Zhigang Tian

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.

Growth inhibition of human hepatocellular carcinoma cells by blocking STAT3 activation with decoy-ODN.

More and more studies show that signal transducer and activator of transcription 3 (STAT3) is frequently constitutively activated in a wide number of malignancies and named as an attractive molecular target for tumor treatment. Here, we employed STAT3-decoy ODN, which specifically block over-activated STAT3, to treat human hepatocellular carcinoma (HCC) cells, and evaluated the cellular proliferation ability and investigated the molecular mechanisms in vitro. The results demonstrated that the proliferation of HCC cells was suppressed significantly by STAT3-decoy ODN, being associated with the increased apoptosis and cell arrest at G0/G1 to S phase transition. Further investigates showed the expression of STAT3-regulated genes including bcl-x1, cyclin D1 and c-myc, which involved in cell apoptosis and cell cycle progression, were down-regulated significantly both at transcription and translation levels. These data suggested that STAT3 may be potentially used as a molecular target in HCC therapy.

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.

Functional characterization of interleukin-15 gene transduction into the human natural killer cell line NKL

BACKGROUND Genetic modification of natural killer (NK) cells is a potential approach to gene-based immunotherapy of cancer. We created human interleukin-15 (hIL-15) gene-modified NKL cells and investigated their functional characterization in vitro. METHODS A recombinant vector (pcDNA3-IL15) or control vector (pcDNA3) was transferred into NKL cells by an electroporation method. Standard reverse transcriptase-polymerase chain reaction (RT-PCR), flow cytometry and MTT methods were performed for NK cell proliferation, apoptosis, cytotoxicity assays and gene expression tests. RESULTS Compared with parental NKL cells, hIL-15 gene modification promoted NK cell proliferation at low doses of IL-2 and inhibited cell apoptosis, which was associated with the up-regulation of anti-apoptosis genes Bcl-2, Bcl-xl and Mcl-1 as well as the down-regulation of apoptosis genes Bim and Noxa. Moreover, the anti-tumor activity of hIL-15 gene-transduced NKL cells against human hepatoma cancer cell line HepG2, H7402 and PLC/PRF-5 cells was enhanced, at least partly, through increasing expression of cytotoxicity-associated genes, including interferon (IFN)-gamma, perforin and FasL. DISCUSSION The hIL-15 genetic modification could improve the proliferation, anti-apoptosis and natural cytotoxicity of NKL cells against hepatocarcinoma cells. These data suggest that hIL-15 gene-modified NKL cells could be useful for clinical cancer immunotherapy in the future.

Opposing effects of interferon‐α and interferon‐γ on the expression of major histocompatibility complex class I chain‐related A in tumors

Natural killer cells are an important component of innate resistance to viruses, bacteria, certain parasites, and tumors. The activating receptor natural killer group 2D (NKG2D) plays a critical role in the elimination of tumor cells by cytotoxic effector cells. It has been shown that the strength of an antitumor immune response might be critically dependent on NKG2D ligard surface levels. Thus, it is essential to regulate the expression of NKG2D ligands in order to ensure effective tumor immunosurveillance and the elimination of pathogen‐infected cells. In the present study, we found that interferon (IFN)‐α and IFNγ exert opposing effects on major histocompatibility complex class I‐related chain A (MICA) expression in human tumor cells. IFNα promotes expression of the NKG2D ligand MICA in tumor cells and therefore enhances their sensitivity to natural killer lysis. In contrast, IFNγ exerts the opposite effect. IFNα promotes MICA expression at the level of transcription by augmenting MICA promoter activity. IFNγ modulates MICA expression not only at the transcriptional level, but also at the post‐translational level by promoting proteolytic cleavage by matrix metalloproteinases. Further study is needed to clarify the precise regulatory mechanisms. The pathways involved in NKG2D ligand induction might represent a promising target for improving immune responses to cancer or infections. (Cancer Sci 2008; 99: 1279–1286)

Small intestinal intraepithelial lymphocytes expressing CD8 and T cell receptor γδ are involved in bacterial clearance during Salmonella enterica serovar Typhimurium infection.

