Guomu Liu

Escherichia coli maltose-binding protein activates mouse peritoneal macrophages and induces M1 polarization via TLR2/4 in vivo and in vitro

Maltose-binding protein (MBP) is a component of the maltose transport system of Escherichia coli. Our previous study found that MBP combined with Bacillus Calmette-Guerin (BCG) increases the percentage of activated macrophages in the spleen and the pinocytic activity of peritoneal macrophages in vivo. However, the effect of MBP alone on macrophages remains unclear. In the present study, the results showed that MBP enhanced LPS-stimulated macrophage activity in vivo. Subsequently, we investigated the regulatory effect of MBP on mouse peritoneal macrophages in vitro and the possible underlying mechanism. The results showed that MBP directly promoted macrophage phagocytic activity and increased the production of NO, IL-1β and IL-6. Notably, macrophage phenotypic analysis showed that MBP significantly increased iNOS, IL-12p70 and CD16/32. In contrast, MBP decreased the secretion of IL-10 and slightly decreased Arg-1 mRNA and CD206 protein expression. These results suggested that MBP activated macrophages and polarized them into M1 macrophages. Further study found that MBP directly bound to macrophages and upregulated TLR2 mRNA expression. This process was accompanied by a clear increase in MyD88 expression and phosphorylation of p38 MAPK and IκB-α, but these effects were largely abrogated by pretreatment with anti-TLR2 or anti-TLR4 antibodies. The effects of MBP on macrophage NO production were also partially inhibited by anti-TLR2 and/or anti-TLR4 antibodies. Furthermore, the effect of MBP on IL-12 and IL-10 secretion was largely influenced by the NF-κB inhibitor PDTC and the p38 MAPK inhibitor SB203580. These results suggest that MBP directly activates macrophages and induces M1 polarization through a process that may involve TLR2 and TLR4.

Cytotoxicity of various types of gold-mesoporous silica nanoparticles in human breast cancer cells.

Recently, gold nanoparticles (AuNPs) have shown promising biological applications due to their unique electronic and optical properties. However, the potential toxicity of AuNPs remains a major hurdle that impedes their use in clinical settings. Mesoporous silica is very suitable for the use as a coating material for AuNPs and might not only reduce the cytotoxicity of cetyltrimethylammonium bromide-coated AuNPs but might also facilitate the loading and delivery of drugs. Herein, three types of rod-like gold-mesoporous silica nanoparticles (termed bare AuNPs, core–shell Au@mSiO2NPs, and Janus Au@mSiO2NPs) were specially designed, and the effects of these AuNPs on cellular uptake, toxic behavior, and mechanism were then systematically studied. Our results indicate that bare AuNPs exerted higher toxicity than the Au@mSiO2NPs and that Janus Au@mSiO2NPs exhibited the lowest toxicity in human breast cancer MCF-7 cells, consistent with the endocytosis capacity of the nanoparticles, which followed the order, bare AuNPs > core–shell Au@mSiO2NPs > Janus Au@mSiO2NPs. More importantly, the AuNPs-induced apoptosis of MCF-7 cells exhibited features that were characteristic of intracellular reactive oxygen species (ROS) generation, activation of c-Jun-N-terminal kinase (JNK) phosphorylation, an enhanced Bax-to-Bcl-2 ratio, and loss of the mitochondrial membrane potential. Simultaneously, cytochrome c was released from mitochondria, and the caspase-3/9 cascade was activated. Moreover, both ROS scavenger (N-acetylcysteine) and JNK inhibitor (SP600125) partly blocked the induction of apoptosis in all AuNPs-treated cells. Taken together, these findings suggest that all AuNPs induce apoptosis through the ROS-/JNK-mediated mitochondrial pathway. Thus, Janus Au@mSiO2NPs exhibit the potential for applications in biomedicine, thus aiding the clinical translation of AuNPs.

Mucin1 mediates autocrine transforming growth factor beta signaling through activating the c-Jun N-terminal kinase/activator protein 1 pathway in human hepatocellular carcinoma cells

