Yizhi Yu

Involvement of ERK, p38 and NF‐κB signal transduction in regulation of TLR2, TLR4 and TLR9 gene expression induced by lipopolysaccharide in mouse dendritic cells

Toll‐like receptors (TLR) are sentinel receptors capable of recognizing pathogen‐associated molecule patterns (PAMP) such as lipopolysaccharide (LPS) and CpG‐containing oligonucleotides (CpG ODN). TLR2 and TLR4 are major receptors for Gram‐positive and Gram‐negative bacterial cell wall components, respectively. TLR9 is necessary for CpG signalling. LPS or CpG ODN can activate immature dendritic cells (DC) and induce DC maturation characterized by production of cytokines, up‐regulation of co‐stimulatory molecules, and increased ability to activate T cells. However, little is known regarding the regulation of TLR gene expression in mouse DC. In this study, we investigated the regulation of TLR2, TLR4 and TLR9 gene expression by LPS in murine immature DC. TLR2, TLR4 and TLR9 mRNA were up‐regulated following LPS stimulation. The up‐regulation of TLR9 expression coincided with significantly increased production of tumour necrosis factor‐α induced by LPS plus CpG ODN. While inhibition of extracellular signal‐related kinase and NF‐κB activation suppressed the up‐regulation of the expression of TLR2, TLR4 and TLR9 mRNA, inhibition of p38 kinase prevented the up‐regulation of TLR2 and TLR4 mRNA expression but enhanced the up‐regulation of TLR9 expression. These results demonstrated that TLR2, TLR4 and TLR9 gene expression was differently regulated by LPS in mouse immature DC. Up‐regulation of TLR2, TLR4 and TLR9 expression by LPS might promote the overall responses of DC to bacteria and help to explain the synergy between LPS and other bacterial products in the induction of cytokine production.

Phosphatase SHP-1 promotes TLR- and RIG-I-activated production of type I interferon by inhibiting the kinase IRAK1

Unbalanced production of proinflammatory cytokines and type I interferons in immune responses may lead to immunopathology; thus, the mechanisms that ensure the beneficial production of proinflammatory cytokines and type I interferons are of particular importance. Here we demonstrate that the phosphatase SHP-1 negatively regulated Toll-like receptor–mediated production of proinflammatory cytokines by inhibiting activation of the transcription factor NF-κB and mitogen-activated protein kinase. Simultaneously, SHP-1 increased the production of type I interferon mediated by Toll-like receptors and the helicase RIG-I by directly binding to and inhibiting activation of the kinase IRAK1. Our data demonstrate that SHP-1 contributes to immune homeostasis by balancing the production of proinflammatory cytokines and type I interferons in the innate immune response.

The Involvement of TNF-α-Related Apoptosis-Inducing Ligand in the Enhanced Cytotoxicity of IFN-β-Stimulated Human Dendritic Cells to Tumor Cells

TNF-α-related apoptosis-inducing ligand (TRAIL) is characterized by its preferential induction of apoptosis of tumor cells but not normal cells. Dendritic cells (DCs), besides their role as APCs, now have been demonstrated to exert cytotoxicity or cytostasis on some tumor cells. Here, we report that both human CD34+ stem cell-derived DCs (CD34DCs) and human CD14+ monocyte-derived DCs (MoDCs) express TRAIL and exhibit cytotoxicity to some types of tumor cells partially through TRAIL. Moderate expression of TRAIL appeared on CD34DCs from the 8th day of culture and was also seen on freshly isolated monocytes. The level of TRAIL expression remained constant until DC maturation. TRAIL expression on immature CD34DCs or MoDCs was greatly up-regulated after IFN-β stimulation. Moreover, IFN-β could strikingly enhance the ability of CD34DCs or MoDCs to kill TRAIL-sensitive tumor cells, but LPS did not have such an effect. The up-regulation of TRAIL on IFN-β-stimulated DCs partially contributed to the increased cytotoxicity of DCs. Pretreatment of TRAIL-sensitive tumor cells with caspase-3 inhibitor could significantly increase their resistance to the cytotoxicity of IFN-β-stimulated DCs. In contrast, NF-κB inhibitor could significantly increase the sensitivity of tumor cells to the killing by nonstimulated or LPS-stimulated DCs. Our studies demonstrate that IFN-β-stimulated DCs are functionally cytotoxic. Thus, an innate mechanism of DC-mediated antitumor immunity might exist in vivo in which DCs act as effectors to directly kill tumor cells partially via TRAIL. Subsequently, DCs act as APCs involved in the uptake, processing, and presentation of apoptotic tumor Ags to cross-prime CD8+ CTL cells.

