Evidence suggests that exosomes can transfer genetic material between cells. However, their roles in hepatitis B virus (HBV) infection remain unclear. Here, we report that exosomes present in the sera of chronic hepatitis B (CHB) patients contained both HBV nucleic acids and HBV proteins, and transferred HBV to hepatocytes in an active manner. Notably, HBV nucleic acids were detected in natural killer (NK) cells from both CHB patients and healthy donors after exposure to HBV-positive exosomes. Through real-time fluorescence microscopy and flow cytometry, 1,1'-dioctadecyl-3,3,3',3',-tetramethylindodicarbocyanine, 4-chlorobenzenesulfnate salt (DiD)-labeled exosomes were observed to interact with NK cells and to be taken up by NK cells, which was enhanced by transforming growth factor-β treatment. Furthermore, HBV-positive exosomes impaired NK-cell functions, including interferon (IFN)-γ production, cytolytic activity, NK-cell proliferation and survival, as well as the responsiveness of the cells to poly (I:C) stimulation. HBV infection suppressed the expression of pattern-recognition receptors, especially retinoic acid inducible gene I (RIG-I), on NK cells, resulting in the dampening of the nuclear factor κB(NF-κB) and p38 mitogen-activated protein kinase pathways. Our results highlight a previously unappreciated role of exosomes in HBV transmission and NK-cell dysfunction during CHB infection.

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NK cells play an important role in early control of HBV infection. The function of NK cells is inhibited in chronic hepatitis B virus (CHB) infection, although the underlying mechanism remains unknown. We found that the expression of STAT3 decreased in peripheral NK cells of CHB patients, and was associated with low levels of degranulation and IFN-secretion. In addition, STAT3 levels were positively correlated with cytolysis-associated molecules and antiviral cytokines, such as CD107a, granzyme B, perforin, and IFN-. HBsAg directly inhibited the expression and activation of STAT3 in NK cells, and knocking down STAT3 expression in NK cells inhibited proliferation, decreased cyclin d1 levels, and suppressed responsiveness to IL-21 stimulation.Furthermore, STAT3 directly bound to the promoter of NKp46, an important activating receptor of NK cells, to regulate its transcription and expression. Taken together, our findings indicate that STAT3 is an important positive regulator of NK cells, and provide a new mechanism of NK cell dysfunction in CHB. K E Y W O R D SCD107a, CHB, granzyme B, IFN-, natural killer cells, NKp46, perforin, STAT3 INTRODUCTIONChronic hepatitis B virus (HBV) infection is a major health problem worldwide, and can trigger a wide spectrum of liver diseases such as liver fibrosis, cirrhosis, and hepatocellular carcinoma (HCC), which are altogether responsible for approximately 1 million deaths annually. 1 The aims of anti-chronic HBV (CHB) therapy are persistent suppression of viral replication, and the prevention of fibrotic progression and oncogenesis. 2 CHB is divided into the hepatitis B envelope Ag (HBeAg)-positive and HBeAg-negative phases. 2 The HBeAg + patients have demonstrated immune tolerance, and HBeAg seroconversion used to be the ideal endpoint of CHB therapy. 3,4 However, recent studies indicate that the HBeAg − CHB patients, including the inactive hepatitis B surface Ag (HBsAg) carriers, have a high risk of progressing to HCC, 5,6 and do not show any immune regulatory mechanism. 2 NK cells are crucial mediators of the innate immune system, and respond to viral infection rapidly without Ag presentation via the Abbreviations: CHB, chronic hepatitis B virus; EGF, epidermal growth factor; HBeAg, hepatitis B envelope Ag; HBsAg, hepatitis B surface Ag; HD, healthy donor; HGF, hepatocyte growth factor; HIES, hyper-IgE syndrome. MHC. 7,8 NK cell functions are regulated by several surface recognition receptors, with both activating and inhibitory functions, 9 which need to be maintained in a balanced state for proper NK cell response. Blocking the activating receptors NKp46 and DNAM-1 impaired NK cell cytotoxicity against cytomegalovirus-infected M s. 10 In CHB patients, the levels of activating receptors such as 2B4 and NKG2D were reduced by TGF-, while that of the inhibitory receptor NKG2A was elevated, resulting in dysfunctional IFN-production and NK cell cytotoxicity. 11-13 HBsAg sero-clearance by pegylated-IFN -2a and nucleos(t)ide analogues restored NK cell function in the HBeAgnegative ...

Hepatitis B virus (HBV) infection is a significant cause of liver disease pathogenesis, which results in the development of hepatic dysfunction, cirrhosis and hepatocellular carcinoma (HCC). Our previous studies showed that oncogene STAT3 might be an ideal target for HCC therapy. Here, we investigated whether targeting blockage of STAT3 signaling is efficient for HBV-related HCC. Based on the refractory of HCC and the persistence of HBV, in this study, we designed shRNAs targeting STAT3. The results showed that blocking STAT3 signaling by shRNAs could promote HBV positive HCC cell apoptosis and induce cell cycle arrest, resulting in HCC cell growth inhibition in vitro. Importantly, STAT3-shRNAs efficiently suppressed HBV replication, which would reduce HBV-derived stimulation to STAT3 signaling and augment STAT3-shRNAs-mediated anti-HCC effect. Finally, STAT3-shRNAs-mediated anti-HBV positive HCC effect was confirmed in xenograft nude mice. This study suggested that targeting STAT3 therapies such as STAT3-shRNAs may be an efficacious strategy for HBV-related HCC.

Many anticancer drugs used in the clinical have potent metal chelating ability. The formed metal complex(es) may exhibit improved (or antagonistic) antitumor activity. However, the underlying mechanism has received limited attention. Therefore, investigation of the mechanism involved in the change upon chelation is required to extend our understanding of the effects of various drugs. In the present study, the proliferation inhibition effect of benzaldehyde nitrogen mustard-2-pyridine carboxylic acid hydrazone (BNMPH) and its copper complex on tumor cell lines was investigated. The copper chelate exhibited almost a 10-fold increase in antitumor activity (with IC50 <5 µM). The results showed that both BNMPH and its copper complex induced reactive oxygen species (ROS) generation, and caused upregulation of caspase 8 and Bax as well as the downregulation of Bcl-2, indicating that apoptosis was involved in the cytotoxic effects. DNA fragmentation noted in the comet assay further supported ROS involvement. The present study indicated that BNMPH and its copper complex effectively induced S phase arrest and the cell cycle arrest was associated with the downregulation of cyclin D1. The formation of acidic vesicular organelles (AVOs) and an increase in cleaved LC3-II demonstrated that autophagy occurred in the HepG2 cells treated with the agents. Taken together, BNMPH and its copper complex exhibited proliferation inhibition via apoptosis, cell cycle arrest and autophagy, which was dependent on ROS. The enhanced antitumor activity of the copper complex was due to its redox-cycling ability, but the mechanism was not altered compared to BNMPH. Our findings may significantly contribute to the understanding of the anti-proliferative effect of BNMPH and its copper complex.