Yinping Lu

Molecular characterization of the type I IFN receptor in two woodchuck species and detection of its expression in liver samples from woodchucks infected with woodchuck hepatitis virus (WHV)

Type I interferons (IFN-α/β) serve as the first line of defense against viral infection and share the same type I IFN receptor (IFNAR) complex, which is composed of IFNAR1 and -2. The Eastern woodchuck (Marmota monax) and Chinese woodchuck (Marmota himalayana) are suitable for studying hepatitis B virus (HBV) infection. Here, the complete or partial sequences of the IFNARs of both species were obtained and analyzed. Small interference RNAs targeting wIFNAR1 and -2 specifically down-regulated the expression of wIFNAR1 and -2 and the IFN-stimulated gene MxA in a woodchuck cell line, respectively. IFNAR2 was significantly up-regulated in primary woodchuck hepatocytes stimulated with IFN-α or -γ. The expression of woodchuck IFNAR1 and -2 was decreased in woodchucks chronically infected with woodchuck hepatitis virus (WHV). These results are essential for studying type I IFN-related innate immunity and therapy in hepadnaviral infection in the woodchuck model.

Upregulation of toll-like receptor 4 on T cells in PBMCs is associated with disease aggravation of HBV-related acute-on-chronic liver failure.

Immune-mediated inflammatory injury is an important feature of the disease aggravation of hepatitis B virus-related acute-on-chronic liver failure (ACLF). Toll-like receptors (TLRs) have been shown previously to play a pivotal role in the activation of innate immunity. The purpose of this study was to characterize the TLR4 expression in peripheral blood mononuclear cells (PBMCs) of ACLF patients and its possible role in the disease aggravation. Twelve healthy subjects, 15 chronic HBV-infected (CHB) patients and 15 ACLF patients were enrolled in this study. The TLR4 expression in PBMCs and T cells of all subjects was examined by real-time PCR and flow cytometry. The correlation of TLR4 expression on T cells with the markers of disease aggravation was evaluated in ACLF patients. The ability of TLR4 ligands stimulation to induce inflammatory cytokine production in ACLF patients was analyzed by flow cytometry. The results showed that TLR4 mRNA level was upregulated in PBMCs of ACLF patients compared to that in the healthy subjects and the CHB patients. Specifically, the expression of TLR4 on CD4+ and CD8+ T cells of PBMCs was significantly increased in ACLF patients. The TLR4 levels on CD4+ and CD8+ T cells were positively correlated with serum total bilirubin (TBIL), direct bilirubin (DBIL), international normalized ratio (INR) levels and white blood cells (WBCs), and negatively correlated with serum albumin (ALB) levels in the HBV-infected patients, indicating TLR4 pathway may play a role in the disease aggravation of ACLF. In vitro TLR4 ligand stimulation on PBMCs of ACLF patients induced a strong TNF-α production by CD4+ T cells, which was also positively correlated with the serum markers for liver injury severity. It was concluded that TLR4 expression is upregulated on T cells in PBMCs, which is associated with the aggravation of ACLF.Immune-mediated inflammatory injury is an important feature of the disease aggravation of hepatitis B virus-related acute-on-chronic liver failure (ACLF). Toll-like receptors (TLRs) have been shown previously to play a pivotal role in the activation of innate immunity. The purpose of this study was to characterize the TLR4 expression in peripheral blood mononuclear cells (PBMCs) of ACLF patients and its possible role in the disease aggravation. Twelve healthy subjects, 15 chronic HBV-infected (CHB) patients and 15 ACLF patients were enrolled in this study. The TLR4 expression in PBMCs and T cells of all subjects was examined by real-time PCR and flow cytometry. The correlation of TLR4 expression on T cells with the markers of disease aggravation was evaluated in ACLF patients. The ability of TLR4 ligands stimulation to induce inflammatory cytokine production in ACLF patients was analyzed by flow cytometry. The results showed that TLR4 mRNA level was upregulated in PBMCs of ACLF patients compared to that in the healthy subjects and the CHB patients. Specifically, the expression of TLR4 on CD4+ and CD8+ T cells of PBMCs was significantly increased in ACLF patients. The TLR4 levels on CD4+ and CD8+ T cells were positively correlated with serum total bilirubin (TBIL), direct bilirubin (DBIL), international normalized ratio (INR) levels and white blood cells (WBCs), and negatively correlated with serum albumin (ALB) levels in the HBV-infected patients, indicating TLR4 pathway may play a role in the disease aggravation of ACLF. In vitro TLR4 ligand stimulation on PBMCs of ACLF patients induced a strong TNF-α production by CD4+ T cells, which was also positively correlated with the serum markers for liver injury severity. It was concluded that TLR4 expression is upregulated on T cells in PBMCs, which is associated with the aggravation of ACLF.

