Cai Zhang

Ectopic expression of microRNA-155 enhances innate antiviral immunity against HBV infection in human hepatoma cells

BackgroundHost innate antiviral immunity is the first line of defense against viral infection, and is precisely regulated by thousands of genes at various stages, including microRNAs. MicroRNA-155 (miR-155) was found to be up-regualted during viral infection, and influence the host immune response. Besides, the expression of miR-155, or its functional orthologs, may also contribute to viral oncogenesis. HBV is known to cause hepatocellular carcinoma, and there is evidence that attenuated intracellular immune response is the main reason for HBV latency. Thus, we assume miR-155 may affect the immune response during HBV infection in human hepatoma cells.ResultsWe found that ectopic expression of miR-155 upregulated the expression of several IFN-inducible antiviral genes in human hepatoma cells. And over-expression of miR-155 suppressed suppressor of cytokine signaling 1 (SOCS1) expression and subsequently enhanced signal transducers and activators of transcription1 (STAT1) and signal transducers and activators of transcription3 (STAT3) phosphorylation. We further demonstrate that ectopic expression of miR-155 inhibits HBV X gene expression to some extent in vitro.ConclusionMiR-155 enhances innate antiviral immunity through promoting JAK/STAT signaling pathway by targeting SOCS1, and mildly inhibits HBV infection in human hepatoma cells.

Interleukin-15 improves cytotoxicity of natural killer cells via up-regulating NKG2D and cytotoxic effector molecule expression as well as STAT1 and ERK1/2 phosphorylation

NK cells are crucial components of the innate immune system, providing a first line of defense against infectious pathogens and tumors. IL-15 is the major physiologic growth factor responsible for NK cell differentiation, survival and cytolytic activity of mature NK cells. However, the exact regulatory mechanism of IL-15 on NK cell function is still unclear. In this study, we try to investigate the mechanism of IL-15 on NK cytolysis. Our results demonstrate that IL-15 treatment increased NKG2D transcripts and surface expression in NK cells. NKG2D or MICA blockade attenuated the up-regulation of IL-15 on NK cytolysis, demonstrating that the up-regulatory effect of IL-15 on NK cytolysis is at least partly dependent of the interaction of NKG2D and MICA. Furthermore, IL-15 augmented the expression of cytotoxic effector molecules (TRAIL and Perforin) and the phosphorylation of STAT1 and ERK1/2, which may also contribute the NK lysis. These results may have therapeutic implications when designing cytokine immunotherapy against cancer.

Interleukin-12 improves cytotoxicity of natural killer cells via upregulated expression of NKG2D.

Natural killer (NK) cells are crucial components of the innate immune system, providing the first line of defense against infectious pathogens and tumors. Interleukin (IL)-12 is an interleukin produced primarily by antigen-presenting cells that play an essential role in the interaction between the innate and adaptive arms of immunity acting upon T and NK cells to generate cytotoxic lymphocytes. In the present study, we explored the effect of IL-12 upregulation on the NK receptor NKG2D and on the promotion of NK cell function. IL-12 enhanced the cytotoxicity of NK cells to different solid and hematological tumor cell lines and promoted interferon-gamma secretion by NK cells. The IL-12-induced cytolytic effect was dependent on the interaction of NKG2D with its ligand, MICA, because blockade of either protein attenuated the effect of IL-12 on NK cytolysis. Reverse transcriptase-polymerase chain reaction and fluorescence-activated cell sorting analyses indicated that IL-12 treatment increased NKG2D transcripts and surface expression in NK cells. Also, IL-12 augmented the expression of cytotoxic effector molecules, TRAIL and perforin, and the phosphorylation of STAT1, STAT4, and ERK1/2, which may also contribute to lysis by NK cells. These results are encouraging for the potential use of IL-12 as part of immunotherapy.

JSI-124 inhibits glioblastoma multiforme cell proliferation through G(2)/M cell cycle arrest and apoptosis augment.

JSI-124(cucurbitacin I) is a selective inhibitor of Janus kinase /Signal transducer and activator of transcription 3(JAK/STAT3) and has been shown to exert anti-proliferative and anti-tumor properties both in vitro and in vivo. As STAT3 activation has been implicated in the development of glioma, we investigated the therapeutic efficacy of JSI-124 on glioblastoma multiforme (GBM) by interfering with STAT3 pathway. In present study, two GBM cell lines, U251 and A172 cells, were treated with JSI-124. The results showed that the cell growth was inhibited significantly in a dose-and time-dependent manner. Further investigation illustrated that the levels of phosphorylated-STAT3 were decreased in GBM cells treated by JSI-124, concomitant with apoptosis augment and cell cycle arrest. Specially, JSI-124 induced G2/M accumulation via down-regulation of cyclin B1 and cdc2 expression. Together these results suggested that inhibition of STAT3 by JSI-124 is a potential strategy for the development of the new glioblastoma multiforme theraputics.