In a previous study, we observed by global gene expression analysis that oncogene mucin1 (MUC1) silencing decreased transforming growth factor beta (TGF-β) signaling in the human hepatocellular carcinoma (HCC) cell line SMMC-7721. In this study, we report that MUC1 overexpression enhanced the levels of phosphorylated Smad3 linker region (p-Smad3L) (Ser-213) and its target gene MMP-9 in HCC cells, suggesting that MUC1 mediates TGF-β signaling. To investigate the effect of MUC1 on TGF-β signaling, we determined TGF-β secretion in MUC1 gene silencing and overexpressing cell lines. MUC1 expression enhanced not only TGF-β1 expression at the mRNA and protein levels but also luciferase activity driven by a TGF-β promoter, as well as elevated the activation of c-Jun N-terminal kinase (JNK) and c-Jun, a member of the activation protein 1 (AP-1) transcription factor family. Furthermore, pharmacological reduction of TGF-β receptor (TβR), JNK and c-Jun activity inhibited MUC1-induced autocrine TGF-β signaling. Moreover, a co-immunoprecipitation assay showed that MUC1 directly bound and activated JNK. In addition, both MUC1-induced TGF-β secretion and exogenous TGF-β1 significantly increased Smad signaling and cell migration, which were markedly inhibited by either TβR inhibitor or small interfering RNA silencing of TGF-β1 gene in HCC cells. The high correlation between MUC1 and TGF-β1 or p-Smad3L (Ser-213) expression was shown in tumor tissues from HCC patients by immunohistochemical staining analysis. Collectively, these results indicate that MUC1 mediates autocrine TGF-β signaling by activating the JNK/AP-1 pathway in HCC cells. Therefore, MUC1 plays a key role in HCC progression and could serve as an attractive target for HCC therapy.

Escherichia coli maltose-binding protein (MBP) directly induces mouse Th1 activation through upregulating TLR2 and downregulating TLR4 expressions.

Maltose-binding protein (MBP), a component of the maltose transport system of Escherichia coli, has been commonly thought to have minimal bioactivity. Our previous studies demonstrated that MBP could significantly enhance Bacillus Calmette-Guerin (BCG)-induced T helper 1 (Th1) cell activation in mice. In the present study, we analyzed the effect of MBP on mouse T cells and found that MBP promoted the proliferation and IFN-γ production of CD4(+) T cells, suggesting that MBP directly induces Th1 activation. To explore the mechanism of Th1 activation, the expression of Toll-like receptor 2/4 (TLR2/4) on purified mouse CD4(+) T cells was detected. The results showed that MBP up-regulated TLR2 while down-regulated TLR4 expression, accompanied by a clear increase in MyD88 expression and IκB phosphorylation. Notably, the addition of anti-TLR2 antibody abrogated the MBP-induced CD4(+) T cells proliferation, IFN-γ secretion and MyD88 expression, whereas the addition of anti-TLR4 antibody exhibited a contradictive effect. Besides, the block of either TLR2 or TLR4 both reduced IκB phosphorylation. These results above suggest that TLR2-mediated MyD88-dependent pathway contributes to MBP-induced Th1 activation, while TLR4 appears to counteract this effect via MyD88-independent pathway.

Impact of Mucin1 knockdown on the phenotypic characteristics of the human hepatocellular carcinoma cell line SMMC-7721

Mucin1 (MUC1) is a transmembrane glycoprotein that plays a key role as an oncogene in the tumorigenesis of many human adenocarcinomas. However, the role of MUC1 in human hepatocellular carcinoma (HCC) progression remains unclear. In the present study, we silenced MUC1 to investigate its effect on the human HCC cell line SMMC-7721 and found that knockdown of MUC1 significantly inhibited cell proliferation, enhanced cell-cell aggregation and induced apoptosis. No significant differences were found in inxa0vitro migration or invasion. We also observed that knockdown of MUC1 decreased the translocation of β‑catenin to the nucleus, reduced the activity of T cell factor and blocked the expression of cyclinxa0D1 and c-Myc. In addition, MUC1 knockdown enhanced the expression of E-cadherin, a molecular chaperone of β‑catenin that plays an important role in cell-cell aggregation. Inxa0vivo assays demonstrated that there was no tumor growth in mice injected with MUC1-silenced cells. Global gene expression analysis showed that a series of genes encoding molecules in the Wnt/β‑catenin, nuclear factor-κB (NF-κB), mitogen-activated protein kinase (MAPK), insulin, transforming growth factorxa0β (TGF-β) and vascular endothelial growth factor (VEGF) signaling pathways were all influenced by the knockdown of MUC1, and these may contribute to the phenotypic alterations observed. Collectively, our results indicate that MUC1 plays a key role in HCC tumorigenesis.

TLR9 played a more important role than TLR2 in the combination of maltose-binding protein and BCG-induced Th1 activation

Our previous study demonstrated that maltose-binding protein (MBP) combined with BCG induced synergistic mouse Th1 activation in vivo. Here, to explore the mechanism of MBP combined with BCG on Th1 activation, mouse purified CD4+ T cells were stimulated with MBP and BCG in vitro. The results showed that MBP combined with BCG synergistically increased IFN-γ production, accompanied with the upregulation of TLR2/9 expressions, suggesting that TLR2/9 were involved in the combination-induced Th1 activation. Next, TLR2 antibodies and TLR9 inhibitor were used to further analyze the effects of TLRs in Th1 activation. Results showed TLR2 antibody partly decreased MBP combined with BCG-induced IFN-γ production, MyD88 expression and IκB phosphorylation, indicating that TLR2-mediated MyD88-dependent pathway was involved in the MBP combined with BCG-induced Th1 activation. Moreover, MBP combined with BCG-induced Th1 activation was completely abrogated by TLR9 inhibitor, suggesting that TLR9-mediated MyD88-dependent pathway played a more important role than TLR2 in the combination-induced Th1 activation. Further study showed that TLR9 inhibitor downregulated TLR2 expression, suggesting that TLR9 signaling regulated TLR2 activation to favor Th1 resonse induced by MBP combined with BCG. Collectively, we demonstrated for the first time that the cross-talk of TLR2 and TLR9 triggered Th1 activation collaboratively and our findings provided valuable information about designing more effective adjuvant for cancer therapy.

