Zhanqing Zhang

HBsAg inhibits TLR9-mediated activation and IFN-α production in plasmacytoid dendritic cells

Plasmacytoid dendritic cells (pDCs), the professional producers of type I interferons (IFN-alpha/beta), play a pivotal role in innate and adaptive immune responses against viral infections. Although functional impairment of circulating pDCs in chronic hepatitis B (CHB) patients has been reported previously, the mechanism responsible for these defects remains unclear. We hypothesize that HBsAg circulating in high amounts during HBV infection may interact with pDC and contribute to pDC dysfunction. In support of this hypothesis we show that pDCs treated with HBsAg secreted much less IFN-alpha than control pDCs. Furthermore, suppression is specific for TLR9, with no effects upon TLR7-mediated IFN-alpha secretion. HBsAg inhibited TLR9-mediated IRF-7 expression and nuclear translocation, which are important for induction of IFN-alpha gene transcription. HBsAg upregulated the SOCS-1 expression and bound to BDCA-2 receptors on the plasma membrane of pDCs, resulting in the inhibition of the IFN-alpha production. In conclusion, the above data suggested that HBsAg may directly interfere with the function of pDC through HBsAg-mediated upregulation of SOCS-1 expression and BDCA-2 ligation, which could partially explain how HBV evades the immune system to establish a persistent infection.

Expression profiles and function of Toll-like receptors 2 and 4 in peripheral blood mononuclear cells of chronic hepatitis B patients.

Toll-like receptors (TLRs) play a central role in sensing and initiating innate antiviral response. In this study, we first investigated the expression of TLR1-10 mRNA transcripts in peripheral blood mononuclear cells (PBMCs) from chronic HBV-infected (CHB) patients and healthy donors by quantitative real-time PCR. The expression of TLR1, TLR2, TLR4 and TLR6 transcripts was significantly lower in PBMCs from CHB patients, and the down-regulation of TLR2 was related to HBV genotype C. Flow cytometric analysis showed that the expression of TLR2 on PBMCs was significantly decreased in CHB patients. Furthermore, impaired cytokine production was observed in PBMCs from CHB patients after challenged with TLR2 and TLR4 ligands and was correlated with the levels of plasma hepatitis B virus surface antigen (HBsAg). In conclusion, our study reveals a possible interaction between HBsAg, TLR signaling and the innate immune response, which may partially explain the mechanism of HBV infection induced immuno-tolerance.

Hepatitis B virus polymerase impairs interferon-α-induced STA T activation through inhibition of importin-α5 and protein kinase C-δ.

Treatment with exogenous interferon (IFN)‐α is not effective in the majority of patients with chronic hepatitis B virus (HBV) infection. Recent evidence suggests that HBV has evolved strategies to block the nuclear translocation of signal transducer and activator of transcription (STAT) 1 to limit IFN‐α–induced cellular antiviral responses. However, it remains unclear whether STAT1 translocation is impaired in chronic hepatitis B patients and what mechanisms are involved. Here we report that the expression of HBV polymerase (Pol) in human hepatic cell lines inhibited induction of IFN‐stimulated genes and resulted in a weakened antiviral activity of IFN‐α. Ectopic expression of Pol suppressed IFN‐α–induced STAT1 serine 727 phosphorylation and STAT1/2 nuclear accumulation, whereas STAT1 tyrosine 701 phosphorylation, and STAT1‐STAT2 heterodimer formation were not affected. Further studies demonstrated that Pol interacted with the catalytic domain of protein kinase C‐δ (PKC‐δ) and perturbed PKC‐δ phosphorylation and its association with STAT1, which resulted in the suppression of STAT1 Ser727 phosphorylation. Moreover, Pol was found to interfere with nuclear transportation of STAT1/2 by competitively binding to the region of importin‐α5 required for STAT1/2 recruitment. Truncation analysis suggested that the terminal protein and RNase H domains of Pol were able to bind to PKC‐δ and importin‐α5, respectively, and were responsible for the inhibition of IFN‐α signaling. More importantly, the inhibition of STAT1 and PKC‐δ phosphorylation were confirmed in a hydrodynamic‐based HBV mouse model, and the blockage of IFN‐α–induced STAT1/2 nuclear translocation was observed in HBV‐infected cells from liver biopsies of chronic HBV patients. Conclusions: These results demonstrate a role for Pol in HBV‐mediated antagonization of IFN‐α signaling and provide a possible molecular mechanism by which HBV resists the IFN therapy and maintains its persistence. (HEPATOLOGY 2013;)

HBsAg inhibits IFN-α production in plasmacytoid dendritic cells through TNF-α and IL-10 induction in monocytes.