Suppression of hepatitis B virus replication in Tupaia hepatocytes by tumor necrosis factor alpha of Tupaia belangeri

Recently, Tupaia belangeri was used to study the full replication cycle of hepatitis B virus (HBV) in the primary hepatocyte cultures. Thus, the Tupaia model represents a suitable model to study the effects of cytokines on HBV infection. Here, Tupaia tumor necrosis factor-alpha (TNF-α) was molecularly cloned and expressed in mammalian cells. A test system for the biological activity of Tupaia TNF-α was established on the basis of its cytotoxic effect to the murine fibrosarcoma cell line L929. Recombinant Tupaia TNF-α was able to suppress HBV replication in primary Tupaia hepatocytes (PTH). However, the formation of HBV covalently closed circular DNA (cccDNA) and viral RNA was not completely prevented. Therefore, Tupaia TNF-α may contribute significantly to the control of HBV infection though it is not able to completely inhibit HBV replication alone. The characterization of this important cytokine allows further studies on its antiviral actions in the Tupaia model.

Pentoxifylline inhibits liver fibrosis via hedgehog signaling pathway

Infection of schistosomiasis japonica may eventually lead to liver fibrosis, and no effective antifibrotic therapies are available but liver transplantation. Hedgehog (HH) signaling pathway has been involved in the process and is a promising target for treating liver fibrosis. This study aimed to explore the effects of pentoxifylline (PTX) on liver fibrosis induced by schistosoma japonicum infection by inhibiting the HH signaling pathway. Phorbol12-myristate13-acetate (PMA) was used to induce human acute mononuclear leukemia cells THP-1 to differentiate into macrophages. The THP-1-derived macrophages were stimulated by soluble egg antigen (SEA), and the culture supernatants were collected for detection of activation of macrophages. Cell Counting Kit-8 (CCK-8) was used to detect the cytotoxicity of the culture supernatant and PTX on the LX-2 cells. The LX-2 cells were administered with activated culture supernatant from macrophages and(or) PTX to detect the transforming growth factor-β gene expression. The mRNA expression of shh and gli-1, key parts in HH signaling pathway, was detected. The mRNA expression of shh and gli-1 was increased in LX-2 cells treated with activated macrophages-derived culture supernatant, suggesting HH signaling pathway may play a key role in the activation process of hepatic stellate cells (HSCs). The expression of these genes decreased in LX-2 cells co-cultured with both activated macrophages-derived culture supernatant and PTX, indicating PTX could suppress the activation process of HSCs. In conclusion, these data provide evidence that PTX prevents liver fibrogenesis in vitro by the suppression of HH signaling pathway.SummaryInfection of schistosomiasis japonica may eventually lead to liver fibrosis, and no effective antifibrotic therapies are available but liver transplantation. Hedgehog (HH) signaling pathway has been involved in the process and is a promising target for treating liver fibrosis. This study aimed to explore the effects of pentoxifylline (PTX) on liver fibrosis induced by schistosoma japonicum infection by inhibiting the HH signaling pathway. Phorbol12-myristate13-acetate (PMA) was used to induce human acute mononuclear leukemia cells THP-1 to differentiate into macrophages. The THP-1-derived macrophages were stimulated by soluble egg antigen (SEA), and the culture supernatants were collected for detection of activation of macrophages. Cell Counting Kit-8 (CCK-8) was used to detect the cytotoxicity of the culture supernatant and PTX on the LX-2 cells. The LX-2 cells were administered with activated culture supernatant from macrophages and(or) PTX to detect the transforming growth factor-β gene expression. The mRNA expression of shh and gli-1, key parts in HH signaling pathway, was detected. The mRNA expression of shh and gli-1 was increased in LX-2 cells treated with activated macrophages-derived culture supernatant, suggesting HH signaling pathway may play a key role in the activation process of hepatic stellate cells (HSCs). The expression of these genes decreased in LX-2 cells co-cultured with both activated macrophages-derived culture supernatant and PTX, indicating PTX could suppress the activation process of HSCs. In conclusion, these data provide evidence that PTX prevents liver fibrogenesis in vitro by the suppression of HH signaling pathway.