Sodium butyrate upregulates expression of NKG2D ligand MICA/B in HeLa and HepG2 cell lines and increases their susceptibility to NK lysis

Natural killer (NK) cells are important effectors in the immune response to tumors. A number of cell-surface inhibitory and activating receptors on NK cells tightly regulate their interaction with target cell ligands. In particular, the strength of an anti-tumor immune response appears to depend critically on surface levels of one activating receptor, NKG2D. Correspondingly, expression of NKG2D ligands on target cells is a requirement for effective tumor immunosurveillance and the elimination of pathogen-infected cells. Sodium butyrate, a potent repressor of histone deacetylase (HDAC), has recently been proposed as a potential agent in cancer treatment based on its ability to modify, in several cancer cell types, the expression of a variety of genes related to cell cycle regulation and apoptosis. Here we report that, in the HeLa and HepG2 tumor cell lines, sodium butyrate upregulated the expression of the MHC class I-related chain molecules A and B (MICA and MICB) at both the mRNA and protein levels, resulting in an enhanced susceptibility of cells in both lines to NK lysis. It also led to an elevated expression of heat shock protein 70 (HSP70) and transcription factor Sp1, and increased the binding of transcription factors Sp1 and heat shock transcription factor 1 (HSF1) to the MICA/B promoter, resulting in increased expression of MICA and MICB. siRNA targeting Sp1 significantly attenuate the enhancement of MICA expression by sodium butyrate. These results suggest that sodium butyrate and other HDAC inhibitors may have therapeutic potential by enhancing the immune response to cancer.

NK cell receptor imbalance and NK cell dysfunction in HBV infection and hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is currently the third leading cause of cancer mortality and a common poor-prognosis malignancy due to postoperative recurrence and metastasis. There is a significant correlation between chronic hepatitis B virus (HBV) infection and hepatocarcinogenesis. As the first line of host defense against viral infections and tumors, natural killer (NK) cells express a large number of immune recognition receptors (NK receptors (NKRs)) to recognize ligands on hepatocytes, liver sinusoidal endothelial cells, stellate cells and Kupffer cells, which maintain the balance between immune response and immune tolerance of NK cells. Unfortunately, the percentage and absolute number of liver NK cells decrease significantly during the development and progression of HCC. The abnormal expression of NK cell receptors and dysfunction of liver NK cells contribute to the progression of chronic HBV infection and HCC and are significantly associated with poor prognosis for liver cancer. In this review, we focus on the role of NK cell receptors in anti-tumor immune responses in HCC, particularly HBV-related HCC. We discuss specifically how tumor cells evade attack from NK cells and how emerging understanding of NKRs may aid the development of novel treatments for HCC. Novel mono- and combination therapeutic strategies that target the NK cell receptor–ligand system may potentially lead to successful and effective immunotherapy in HCC.Cellular & Molecular Immunology advance online publication, 6 October 2014; doi:10.1038/cmi.2014.91

Reversal of hepatitis B virus-induced immune tolerance by an immunostimulatory 3p-HBx-siRNAs in a retinoic acid inducible gene I-dependent manner.

It is extensively accepted that hepatitis B virus (HBV) escapes from innate immunity by inhibiting type I interferon (IFN) production, but efficient intervention to reverse the immune tolerance is still not achieved. Here, we report that 5′‐end triphosphate hepatitis B virus X gene (HBx)‐RNAs (3p‐HBx‐short interfering [si]RNAs) exerted significantly stronger inhibitory effects on HBV replication than regular HBx‐siRNAs in stably HBV‐expressing hepatoplastoma HepG2.2.15 cells through extremely higher expression of type I IFNs, IFN‐induced genes and proinflammatory cytokines, and retinoic acid inducible gene I (RIG‐I) activation. Also, 3p‐HBx‐siRNA were more efficient to stimulate type I IFN response than HBx sequence‐unrelated 3p‐scramble‐siRNA in HepG2.2.15 cells, indicating that a stronger immune‐stimulating effect may partly result from the reversal of immune tolerance through decreasing HBV load. In RIG‐I‐overexpressed HepG2.2.15 cells, 3p‐HBx‐siRNAs exerted stronger inhibitory effects on HBV replication with greater production of type I IFNs; on the contrary, in RIG‐I‐silenced HepG2.2.15 cells or after blockade of IFN receptor by monoclocnal antibody, inhibitory effect of 3p‐HBx‐siRNAs on HBV replication was largely attenuated, indicating that immunostimulatory function of 3p‐HBx‐siRNAs was RIG‐I and type I IFN dependent. Moreover, in HBV‐carrier mice, 3p‐HBx‐siRNA more strongly inhibited HBV replication and promoted IFN production than HBx‐siRNA in primary HBV+ hepatocytes and, therefore, significantly decreased serum hepatitis B surface antigen and increased serum IFN‐β. Conclusion: 3p‐HBx‐siRNAs may not only directly inhibit HBV replication, but also stimulate innate immunity against HBV, which are both beneficial for the inversion of HBV‐induced immune tolerance. (HEPATOLOGY 2011;)

Histone deacetylase inhibitor SAHA epigenetically regulates miR-17-92 cluster and MCM7 to upregulate MICA expression in hepatoma.

