and 2A5), both involved in aflatoxin B1, metabolism, but minor changes or no changes for others (2B, 2C, 2E, 3A). There was a fivefold decrease in the total liver P450 microsomal content 3 days' post-CTL injection with the result that the relative proportion of CYP2A5 and 1A compared with other P450s is increased. Individual microsomal P450 enzyme contents estimated by Western blotting; Northern blot analysis of liver CYP messenger RNA (mRNA) levels as well as in vitro metabolism of specific substrates for different P450 isoenzymes were consistent with the immunohistochemical data. Immunohistochemical staining with antibodies to cytosolic pi class GST was increased 1 and 3 days postinjection followed by a progressive decrease at later time points (the same phenomenon was observed to a lesser extent for GST alpha). The activity of hepatic cytosols toward substrates specific for different subclasses of GST (mu, pi, alpha) showed that while GST mu was not changed in the CTL-injected HBV transgenic mice, GST pi and, to a lesser extent, alpha were increased as compared with controls. These results suggest that liver cell injury induced by a process of acute fulminant-like hepatitis can lead to the induction of some carcinogen metabolizing enzymes notably, Cyp 1A, 2A5 and GST pi in the mouse.

Hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) occurs more often in men than in women. Male HBV carriers usually have higher viral loads, which is a well-known risk factor for HCC. Whether and how the male androgen axis regulates HBV transcription and replication is investigated here. We used HBV transgenic mice to evaluate any sex disparity of serum hepatitis B surface antigen and HBV titers as well as the castration effect on this disparity. Compared to females, HBV transgenic male mice showed higher hepatitis B surface antigen and viral titers, which were lessened by castration of the males. In a cell culture system, HepG2 cells transfected with HBV and androgen receptor (AR) constructs were used to study the effect of the androgen pathway on viral transcription and replication. We found the ligand-stimulated AR could increase the transcription of HBV RNAs through its transcription activation domain. A genomic region within HBV enhancer I was identified that is responsible for the transcriptional activation of AR. The results from chromatin immunoprecipitation and in vitro binding assays further demonstrated a direct binding of AR to this region, in a ligand-dependent manner. Two androgen-responsive element motifs in this region were identified, and their mutations can significantly abolish the AR effects. This study demonstrated that the androgen pathway can increase the transcription of HBV through direct binding to the androgen-responsive element sites in viral enhancer I. This may explain a higher HBV titer in male carriers and an increased risk of HCC.

Targeting Innate Immunity: A New Step in the Development of Combination Therapy for Chronic Hepatitis B