Local Stimulation of Liver Sinusoidal Endothelial Cells with a NOD1 Agonist Activates T Cells and Suppresses Hepatitis B Virus Replication in Mice

Functional maturation of liver sinusoidal endothelial cells (LSECs) induced by a NOD1 ligand (diaminopimelic acid [DAP]) during viral infection has not been well defined. Thus, we investigated the role of DAP-stimulated LSEC maturation during hepatitis B virus (HBV) infection and its potential mechanism in a hydrodynamic injection (HI) mouse model. Primary LSECs were isolated from wild-type C57BL/6 mice and stimulated with DAP in vitro and in vivo and assessed for the expression of surface markers as well as for their ability to promote T cell responses via flow cytometry. The effects of LSEC maturation on HBV replication and expression and the role of LSECs in the regulation of other immune cells were also investigated. Pretreatment of LSECs with DAP induced T cell activation in vitro. HI-administered DAP induced LSEC maturation and subsequently enhanced T cell responses, which was accompanied by an increased production of intrahepatic cytokines, chemokines, and T cell markers in the liver. The HI of DAP significantly reduced the HBsAg and HBV DNA levels in the mice. Importantly, the DAP-induced anti-HBV effect was impaired in the LSEC-depleted mice, which indicated that LSEC activation and T cell recruitment into the liver were essential for the antiviral function mediated by DAP application. Taken together, the results showed that the Ag-presenting ability of LSECs was enhanced by DAP application, which resulted in enhanced T cell responses and inhibited HBV replication in a mouse model.

Construction and characterization of a hepatitis B virus replicon

To establish a replication cellular model of hepatitis B virus (HBV) and determine its application in antiviral drug evaluation, we constructed an expression plasmid which contained 1.3 copies of the HBV genome, and measured the level of viral replication after transient transfection in Huh7 cells. We then observed the effect of antiviral drug administration. 1.3 fold of the HBV(ayw) gene fragment was cloned into pCR2.1 by PCR and restriction endonuclease digestion. The recombinant plasmid was transient transfected into Huh7 cells, HBsAg, HBeAg and HBV DNA in supernatant of Huh7 cells were measured by ELISA and real-time PCR respectively; intracellular HBV replicative intermediates and intracellular HBV transcripts were detected by Southern blot and Northern blot respectively. The antiviral effect of adefovir, a novel anti-HBV nucleotide analogue, was evaluated in this cellular model system. The results indicated that a recombinant plasmid of HBV replicon was constructed successfully; the HBV genome carried in plasmid pHBV1.3 could efficiently replicate and be expressed in Huh 7 cells, adefovir could inhibit HBV replication in this cellular model, and the inhibition was dosage-dependent. The conclusion is HBV replicon, which can initiate viral replication efficiently in hepatoma cells, may be a useful tool in the study of HBV replication and antiviral drug.

TLR2 Expression in Peripheral CD4+ T Cells Promotes Th17 Response and Is Associated with Disease Aggravation of Hepatitis B Virus-Related Acute-On-Chronic Liver Failure

Th17 responses have been shown to play crucial roles in the pathogenesis of hepatitis B virus (HBV)-associated acute-on-chronic liver failure (ACLF). The mechanism underlying the enhanced Th17 responses in these patients remains largely unclear. Here we investigated toll-like receptors (TLRs) expression in peripheral T cells and their roles in Th17 cell differentiation and disease aggravation in ACLF patients. 18 healthy subjects (HS), 20 chronic HBV-infected (CHB) patients, and 26 ACLF patients were enrolled and examined for TLRs expression in peripheral blood mononuclear cells (PBMCs). The correlations of T cell TLR2 expression with the antigen non-specific Th17 responses and disease aggravation, as well as the Th17 response to TLR2 ligand stimulation were evaluated in ACLF patients. Compared to HS and CHB patients, ACLF patients showed a distinct TLRs expression pattern in PBMCs. Significantly increased TLR2 expression in T cells was observed in ACLF patients. The TLR2 expression in CD4+ T cells was correlated with the Th17 responses and the clinical markers for disease aggravation in ACLF patients. Moreover, TLR2 ligands stimulation promoted Th17 cell differentiation and response in PBMCs of ACLF patients. These findings implicate that TLR2 signaling plays critical roles in Th17 cell differentiation and disease aggravation of HBV-related ACLF.