Type I Interferon (IFN) is one of the first lines of defense against viral infection. Plasmacytoid dendritic cells (pDCs) are professional IFN-α-producing cells that play an important role in the antiviral immune response. Previous studies have reported that IFN-α production is impaired in chronic hepatitis B (CHB) patients. However, the mechanisms underlying the impairment in IFN-α production are not fully understood. Here, we report that plasma-derived hepatitis B surface antigen (HBsAg) and HBsAg expressed in CHO cells can significantly inhibit toll like receptor (TLR) 9-mediated Interferon-α (IFN-α) production in peripheral blood mononuclear cells (PBMCs) from healthy donors. Further analysis indicated that monocytes participate in the inhibitory effect of HBsAg on pDCs through the secretion of TNF-α and IL-10. Furthermore, TLR9 expression on pDCs was down-regulated by TNF-α, IL-10 and HBsAg treatment. This down-regulation may partially explain the inhibition of IFN-α production in pDCs. In conclusion, we determined that HBsAg inhibited the production of IFN-α by pDCs through the induction of monocytes that secreted TNF-α and IL-10 and through the down-regulation of TLR9 expression on pDCs. These data may aid in the development of effective antiviral treatments and lead to the immune control of the viral infections.

In situ analysis of intrahepatic virological events in chronic hepatitis B virus infection

Persistent hepatitis B virus (HBV) infection is established by the formation of an intranuclear pool of covalently closed circular DNA (cccDNA) in the liver. Very little is known about the intrahepatic distribution of HBV cccDNA in infected patients, particularly at the single-cell level. Here, we established a highly sensitive and specific ISH assay for the detection of HBV RNA, DNA, and cccDNA. The specificity of our cccDNA probe set was confirmed by its strict intranuclear signal and by a series of Southern blot analyses. Use of our in situ assay in conjunction with IHC or immunofluorescence uncovered a surprisingly mosaic distribution of viral antigens and nucleic acids. Most strikingly, a mutually exclusive pattern was found between HBV surface antigen-positive (HBsA-positive) and HBV DNA- and cccDNA-positive cells. A longitudinal observation of patients over a 1-year period of adeforvir therapy confirmed the persistence of a nuclear reservoir of viral DNA, although cytoplasmic DNA was effectively depleted in these individuals. In conclusion, our method for detecting viral nucleic acids, including cccDNA, with single-cell resolution provides a means for monitoring intrahepatic virological events in chronic HBV infection. More important, our observations unravel the complexity of the HBV life cycle in vivo.

Atomic-scale mapping of dipole frustration at 90 degrees charged domain walls in ferroelectric PbTiO3 films

The atomic-scale structural and electric parameters of the 90° domain-walls in tetragonal ferroelectrics are of technological importance for exploring the ferroelectric switching behaviors and various domain-wall-related novel functions. We have grown epitaxial PbTiO3/SrTiO3 multilayer films in which the electric dipoles at 90° domain-walls of ferroelectric PbTiO3 are characterized by means of aberration-corrected scanning transmission electron microscopy. Besides the well-accepted head-to-tail 90° uncharged domain-walls, we have identified not only head-to-head positively charged but also tail-to-tail negatively charged domain-walls. The widths, polarization distributions, and strains across these charged domain-walls are mapped quantitatively at atomic scale, where remarkable difference between these domain-walls is presented. This study is expected to provide fundamental information for understanding numerous novel domain-wall phenomena in ferroelectrics.

Comparison of circulating, hepatocyte specific messenger RNA and microRNA as biomarkers for chronic hepatitis B and C.

Circulating microRNAs have been widely recognized as a novel category of biomarker in a variety of physiological and pathological conditions. Other reports revealed that fragments of organ specific messenger RNAs are also detectable in serum/plasma and can be utilized as sensitive indicators of liver pathology and cancer. In order to assess the sensitivity and reliability of these two class of RNAs as marker of hepatitis B or C induced chronic liver disease, we collected plasma samples from 156 chronic hepatitis B or C patients (HBV active n = 112, HBV carrier n = 19, hepatitis C n = 25) and 22 healthy donors and quantified their circulating mRNA for albumin, HP (haptoglobin), CYP2E1 (cytochrome P450, family 2, subfamily E) and ApoA2 (Apolipoprotein A2) in conjunction with microRNA-122, a well established marker for acute and chronic liver injury. We found that plasma microRNA-122 level is significantly elevated in patients with active HBV but not in HBV carriers. Furthermore, microRNA-122 is not elevated in HCV patients even though their median serum alanine aminotransferase (sALT) was three fold of the healthy donors. Nevertheless, circulating mRNAs, especially albumin mRNA, showed much more sensitivity in distinguishing active hepatitis B, hepatitis B carrier or HCV patientsfrom healthy control. Correlation and multiple linear regression analysis suggested that circulating mRNAs and miRNAs are much more related to HBsAg titre than to sALT. Immunoprecipitation of HBsAg in HBV patients’ plasma resulted in enrichment of albumin and HP mRNA suggesting that fragments of liver specific transcripts can be encapsidated into HBsAg particles. Taken together, our results suggest that hepatocyte specific transcripts in plasma like albumin mRNA showed greater sensitivity and specificity in differentiating HBV or HCV induced chronic liver disease than microRNA-122. Circulating mRNA fragments merit more attention in the quest of next generation biomarkers for various maladies.

