Xiaohong Liang

T cell immunoglobulin- and mucin-domain-containing molecule-3 (Tim-3) mediates natural killer cell suppression in chronic hepatitis B

BACKGROUND & AIMS T cell immunoglobulin- and mucin-domain-containing molecule-3 (Tim-3) has been shown to influence autoimmune diseases; however, its function in viral infection has not been well-defined. We therefore investigated the expression and regulatory function of Tim-3 in natural killer (NK) cells in chronic Hepatitis B (CHB) infection. METHODS Seventy-six CHB patients, 38 healthy controls, and 18 patients with fatty liver disease (FLD) were tested for Tim-3 expression on peripheral blood mononuclear cells (PBMCs) and in the liver tissue by flow cytometry and immunohistochemical stainning. The effects of HBV infection on Tim-3 expression in NK cells and the roles of Tim-3 in regulation of NK-cell function were also studied. RESULTS There was a significant increase of Tim-3 expression in PBMCs, circulating NK cells and liver infiltrating lymphocytes (LILs) from CHB patients compared to that of healthy controls and FLD patients. Increased Tim-3 expression was also detected in NK92 cells that had been transfected with a HBV expression vector and NK cells isolated from the liver of HBV transgenic mice. Importantly, blockage of Tim-3 signaling with anti-Tim-3 antibodies or Tim-3-Fc fusion proteins resulted in an increased cytotoxicity for NK92 cells compared to HepG2 and HepG2.2.15 cells, as well as an elevated interferon-gamma (IFN-gamma) production. Similarly, enhanced cytotoxicity was also observed in PBMCs or NK cells from CHB patients treated with the Tim-3 blockade ex vivo. CONCLUSION HBV infection can up-regulate Tim-3 expression in NK cells, which may in turn suppress NK-cell functions in CHB patients.

Hepatitis B Virus Sensitizes Hepatocytes to TRAIL-Induced Apoptosis through Bax

Hepatitis B virus (HBV) infection afflicts >300 million people worldwide and is a leading cause of hepatocyte death, cirrhosis, and hepatocellular carcinoma. While the morphological characteristics of dying hepatocytes are well documented, the molecular mechanisms leading to the death of hepatocytes during HBV infection are not well understood. TRAIL, the TNF-related apoptosis-inducing ligand, has recently been implicated in the death of hepatocytes under certain inflammatory but not normal conditions. To determine the potential roles of TRAIL in HBV-induced hepatitis, we examined the effects of HBV and its X protein (HBx) on TRAIL-induced hepatocyte apoptosis both in vivo and in vitro. We found that hepatitis and hepatic cell death in HBV transgenic mice were significantly inhibited by a soluble TRAIL receptor that blocks TRAIL function. We also found that HBV or HBx transfection of a hepatoma cell line significantly increased its sensitivity to TRAIL-induced apoptosis. The increase in TRAIL sensitivity were associated with a dramatic up-regulation of Bax protein expression. Knocking down Bax expression using Bax-specific small interference RNA blocked HBV-induced hepatitis and hepatocyte apoptosis. The degradation of caspases 3 and 9, but not that of Bid or caspase-8, was preferentially affected by Bax knockdown. These results establish that HBV sensitizes hepatocytes to TRAIL-induced apoptosis through Bax and that Bax-specific small interference RNA can be used to inhibit HBV-induced hepatic cell death.

Roles of TIPE2 in hepatitis B virus-induced hepatic inflammation in humans and mice.

Hepatitis B virus (HBV)-induced hepatic inflammation afflicts hundreds of millions of people worldwide and is a leading cause of hepatic cancer. While the deleterious effect of the chronic hepatitis is well recognized, the molecular mechanisms underlying the pathogenesis of HBV-induced hepatic inflammation are not well understood. We report here that the tumor necrosis factor-alpha-induced protein-8 like-2 (TIPE2 or TNFAIP8L2), a newly identified regulator of immune receptor signaling, plays an important role in controlling HBV-induced hepatitis. Patients with chronic hepatitis B had significantly reduced levels of TIPE2 expression in their peripheral blood mononuclear cells (PBMCs) as compared to healthy individuals. The TIPE2 expression negatively correlated with the blood levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total bilirubin (Tbil) as well as the HBV load of the patients. Importantly, using a murine model of HBV-induced hepatitis, we found that TIPE2-deficient mice developed significantly more severe hepatic inflammation than wild type mice. These results indicate that TIPE2 plays an important role in taming HBV-induced hepatic inflammation.