Background:Epigenetic therapy using histone deacetylase inhibitors (HDACi) has shown promise in clinical trials for the treatment of human malignancies. In addition to the immediate effects on the tumour cell growth, HDACi upregulates the expression of MHC class I-related chain molecules A and B (MICA and MICB), resulting in an enhanced susceptibility of tumour cells to natural killer cell-mediated lysis. The molecular mechanism underlying is still unclear.Methods:The transcriptional regulation mechanism underlying suberoylanilide hydroxamic acid (SAHA)-mediated regulation of MICA and related miRNA expression was investigated using promoter acetylation assays, bioinformatics analysis and chromatin immunoprecipitation assay.Results:SAHA upregulates the transcription of MICA/B by promoting MICA-associated histone acetylation while suppressing the MICA/B-targeting miRNAs miR-20a, miR-93 and miR-106b. The mechanism by which SAHA repressed miRNAs transcription involved repression of their host genes (miR-17-92 cluster and MCM7). SAHA downregulated the miR-17-92 cluster by abolishing tyrosine phosphorylation of STAT3 and decreased MCM7 transcription through localised histone deacetylation.Conclusions:The HDACi SAHA epigenetically upregulates MICA expression through regulating the expression of miR-17-92 cluster and MCM7 in hepatoma, thus enhancing the sensitivity of HCC to natural killer cell-mediated lysis. This novel mechanism of action provides promise for HDACi in therapy of HCC.

The TLR7 agonists imiquimod and gardiquimod improve DC-based immunotherapy for melanoma in mice

Toll-like receptors (TLRs) are a family of highly conserved germline-encoded pattern-recognition receptors that are essential for host immune responses. TLR ligands represent a promising class of immunotherapeutics or vaccine adjuvants with the potential to generate an effective antitumor immune response. The TLR7/8 agonists have aroused interest because they not only activate antigen-presenting cells but also promote activation of T and natural killer (NK) cells. However, the exact mechanism by which stimulation of these TLRs promotes immune responses remains unclear, and different TLR7/8 agonists have been found to induce different responses. In this study, we demonstrate that both gardiquimod and imiquimod promote the proliferation of murine splenocytes, stimulate the activation of splenic T, NK and natural killer T (NKT) cells, increase the cytolytic activity of splenocytes against B16 and MCA-38 tumor cell lines, and enhance the expression of costimulatory molecules and IL-12 by macrophages and bone marrow-derived dendritic cells (DCs). In a murine model, both agonists improved the antitumor effects of tumor lysate-loaded DCs, resulting in delayed growth of subcutaneous B16 melanoma tumors and suppression of pulmonary metastasis. Further, we found that gardiquimod demonstrated more potent antitumor activity than imiquimod. These results suggest that TLR7/8 agonists may serve as potent innate and adaptive immune response modifiers in tumor therapy. More importantly, they can be used as vaccine adjuvants to potentiate the efficiency of DC-based tumor immunotherapy.

Therapeutic recovery of hepatitis B virus (HBV)-induced hepatocyte-intrinsic immune defect reverses systemic adaptive immune tolerance†‡

Hepatitis B virus (HBV) persistence aggravates hepatic immunotolerance, leading to the failure of cell‐intrinsic type I interferon and antiviral response, but whether and how HBV‐induced hepatocyte‐intrinsic tolerance influences systemic adaptive immunity has never been reported, which is becoming the major obstacle for chronic HBV therapy. In this study, an HBV‐persistent mouse, established by hydrodynamic injection of an HBV‐genome‐containing plasmid, exhibited not only hepatocyte‐intrinsic but also systemic immunotolerance to HBV rechallenge. HBV‐specific CD8+ T‐cell and anti‐HBs antibody generation were systemically impaired by HBV persistence in hepatocytes. Interestingly, HBV‐induced hepatocyte‐intrinsic immune tolerance was reversed when a dually functional vector containing both an immunostimulating single‐stranded RNA (ssRNA) and an HBx‐silencing short hairpin RNA (shRNA) was administered, and the systemic anti‐HBV adaptive immune responses, including CD8+ T‐cell and anti‐HBs antibody responses, were efficiently recovered. During this process, CD8+ T cells and interferon‐gamma (IFN‐γ) secreted play a critical role in clearance of HBV. However, when IFN‐α/β receptor was blocked or the Toll‐like receptor (TLR)7 signaling pathway was inhibited, the activation of CD8+ T cells and clearance of HBV was significantly impaired. Conclusion: These results suggest that recovery of HBV‐impaired hepatocyte‐intrinsic innate immunity by the dually functional vector might overcome systemic adaptive immunotolerance in an IFN‐α‐ and TLR7‐dependent manner. The strategy holds promise for therapeutic intervention of chronic persistent virus infection and associated cancers. (Hepatology 2013;)