Plasma microRNA profile as a predictor of early virological response to interferon treatment in chronic hepatitis B patients.

BACKGROUND Interferon (IFN) and pegylated interferon (PEG-IFN) treatment of chronic hepatitis B leads to a sustained virological response in a limited proportion of patients and has considerable side effects. To find novel markers associated with prognosis of IFN therapy, we investigated whether a pretreatment plasma microRNA profile could be used to predict early virological response to IFN. METHODS We performed microRNA microarray analysis of plasma samples from 94 patients with chronic hepatitis B who received IFN therapy. The microRNA profiles from 13 liver biopsy samples were also measured. The OneR feature ranking and incremental feature selection method were used to rank and optimize the number of features in the model. Support vector machine prediction engine and jack-knife cross-validation were used to generate and evaluate the prediction model. RESULTS The optimized model consisting of 11 microRNAs yielded a 74.2% overall accuracy in the training group and was independently confirmed in the test group (71.4% accuracy). Univariate and multivariate logistic regression analyses confirmed its independent association with early virological response (OR=7.35; P=2.12×10(-5)). Combining the microRNA profile with the alanine aminotransferase level improved the overall accuracy from 73.4% to 77.3%. Co-transfection of an HBV replicative construct with microRNA mimics revealed that let-7f, miR-939 and miR-638 were functionally associated with the HBV life cycle. CONCLUSIONS The 11 microRNA signatures in plasma, together with basic clinical variables, might provide an accurate method to assist in medication decisions and improve the overall sustained response to IFN treatment.

Characterization of gene expression profiles in HBV-related liver fibrosis patients and identification of ITGBL1 as a key regulator of fibrogenesis

Although hepatitis B virus (HBV) infection is the leading cause of liver fibrosis (LF), the mechanisms underlying liver fibrotic progression remain unclear. Here, we investigated the gene expression profiles of HBV-related LF patients. Whole genome expression arrays were used to detect gene expression in liver biopsy samples from chronically HBV infected patients. Through integrative data analysis, we identified several pathways and key genes involved in the initiation and exacerbation of liver fibrosis. Weight gene co-expression analysis revealed that integrin subunit β-like 1 (ITGBL1) was a key regulator of fibrogenesis. Functional experiments demonstrated that ITGBL1 was an upstream regulator of LF via interactions with transforming growth factor β1. In summary, we investigated the gene expression profiles of HBV-related LF patients and identified a key regulator ITGBL1. Our findings provide a foundation for future studies of gene functions and promote the development of novel antifibrotic therapies.

MicroRNA-939 restricts Hepatitis B virus by targeting Jmjd3-mediated and C/EBPα-coordinated chromatin remodeling.

Multi-layered mechanisms of virus host interaction exist for chronic hepatitis B virus (HBV) infection, which have been typically manifested at the microRNA level. Our previous study suggested that miRNA-939 (miR-939) may play a potential role in regulating HBV replication. Here we further investigated the mechanism by which miR-939 regulates HBV life cycle. We found that miR-939 inhibited the abundance of viral RNAs without direct miRNA-mRNA base pairing, but via host factors. Expression profiling and functional validation identified Jmjd3 as a target responsible for miR-939 induced anti-HBV effect. Jmjd3 appeared to enhance the transcription efficiency of HBV enhancer II/core promoter (En II) in a C/EBPα-dependent manner. However, the demethylase activity of Jmjd3 was not required in this process. Rather, Jmjd3’s transactivation activity depended on its interaction with C/EBPα. This coordinated action further recruited the Brm containing SWI/SNF chromatin remodeling complex which promoted the transcription of HBV RNAs. Taken together, we propose that the miR-939-Jmjd3 axis perturbs the accessibility of En II promoter to essential nuclear factors (C/EBPα and SWI/SNF complex) therefore leading to compromised viral RNA synthesis and hence restricted viral multiplication.