ABSTRACT The intestinal immune system is crucial for the maintenance of mucosal homeostasis and has evolved under the dual pressure of protecting the host from pathogenic infection and coexisting with the dense and diverse commensal organisms in the lumen. Intestinal intraepithelial lymphocytes (iIELs) are the first element of the host T cell compartment available to respond to oral infection by pathogens. This study demonstrated that oral infection by Salmonella enterica serovar Typhimurium promoted the expansion of iIELs, particularly CD8+ TCRγδ+ IELs, enhanced expression of NKG2D on iIELs, increased expression of MULT1, and decreased expression of Qa-1 by intestinal epithelial cells (IECs), leading to activation of, particularly, CD8+ TCRγδ+ iIELs and cytolytic activity against S. Typhimurium -infected IECs. Blockade of NKG2D recognition or depletion of TCRγδ+ cells using a depleting monoclonal antibody significantly attenuated the clearance of S. Typhimurium in the intestine and other tissues. This study suggests that iIELs, particularly CD8+ TCRγδ+ iIELs, play important roles in the detection of pathogenic bacteria and eradication of infected epithelial cells and, thus, provide protection against invading pathogens. These data further our understanding of the mechanisms by which the immune system of the intestinal mucosa discriminates between pathogenic and commensal organisms.

Comparison in the effects of IL-2, IL-12, IL-15 and IFNα on gene regulation of granzymes of human NK cell line NK-92

Serine protease granzymes (GZM) are key effector molecules of granule-mediated cytotoxicity of NK cell, but the requirement for their expression regulation remains largely undefined. In this study, it was showed that IL-2, IL-12, IL-15 and IFNalpha differently regulated the expression of granzymes in NK cells. Although IL-2, IL-12, IL-15 and IFNalpha exerted overlapping positive effects on transcripts of GZM-A and GZM-B, the augment extent and the peaked time course were different for each GZM member after stimulation with these cytokines. Additionally, only IFNalpha up-regulated the transcription of GZM-K, while IL-15 dramatically down-regulated the GZM-H mRNA level. The protein levels of GZM-B and cytotoxicity of NK-92 cells in response to IL-2 or IFNalpha were analyzed, the results showed a positive relationship between cytotoxicity and GZM-B expression level. Together, these findings illustrated that IL-2, IL-12, IL-15 and IFNalpha exerted differential regulating effects on granzyme genes of NK cells.

hIL-15 gene-modified human natural killer cells (NKL-IL15) augments the anti-human hepatocellular carcinoma effect in vivo

Genetic modification of NK cells may provide new possibilities for developing effective cancer immunotherapy by improving NK cell function and specificity. We previously established human interleukin-15 (hIL-15) gene-modified NKL cells (NKL-IL15) and demonstrated their therapeutic efficiency against human hepatocellular carcinoma (HCC) in vitro. To further assess the applicability of NKL-IL15 cells in adoptive cellular immunotherapy, we further investigated their natural cytotoxicity against HCC in vivo in the present study. NKL-IL15 cells exhibited strong inhibition on the growth of transplanted human HCC tumors in xenograft nude mouse models. Further investigation showed that NKL-IL15 cells expressed much higher levels of cytolysis-related molecules, including NKp80, TRAIL, granzyme B, IFN-γ, and TNF-α, than parental NKL cells in response to HCC stimulation. Moreover, soluble mediators secreted by NKL-IL15 cells decreased HCC cell proliferation; in particular, NKL-IL15-derived TNF-α and IFN-γ induced higher NKG2D ligand expression on target cells and resulted in the increased susceptibility of HCCs to NKL-mediated cytolysis. These results show that hIL-15 gene-modified human NK cells can augment the anti-tumor effect of NK cells on human HCC in vivo and suggest their promising applicability as a new candidate for adoptive immunotherapy against HCCs in the future.