Molecular characterization of woodchuck IFI16 and AIM2 and their expression in woodchucks infected with woodchuck hepatitis virus (WHV)

IFI16 and AIM2 are important DNA sensors in antiviral immunity. To characterize these two molecules in a woodchuck model, which is widely used to study hepatitis B virus (HBV) infection, we cloned and analyzed the complete coding sequences (CDSs) of woodchuck IFI16 and AIM2, and found that AIM2 was highly conserved in mammals, whereas the degree of sequence identity between woodchuck IFI16 and its mammalian orthologues was low. IFI16 and IFN-β were upregulated following VACV ds 70 mer transfection, while AIM2 and IL-1β were upregulated following poly (dA:dT) transfection, both in vitro and in vivo; IFI16-targeted siRNA decreased the transcription of IFI16 and IFN-β stimulated by VACV ds 70 mer, and AIM2 siRNA interference downregulated AIM2 and IL-1β transcripts stimulated by poly (dA:dT), in vitro, suggesting that woodchuck IFI16 and AIM2 may play pivotal roles in the DNA-mediated induction of IFN-β and IL-1β, respectively. IFI16 and AIM2 transcripts were upregulated in the liver and spleen following acute WHV infection, while IFI16 was downregulated in the liver following chronic infection, implying that IFI16 and AIM2 may be involved in WHV infection. These data provide the basis for the study of IFI16- and AIM2-mediated innate immunity using the woodchuck model.

LSECs express functional NOD1 receptors: A role for NOD1 in LSEC maturation-induced T cell immunity in vitro

HighlightsLSECs stimulated with MDP only can induce the upregulation of the co‐inhibitory molecule PD‐L1.DAP stimulation in vitro could promote LSEC maturation and activate HBV‐specific T cell responses.T cells pre‐primed by DAP‐treated LSECs can inhibit HBV expression and replication in vivo.These results are of particular relevance for the regulation of the local innate immune response against HBV infections. &NA; Liver sinusoidal endothelial cells (LSECs) are organ resident APCs capable of antigen presentation and subsequent tolerization of T cells under physiological conditions. In this study, we investigated whether LSEC pretreatment with NOD‐like receptor (NLR) agonists can switch the cells from a tolerogenic to an immunogenic state and promote the development of T cell immunity. LSECs constitutively express NOD1, NOD2 and RIPK2. Stimulation of LSECs with DAP induced the activation of NF‐&kgr;B and MAP kinases and upregulated the expression of chemokines (CXCL2/9, CCL2/7/8) and cytokines (IFN‐&ggr;, TNF‐&agr; and IL‐2). Pretreatment of LSECs with DAP induced significantly increased IFN‐&ggr; and IL‐2‐production by HBV‐stimulated CD8+ T cells primed by DAP‐treated LSECs. Consistently, a significant reduction in the HBV DNA and HBsAg level occurred in mice receiving T cells primed by DAP‐treated LSECs. MDP stimulation had no impact on LSECs or HBV‐stimulated CD8+ T cells primed with MDP‐treated LSECs except for the upregulation of PD‐L1. DAP stimulation in vitro could promote LSEC maturation and activate HBV‐specific T cell responses. These results are of particular relevance for the regulation of the local innate immune response against HBV infections.

Differential escape of HCV from CD8+ T cell selection pressure between China and Germany depends on the presenting HLA class I molecule

Adaptation of hepatitis C virus (HCV) to CD8+ T cell selection pressure is well described; however, it is unclear if HCV differentially adapts in different populations. Here, we studied HLA class I‐associated viral sequence polymorphisms in HCV 1b isolates in a Chinese population and compared viral substitution patterns between Chinese and German populations. We identified three HLA class I‐restricted epitopes in HCV NS3 with statistical support for selection pressure and found evidence for differential escape pathways between isolates from China and Germany depending on the HLA class I molecule. The substitution patterns particularly differed in the epitope VTLTHPITK1635‐1643, which was presented by HLA‐A*03 as well as HLA‐A*11, two alleles with highly different frequencies in the two populations. In Germany, a substitution in position seven of the epitope was the most frequent substitution in the presence of HLA‐A*03, functionally associated with immune escape and nearly absent in Chinese isolates. In contrast, the most frequent substitution in China was located at position two of the epitope and became the predominant consensus residue. Moreover, substitutions in position one of the epitope were significantly enriched in HLA‐A*11‐positive individuals in China and associated with different patterns of CD8+ T cell reactivity. Our study confirms the differential escape pathways selected by HCV that depended on different HLA class I alleles in Chinese and German populations, indicating that HCV differentially adapts to distinct HLA class I alleles in these populations. This result has important implications for vaccine design against highly variable and globally distributed pathogens, which may require matching antigen sequences to geographic regions for T cell‐based vaccine strategies.