Dopamine Inhibits the Expression of Hepatitis B Virus Surface and e Antigens by Activating the JAK/STAT Pathway and Upregulating Interferon-stimulated Gene 15 Expression.

Background and Aims: Hepatitis B virus (HBV) infection is a major risk factor for cirrhosis and liver cancer, and its treatment continues to be difficult. We previously demonstrated that a dopamine analog inhibited the packaging of pregenomic RNA into capsids. The present study aimed to determine the effect of dopamine on the expressions of hepatitis B virus surface and e antigens (HBsAg and HBeAg, respectively) and to elucidate the underlying mechanism. Methods: We used dopamine-treated HBV-infected HepG2.2.15 and NTCP-G2 cells to monitor HBsAg and HBeAg expression levels. We analyzed interferon-stimulated gene 15 (ISG15) expression in dopamine-treated cells. We knocked down ISG15 and then monitored HBsAg and HBeAg expression levels. We analyzed the expression of Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway factors in dopamine-treated cells. We used dopamine hydrochloride-treated adeno-associated virus/HBV-infected mouse model to evaluate HBV DNA, HBsAg, and HBeAg expression. HBV virus was collected from HepAD38.7 cell culture medium. Results: Dopamine inhibited HBsAg and HBeAg expression and upregulated ISG15 expression in HepG2.2.15 and HepG2-NTCP cell lines. ISG15 knockdown increased HBsAg and HBeAg expression in HepG2.2.15 cells. Dopamine-treated cells activated the JAK/STAT pathway, which upregulated ISG15 expression. In the adeno-associated virus-HBV murine infection model, dopamine downregulated HBsAg and HBeAg expression and activated the JAK-STAT/ISG15 axis. Conclusions: Dopamine inhibits the expression of HBsAg and HBeAg by activating the JAK/STAT pathway and upregulating ISG15 expression.

Erythroid-transdifferentiated myeloid cells promote portal vein tumor thrombus in hepatocellular carcinoma.

Rationale: Hepatocellular carcinoma (HCC) is primarily characterized by a high incidence of vascular invasion. However, the specific mechanism underlying portal vein tumor thrombus (PVTT) in HCC remains unclear. As a consequence of myeloid cell developmental arrest, CD71+ erythroid progenitor cells (EPCs) and myeloid-derived suppressor cells play important roles in HCC; however, their roles in PVTT remain unclear. Methods: The role of CD71+ EPCs in the HCC tumor microenvironment (TME) was evaluated via morphological, RNA-sequencing, enzyme-linked immunosorbent assay, and flow cytometric analyses. Co-culture techniques were employed to assess the CD45+ EPCs and their vascular compromising effect. Additionally, the PVTT-promoting function of CD45+ EPCs was explored in vivo in a murine model. Results: The CD45+EPCs in HCC tissues exhibited increased myeloid cell features, including morphology, surface markers, transforming growth factor (TGF)-ß generation, and gene expression, compared with those in circulation. Hence, a large proportion of CD45+EPCs, particularly those in TMEs, comprise erythroid-transdifferentiated myeloid cells (EDMCs). Additionally, the expression of C-C chemokine receptor type 2 (CCR2) mRNA was upregulated in CD45+EPCs within the TME. Tumor macrophages from HCC tissues induced substantial migration of CD45+EPCs in a dose-dependent manner. Meanwhile, results from immunofluorescence analyses revealed that these two cell types are positively associated in the TME and circulation. That is, EDMCs are chemoattracted by HCC macrophages mainly via CCR2 from CD45+ EPCs in the circulation. Additionally, the expressions of FX, FVII, FGB, C4b, CFB, and CFH were elevated in CD45+EPCs within the TME compared with those in the spleen. The CD45+EPCs from the HCC TME promoted vessel endothelial cell migration and compromised tube formation through TGF-ß and FGB, respectively. Additionally, CD45+EPCs from the TME induced HCC cell migration. HCC macrophage-induced CD45+EPCs to exhibit higher levels of FX, FVII, FGB, and TGF-ß. Meanwhile, upregulation of CCAAT/enhancer binding protein beta expression induced FGB and TGF-ß generation in CD45+EPCs in the TME. WTAP, a major RNA m6A writer, stabilized FX and FVII mRNA and enhanced their nuclear export in CD45+EPCs from the TME. CD45+EPCs from the TME were positively associated with PVTT and poor prognosis. Splenectomy reduced the level of CD45+EPCs in the circulation and TME, as well as the incidence of microvascular invasion. The incidence of microvascular invasion increased following the transfer of HCC tissue CD45+EPCs to splenectomized HCC-bearing mice. Conclusions: The CD45+EPCs enriched in the HCC microenvironment are EDMCs, which are induced by HCC macrophages to migrate from the circulation to the TME. Subsequently, EDMCs promote PVTT by compromising the blood vessel endothelium, aggravating coagulation, and promoting HCC cell migration.