Efficient induction of antitumor T cell immunity by exosomes derived from heat‐shocked lymphoma cells

Exosomes secreted by tumor cells could serve as a promising immunotherapeutic tumor vaccine. Heat shock proteins (HSP) induced in tumor cells by heat shock are molecular chaperones with potent adjuvant activity in the induction of antigen‐specific T cell responses. To improve exosome‐based tumor vaccines, we have investigated the efficacy of exosomes derived from heat‐shocked mouse B lymphoma cells (HS‐Exo) in the induction of antitumor immune responses. We found that HS‐Exo, compared with control exosomes derived from the same cells (Exo), contain more HSP60 and HSP90 and increased amounts of molecules involved in immunogenicity including MHC class I, MHC class II, CD40, CD86, RANTES and IL‐1β. Furthermore, HS‐Exo induce both phenotypic and functional maturation of dendritic cells more efficiently. HS‐Exo immunization activates T cell responses more potently. Importantly, HS‐Exo induce dramatically increased antitumor immune responses compared to control exosomes from the same cells in prophylaxis and therapeutic in vivo lymphoma models. We further demonstrate that CD8+ T cells are the predominant T cell subset responsible for the antitumor effect of HS‐Exo and that CD4+ T cells are necessary in the induction phase of tumor rejection in a prophylaxis model. These findings provide a novel strategy to improve the efficacy of exosome‐based tumor vaccines.

Circular RNA circMTO1 acts as the sponge of microRNA‐9 to suppress hepatocellular carcinoma progression

Noncoding RNAs play important roles in cancer biology, providing potential targets for cancer intervention. As a new class of endogenous noncoding RNAs, circular RNAs (circRNAs) have been recently identified in cell development and function, and certain types of pathological responses, generally acting as a microRNA (miRNA) sponge to regulate gene expression. Identifying the deregulated circRNAs and their roles in cancer has attracted much attention. However, the expression profile and function of circRNAs in human hepatocellular carcinoma (HCC) remain to be investigated. Here, we analyzed the expression profile of human circRNAs in HCC tissues and identified circMTO1 (mitochondrial translation optimization 1 homologue; hsa_circRNA_0007874/hsa_circRNA_104135) as one circRNA significantly down‐regulated in HCC tissues. HCC patients with low circMTO1 expression had shortened survival. By using a biotin‐labeled circMTO1 probe to perform RNA in vivo precipitation in HCC cells, we identified miR‐9 as the circMTO1‐associated miRNA. Furthermore, silencing of circMTO1 in HCC could down‐regulate p21, the target of oncogenic miR‐9, resulting in the promotion of HCC cell proliferation and invasion. In addition, the tumor‐promoting effect of circMTO1 silencing was blocked by miR9 inhibitor. Intratumoral administration of cholesterol‐conjugated circMTO1 small interfering RNA promoted tumor growth in HCC‐bearing mice in vivo. Conclusion: circMTO1 suppresses HCC progression by acting as the sponge of oncogenic miR‐9 to promote p21 expression, suggesting that circMTO1 is a potential target in HCC treatment. The decrease of circMTO1 in HCC tissues may serve as a prognosis predictor for poor survival of patients. (Hepatology 2017;66:1151‐1164).