Tim-3 fosters HCC development by enhancing TGF-β-mediated alternative activation of macrophages

Objective Tumour-associated macrophages (TAMs) and their alternative activation contribute greatly to the development of hepatocellular carcinoma (HCC). Tim-3 is highly expressed on macrophages and regulates macrophage functions in several conditions. However, whether Tim-3 is involved in the activation and the function of TAMs has not been reported. Design Tim-3 expression in HCC samples was evaluated by flow cytometry, immunohistochemistry and confocal analysis. We analysed the effects of Tim-3 knockdown on macrophages in growth of H22 tumour homografts in BALB/c mice. Tim-3 interference was performed by neutralising antibody, small interfering RNA or short hairpin RNA-expressing lentivirus. Cytokine production was evaluated by reverse transcription PCR, ELISA or Cytometric Bead Array. The effects of Tim-3 interference in macrophages were examined with regard to alternative activation of macrophages and proliferation and migration of Hepa1-6 cells. Cell growth curve, colony formation and transwell assays were involved to estimate cell proliferation and migration. Results Tim-3 expression was significantly increased in both peripheral blood monocytes and TAMs in patients with HCC. The Tim-3 expression in monocytes/TAMs strongly correlated with higher tumour grades and the poor survival of patients with HCC. Consistently, HCC conditioned medium or transforming growth factor-β fostered Tim-3 expression and the alternative activation of macrophages. Moreover, Tim-3 interference in macrophages significantly inhibited the alternative activation of macrophages and suppressed HCC cell growth both in vitro and in vivo. Blocking interleukin 6 reversed the Tim-3-mediated effects on HCC cell growth in vitro. Conclusions Tim-3 displays critical roles in microenvironment-induced activation and protumoral effects of TAMs in HCC. Interference of Tim-3 might be great potential in HCC therapy.

Hepatitis B virus core protein inhibits TRAIL-induced apoptosis of hepatocytes by blocking DR5 expression

Hepatitis B virus (HBV) causes chronic hepatitis in hundreds of millions of people worldwide, which can eventually lead to hepatocellular carcinoma (HCC). The molecular mechanisms underlying HBV persistence are not well understood. TRAIL, the TNF-related apoptosis-inducing ligand, has recently been implicated in hepatocyte death during HBV infection. We report here that the HBV core protein (HBc) is a potent inhibitor of TRAIL-induced apoptosis. Overexpressing HBc significantly decreased TRAIL-induced apoptosis of human hepatoma cells, whereas knocking-down HBc expression in hepatoma cells transfected with HBV genome enhanced it. When present in the same cell, HBc blocked the pro-apoptotic effect of the HBV X protein (HBx). The resistance of HBc-expressing cells to TRAIL-induced apoptosis was associated with a significant reduction in death receptor 5 (DR5) expression. Upon transfection, HBc significantly repressed the promoter activity of the human DR5 gene. Importantly, HBc gene transfer inhibited hepatocyte death in a mouse model of HBV-induced hepatitis; and in patients with chronic hepatitis, DR5 expression in the liver was significantly reduced. These results indicate that HBc may prevent hepatocytes from TRAIL-induced apoptosis by blocking DR5 expression, which in turn contributes to the development of chronic hepatitis and HCC. They also call into question the potential side effects of HBc-based vaccines.

Blockade of Tim-3 Pathway Ameliorates Interferon- γ Production from Hepatic CD8 + T Cells in a Mouse Model of Hepatitis B Virus Infection

T cell immunoglobulin- and mucin-domain-containing molecule-3 (Tim-3) has been reported to participate in the pathogenesis of inflammatory diseases. However, whether Tim-3 is involved in hepatitis B virus (HBV) infection remains unknown. Here, we studied the expression and function of Tim-3 in a hydrodynamics-based mouse model of HBV infection. A significant increase of Tim-3 expression on hepatic T lymphocytes, especially on CD8+ T cells, was demonstrated in HBV model mice from day 7 to day 18. After Tim-3 knockdown by specific shRNAs, significantly increased IFN-γ production from hepatic CD8+ T cells in HBV model mice was observed. Very interestingly, we found Tim-3 expression on CD8+ T cells was higher in HBV model mice with higher serum anti-HBs production. Moreover, Tim-3 knockdown influenced anti-HBs production in vivo. Collectively, our data suggested that Tim-3 might act as a potent regulator of antiviral T-cell responses in HBV infection.

Increased Tim-3 expression on peripheral lymphocytes from patients with rheumatoid arthritis negatively correlates with disease activity.