LAG3+ erythroid progenitor cells inhibit HBsAg seroclearance during finite pegylated interferon treatment through LAG3 and TGF-ß.

HBsAg seroclearance, the ideal aim of anti-hepatitis B virus (HBV) treatment, cannot be achieved easily. Anemia is another common issue for chronic hepatitis B (CHB) patients, which leads to elevation of erythroid progenitor cells (EPCs) and immune suppression in cancer. This study investigated the role of EPCs in HBsAg seroclearance following pegylated interferon-α (PEG-IFN) treatment. CD45+EPC accumulation in CHB patients and an AAV/HBV mice model was found in the circulation and liver by flow cytometry and immunofluorescence tests. Wright-Giemsa staining showed that these pathological CD45+EPCs presented elevated erythroid cells with relative immature morphologies and atypical cells compared with the control cells. CD45+EPCs were associated with immune tolerance and decreased HBsAg seroclearance during finite PEG-IFN treatment. CD45+EPCs suppressed antigen non-specific T cell activation and HBV-specific CD8+T cells, partially through transforming growth factor ß (TGF-ß). RNA-seq revealed that CD45+EPCs in patients with CHB presented a distinct gene expression profile compared with CD45-EPCs and CD45+EPCs from cord blood. Notably, CD45+EPCs from patients with CHB expressed high level of Lymphocyte-activation gene 3 (LAG3), an immune checkpoint molecule, and were then defined as LAG3+EPCs. LAG3+EPCs diminished the function of antigen presenting cells through LAG3, which was another mechanism by which LAG3+EPCs' suppressed HBV-specific CD8+T cells. Anti-LAG3 and anti-TGF-ß combination treatment decreased serum HBeAg, HBV DNA levels and HBsAg level, as well as HBsAg-expression in hepatocytes during PEG-IFN treatment in the AAV/HBV mice model. Conclusions: LAG3+EPCs inhibited the efficacy of PEG-IFN treatment on HBsAg seroclearance induced by LAG3 and TGF-ß. Anti-LAG3, anti-TGF-ß and PEG-IFN combination treatment might facilitate HBV clearance.

Reduction in Intrahepatic cccDNA and Integration of HBV in Chronic Hepatitis B Patients with a Functional Cure.

Background and Aims: Functional cure (FC) is characterized by the clearance of the hepatitis B surface antigen from the serum of patients with chronic hepatitis B (CHB). However, the level of intrahepatic covalently closed circular DNA (cccDNA) and hepatitis B virus (HBV) integration remains unclear. We conducted this study to determine them and reveal their value in the treatment of CHB. Methods: There were two sessions to elucidate the changes in intrahepatic cccDNA and HBV integration after antiviral therapy. In the first session, 116 patients were enrolled and divided into FC, non-functional cure (NFC), and CHB groups, including 48 patients with functionally cured CHB, 27 with CHB without functional cure after antiviral treatment, and 41 with treatment-naïve CHB. Patients were tested for both intrahepatic cccDNA and other viral markers. All patients in the FC group were followed up for at least 24 weeks to observe relapse. In the second session, another ten patients were included for in-depth whole-genome sequencing to analyze HBV integration. Results: Thirteen patients in the FC group were negative for intrahepatic cccDNA. Intrahepatic cccDNA was much higher in the CHB group compared with the FC group. Seven patients had HBsAg seroreversion, including two with virological relapse. Integration of HBV was detected in one (33.3%) functionally cured patients and in seven (100%) with CHB. 28.0% of the HBV breakpoints were assigned in the 1,500 nt to 1,900 nt range of the HBV genome. Conclusions: After achieving an FC, the rate of intrahepatic cccDNA and HBV integration was significantly reduced in patients with CHB. For those patients who cleared intrahepatic cccDNA, the chances of developing virological relapse were even lower.

Unsupervised Clustering Reveals Distinct Subtypes of Biliary Atresia Based on Immune Cell Types and Gene Expression.