Up-regulation of TLR9 gene expression by LPS in mouse macrophages via activation of NF-κB, ERK and p38 MAPK signal pathways

Toll-like receptors (TLR) are critical in the activation of macrophages by bacterial products. It has been shown that TLR2 and TLR4 mediate lipopolysaccharide (LPS) and lipoproteins signal transduction, respectively. Regulation of TLR2 and TLR4 expression by LPS was considered to be one of the mechanisms to control the overall responses of immune cells to bacteria. However, little is known about whether the other members of TLR family are regulated by LPS. Recently, TLR9 was demonstrated to be essential for CpG DNA signaling. Given the effective immune modulation by CpG DNA, regulation of TLR9 expression might play important role in controlling the overall responses of immune cells to bacteria. In this study, regulation of TLR9 gene expression in mouse macrophage cell line RAW264.7 by LPS was investigated. Semiquantitative RT-PCR was performed to determine gene expression of TLR9. Following LPS stimulation, TLR9 gene expression was upregulated within 1 h and reached peak level at about 3 h. LPS stimulation activated NF-kappaB, ERK and p38 MAPK signal pathways. Pretreatment of macrophages with inhibitors of NF-kappaB, ERK and p38 MAPK signal pathways inhibited LPS-induced upregulation of TLR9 mRNA expression. Our results demonstrated that LPS stimulation could upregulate gene expression of TLR9 via NF-kappaB, ERK, and p38 MAPK signal pathways in macrophages, indicating that macrophages with increased TLR9 expression induced by LPS might respond to invading bacteria more effectively.

Molecular cloning and characterization of a novel CXC chemokine macrophage inflammatory protein-2 gamma chemoattractant for human neutrophils and dendritic cells.

Chemokines play important roles in leukocyte trafficking as well as function regulation. In this study, we described the identification and characterization of a novel CXC chemokine from a human dendritic cell (DC) cDNA library, the full-length cDNA of which contains an open reading frame encoding 111 aa with a putative signal peptide of 34 aa. This CXC chemokine shares greatest homology with macrophage inflammatory protein (MIP)-2αβ, hence is designated as MIP-2γ. Mouse MIP-2γ was identified by electrocloning and is highly homologous to human MIP-2γ. Northern blotting revealed that MIP-2γ was constitutively and widely expressed in most normal tissues with the greatest expression in kidney, but undetectable in most tumor cell lines except THP-1 cells. In situ hybridization analysis demonstrated that MIP-2γ was mainly expressed by the epithelium of tubules in the kidney and hepatocytes in the liver. Although no detectable expression was observed in freshly isolated or PMA-treated monocytes, RT-PCR analysis revealed MIP-2γ expression by monocyte-derived DC. Recombinant MIP-2γ from 293 cells is about 9.5 kDa in size and specifically detectable by its polyclonal Ab developed by the immunization with its 6His-tagged fusion protein. The eukaryotically expressed MIP-2γ is a potent chemoattractant for neutrophils, and weaker for DC, but inactive to monocytes, NK cells, and T and B lymphocytes. Receptor binding assays showed that MIP-2γ does not bind to CXCR2. This implies that DC might contribute to the innate immunity through the production of neutrophil-attracting chemokines and extends the knowledge about the regulation of DC migration.

Fas Signal Promotes Lung Cancer Growth by Recruiting Myeloid-Derived Suppressor Cells via Cancer Cell-Derived PGE2