Tim-3 has been reported as an important regulatory molecule and plays a pivotal role in several autoimmunity diseases. Here, we demonstrated the increased expression of Tim-3 on peripheral CD4(+) T, CD8(+) T, NKT cells and monocytes from RA patients compared to those from healthy controls. Percentage of Tim-3(+) cells in peripheral blood mononuclear cells (PBMCs) showed an inverse correlation with disease activity score 28 (DAS28) and plasma TNF-α level. Similar negative correlations were found between disease activity and Tim-3 levels on CD4(+) T, CD8(+) T and NKT cells. Consistently, Tim-3 expression on CD3(+) T cells was further increased in patients with disease remission after treatment. Tim-3 expression on CD8(+) T and NKT cells negatively correlates with plasma TNF-α. Our results suggest that Tim-3 might participate in the proceeding of RA by its negative regulation on various T cell subsets. Tim-3 might be a potential new marker for assessing severity of RA.

Zinc Fingers and Homeoboxes 2 Inhibits Hepatocellular Carcinoma Cell Proliferation and Represses Expression of Cyclins A and E

BACKGROUND & AIMS Zinc-fingers and homeoboxes 2 (ZHX2) represses transcription of several genes associated with liver cancer. However, little is known about the role of ZHX2 in the development of hepatocellular carcinoma (HCC). We investigated the mechanisms by which ZHX2 might affect proliferation of HCC cells. METHODS We overexpressed and knocked down ZHX2 in HCC cells and analyzed the effects on proliferation, colony formation, and the cell cycle. We also analyzed the effects of ZHX2 overexpression in growth of HepG2.2.15 tumor xenografts in nude mice. Chromatin immunoprecipitation and luciferase reporter assays were used to measure binding of ZHX2 target promoters. Levels of ZHX2 in HCC samples were evaluated by immunohistochemistry. RESULTS ZHX2 overexpression significantly reduced proliferation of HCC cells and growth of tumor xenografts in mice; it led to G1 arrest and reduced levels of Cyclins A and E in HCC cell lines. ZHX2 bound to promoter regions of CCNA2 (which encodes Cyclin A) and CCNE1 (which encodes Cyclin E) and inhibited their transcription. Knockdown of Cyclin A or Cyclin E reduced the increased proliferation mediated by ZHX2 knockdown. Nuclear localization of ZHX2 was required for it to inhibit proliferation of HCC cells in culture and in mice. Nuclear localization of ZHX2 was reduced in human HCC samples, even in small tumors (diameter, <5 cm), compared with adjacent nontumor tissues. Moreover, reduced nuclear levels of ZHX2 correlated with reduced survival times of patients, high levels of tumor microvascularization, and hepatocyte proliferation. CONCLUSIONS ZHX2 inhibits HCC cell proliferation by preventing expression of Cyclins A and E, and reduces growth of xenograft tumors in mice. Loss of nuclear ZHX2 might be an early step in the development of HCC.

Acquisition of anoikis resistance reveals a synoikis-like survival style in BEL7402 hepatoma cells.

Resistance to anoikis is a hallmark of human malignancies. Our results showed that hepatoma cells resisted anoikis by non-proliferation, non-apoptosis and cell cycle arrest which were termed synoikis-like. These synoikis-like cells are more resistant to extracellular stimuli and could spontaneously attach and proliferate again under suitable conditions, which indicate a reversible property of these cells. Microarray expression profile reveals the change of molecules involved in the synoikis-like hepatoma cells and our data indicated that ANGPTL4 contributed to anoikis resistance of hepatoma cells. These results demonstrated that hepatoma cells might resist anoikis through a synoikis-like survival style, which may facilitate tumor metastasis.

sTRAIL levels and TRAIL gene polymorphisms in Chinese patients with fatty liver disease

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily, and has been identified as a novel mediator of fatty liver disease (FLD). The aim of our study was to further investigate the relationship between TRAIL and FLD. We found that soluble TRAIL (sTRAIL) concentrations in non-alcoholic FLD (NAFLD) patients were significantly higher than those of controls, and that sTRAIL levels positively correlated with triglyceride concentrations in NAFLD patients. Our results also indicated that the AA/TT genotypes of TRAIL at 1525/1595 engendered a lower risk of FLD attack and a less severe form of steatosis for NAFLD patients in Chinese population. This study provides a means to test for susceptibility to FLD and may assist in the diagnosis of FLD. In addition, we found that 1525G/A and 1595C/T sites were in complete linkage disequilibrium in Chinese population. This might indicate a haplotype with high genetic frequency of TRAIL.