Background: Biliary atresia (BA) is a severe cholangiopathy of early infancy that destroys cholangiocytes, obstructs ductular pathways and if left untreated, culminates to liver cirrhosis. Mechanisms underlying the etiological heterogeneity remain elusive and few studies have attempted phenotyping BA. We applied machine learning to identify distinct subtypes of BA which correlate with the underlying pathogenesis. Methods: The BA microarray dataset GSE46995 was downloaded from the Gene Expression Omnibus (GEO) database. Unsupervised hierarchical cluster analysis was performed to identify BA subtypes. Then, functional enrichment analysis was applied and hub genes identified to explore molecular mechanisms associated with each subtype. An independent dataset GSE15235 was used for validation process. Results: Based on unsupervised cluster analysis, BA patients can be classified into three distinct subtypes: Autoimmune, Viral and Embryonic subtypes. Functional analysis of Subtype 1 correlated with Fc Gamma Receptor (FCGR) activation and hub gene FCGR2A, suggesting an autoimmune response targeting bile ducts. Subtype 2 was associated with immune receptor activity, cytokine receptor, signaling by interleukins, viral protein interaction, suggesting BA is associated with viral infection. Subtype 3 was associated with signaling and regulation of expression of Robo receptors and hub gene ITGB2, corresponding to embryonic BA. Moreover, Reactome pathway analysis showed Neutrophil degranulation pathway enrichment in all subtypes, suggesting it may result from an early insult that leads to biliary stasis. Conclusions: The classification of BA into different subtypes improves our current understanding of the underlying pathogenesis of BA and provides new insights for future studies.

IFI16 is involved in HBV-associated acute-on-chronic liver failure inflammation.

BACKGROUND: Hepatitis B virus (HBV) is a hepatotropic DNA virus, and its DNA may be a potent inflammatory molecule. Interferon-inducible protein 16 (IFI16), a newly discovered DNA sensor, plays an important role in the process of inflammation in viral infections. Our study sought to identify a correlation between IFI16 expression and inflammation in patients with chronic hepatitis B (CHB) and HBV-associated acute-on-chronic liver failure (HBV-ACLF). METHODS: We performed flow cytometry to measure IFI16 levels in peripheral blood mononuclear cells (PBMC) and used immunohistochemistry and western blotting to measure IFI16 protein levels in liver tissues. The cellular source of IFI16 was detected using double immunofluorescence. All datum were analyzed using SPSS 13.0 and GraphPad Prism 6. RESULTS: The number of IFI16+ cells was significantly associated with the degree of inflammation. In detail, the number of IFI16+ cells was higher in livers but lower in PBMCs in HBV-ACLF patients than those in CHB patients and healthy controls. There was no significant difference between CHB patients and healthy controls in numbers of IFI6+ cells in livers and PBMCs. There was no significant relationship between IFI16 expression levels and HBV parameters. Furthermore, IFI16 was expressed in the nucleus of Kupffer cells (KCs), endothelial cells, natural killer cells, dendritic cells, and hepatic stellate cells in healthy donors and CHB patients, but only in the cytoplasm of KCs in the livers of HBV-ACLF patients. CONCLUSIONS: IFI16 was closely related to the degree of inflammation in CHB and HBV-ACLF patients and may serve as a vital contributor to the pathogeneses of liver damage in HBV-ACLF.

Soluble ST2 plasma concentrations predict mortality in HBV-related acute-on-chronic liver failure.

Hepatitis B virus-related acute-on-chronic liver failure (HBV-ACLF) is a rapidly progressing and frequently fatal condition. The aim of this study was to determine whether interleukin- (IL-) 33 and soluble ST2 (sST2) were associated with disease severity and mortality in HBV-ACLF. We found that plasma levels of sST2 but not IL-33 were higher in HBV-ACLF patients compared with chronic hepatitis B (CHB) patients and healthy controls. However, plasma levels of IL-33, TNF-α, IFN-γ, and IL-10 did not correlate with sST2 levels. Similarly, immunohistochemistry revealed low IL-33 expression and high ST2 expression in liver sections of patients with HBV-ACLF. Evaluation of dynamic changes of sST2 in HBV-ACLF showed that plasma sST2 levels increased over time in patients who died during the 180-day follow-up but decreased in those who survived. In addition, plasma sST2 level after week 1 correlated with disease severity, as assessed by total bilirubin, prothrombin time, and model for end-stage liver disease score. Results of Kaplan-Meier survival analysis showed that higher sST2 concentration (≥87 ng/mL) at week 3 was associated with poor survival. These findings indicate the potential usefulness of sST2 as a predictor of disease severity and in making treatment decisions for patients with HBV-ACLF.