Fas/FasL system has been extensively investigated with respect to its capacity to induce cellular apoptosis. However, accumulated evidences show that Fas signaling also exhibits nonapoptotic functions, such as induction of cell proliferation and differentiation. Lung cancer is one of cancer’s refractory to the immunotherapy, however, the underlying mechanisms remain to be fully understood. In this study, we show that Fas overexpression does not affect in vitro growth of 3LL cells, but promotes lung cancer growth in vivo. However, such tumor-promoting effect is not observed in FasL-deficient (gld) mice, and also not observed in the immune competent mice once inoculation with domain-negative Fas-overexpressing 3LL cells, suggesting the critical role of Fas signal in the promotion of lung cancer growth in vivo. More accumulation of myeloid-derived suppressor cells (MDSC) and Foxp3+ regulatory T cells is found in tumors formed by inoculation with Fas-overexpressing 3LL cells, but not domain-negative Fas-overexpressing 3LL cells. Accordingly, Fas-ligated 3LL lung cancer cells can chemoattract more MDSC but not regulatory T cells in vitro. Furthermore, Fas ligation induces 3LL lung cancer cells to produce proinflammatory factor PGE2 by activating p38 pathway, and in turn, 3LL cells-derived PGE2 contribute to the Fas ligation-induced MDSC chemoattraction. Furthermore, in vivo administration of cyclooxygenase-2 inhibitor can significantly reduce MDSC accumulation in the Fas-overexpressing tumor. Therefore, our results demonstrate that Fas signal can promote lung cancer growth by recruiting MDSC via cancer cell-derived PGE2, thus providing new mechanistic explanation for the role of inflammation in cancer progression and immune escape.

Hepatic RIG-I Predicts Survival and Interferon-α Therapeutic Response in Hepatocellular Carcinoma

In hepatocellular carcinoma (HCC), biomarkers for prediction of prognosis and response to immunotherapy such as interferon-α (IFN-α) would be very useful in the clinic. We found that expression of retinoic acid-inducible gene-I (RIG-I), an IFN-stimulated gene, was significantly downregulated in human HCC tissues. Patients with low RIG-I expression had shorter survival and poorer response to IFN-α therapy, suggesting that RIG-I is a useful prognosis and IFN-α response predictor for HCC patients. Mechanistically, RIG-I enhances IFN-α response by amplifying IFN-α effector signaling via strengthening STAT1 activation. Furthermore, we found that RIG-I deficiency promotes HCC carcinogenesis and that hepatic RIG-I expression is lower in men than in women. RIG-I may therefore be a tumor suppressor in HCC and contribute to HCC gender disparity.

Notch1 Signaling Sensitizes Tumor Necrosis Factor-related Apoptosis-inducing Ligand-induced Apoptosis in Human Hepatocellular Carcinoma Cells by Inhibiting Akt/Hdm2-mediated p53 Degradation and Up-regulating p53-dependent DR5 Expression

Notch signaling plays a critical role in regulating cell proliferation, differentiation, and apoptosis. Our previous study showed that overexpression of Notch1 could inhibit human hepatocellular carcinoma (HCC) cell growth by arresting the cell cycle and inducing apoptosis. HCC cells are resistant to apoptotic induction by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), so new therapeutic approaches have been explored to sensitize HCC cells to TRAIL-induced apoptosis. We are wondering whether and how Notch1 signaling can enhance the sensitivity of HCC cells to TRAIL-induced apoptosis. In this study, we found that overexpression of ICN, the constitutive activated form of Notch1, up-regulated p53 protein expression in HCC cells by inhibiting proteasome degradation. p53 up-regulation was further observed in human primary hepatocellular carcinoma cells after activation of Notch signaling. Inhibition of the Akt/Hdm2 pathway by Notch1 signaling was responsible for the suppression of p53 proteasomal degradation, thus contributing to the Notch1 signaling-mediated up-regulation of p53 expression. Accordingly, Notch1 signaling could make HCC cells more sensitive to TRAIL-induced apoptosis, whereas Notch1 signaling lost the synergistic promotion of TRAIL-induced apoptosis in p53-silenced HepG2 HCC cells and p53-defective Hep3B HCC cells. The data suggest that enhancement of TRAIL-induced apoptosis by Notch1 signaling is dependent upon p53 up-regulation. Furthermore, Notch1 signaling could enhance DR5 expression in a p53-dependent manner. Taken together, Notch1 signaling sensitizes TRAIL-induced apoptosis in HCC cells by inhibiting Akt/Hdm2-mediated p53 degradation and up-regulating p53-dependent DR5 expression. Thus, our results suggest that activation of Notch1 signaling may be a promising approach to improve the therapeutic efficacy of TRAIL-resistant HCC.