Escherichia coli Maltose-Binding Protein Induces M1 Polarity of RAW264.7 Macrophage Cells via a TLR2- and TLR4-Dependent Manner

Maltose-binding protein (MBP) is a critical player of the maltose/maltodextrin transport system in Escherichia coli. Our previous studies have revealed that MBP nonspecifically induces T helper type 1 (Th1) cell activation and activates peritoneal macrophages obtained from mouse. In the present study, we reported a direct stimulatory effect of MBP on RAW264.7 cells, a murine macrophage cell line. When stimulated with MBP, the production of nitric oxide (NO), IL-1β, IL-6 and IL-12p70, and the expressions of CD80, MHC class II and inducible nitric oxide synthase (iNOS) were all increased in RAW264.7 cells, indicating the activation and polarization of RAW264.7 cells into M1 macrophages induced by MBP. Further study showed that MBP stimulation upregulated the expression of TLR2 and TLR4 on RAW264.7 cells, which was accompanied by subsequent phosphorylation of IκB-α and p38 MAPK. Pretreatment with anti-TLR2 or anti-TLR4 antibodies largely inhibited the phosphorylation of IκB-α and p38 MAPK, and greatly reduced MBP-induced NO and IL-12p70 production, suggesting that the MBP-induced macrophage activation and polarization were mediated by TLR2 and TLR4 signaling pathways. The observed results were independent of lipopolysaccharide contamination. Our study provides a new insight into a mechanism by which MBP enhances immune responses and warrants the potential application of MBP as an immune adjuvant in immune therapies.

The Combination of MBP and BCG-Induced Dendritic Cell Maturation through TLR2/TLR4 Promotes Th1 Activation In Vitro and Vivo

To explore whether TLR2/TLR4 could be involved in the maturation of dendritic cells and polarization of CD4+ T cells induced by dendritic cells stimulated with MBP and BCG, in vitro and in vivo experiments using TLR2−/− or TLR4−/− mice were employed. MBP and BCG elevated CD80, CD86 and MHC class II expressed on dendritic cells and increased IL-12 protein, induced DC maturation, and indirectly promoted Th1 activation. Moreover, MBP and BCG upregulated costimulatory molecules on DCs in a TLR2- and TLR4-dependent manner. The levels of IFN-γ, IL-4, and IL-10 in CD4+ T cells cocultured with dendritic cells from different types of mice were determined with ELISPOT or ELISA method. TLR2/TLR4 is important in the maturation and activation of dendritic cells and the activation of Th1 cells induced by stimulation with MBP and BCG. In conclusion, TLR2 and TLR4 play an important role in the upregulation of costimulatory molecules and MHC class II molecules on dendritic cells and the activation of Th1 cells induced by stimulation with MBP and BCG. The results above indicate that the combination of MBP and BCG induced the maturation and activation of dendritic cells and promoted Th1 activation via TLR2/TLR4.

Escherichia coli maltose-binding protein (MBP) activates mouse Th1 through TLR2-mediated MyD88-dependent pathway and TLR4-mediated TRIF-dependent pathway

Abstracts MBP (maltose‐binding protein) is a component of Escherichia coli. Our previous study found that MBP directly induces the activation of Th1 (T helper type 1), but the molecular mechanism remains unclear. In the present study, CD4+T cells were purified from the spleens of normal mice using antibody‐coated immunomagnetic beads by negative selection. CD4+T cells activated with a CD3/CD28 antibody were stimulated with MBP. The results indicated that MBP elevated IFN‐&ggr; mRNA levels in activated CD4+T cells and promoted IFN‐&ggr; production from activated CD4+T cells. To explore TLR2/TLR4 signaling involved in the mechanism of MBP‐induced activation of Th1, we further detected downstream molecules of TLR2/TLR4 signaling. We found that MBP increased the mRNA levels of MyD88, TRAF6, TRIF and TRAF3 expressed in CD4+T cells. The results suggested that downstream molecules of TLR2/TLR4 signaling may be involved in MBP‐induced activation of CD4+T cells. Furthermore, MyD88, TRIF, TRAF3 and TRAF6 expressed in activated CD4+T cells blocked with anti‐TLR2 antibody or anti‐TLR4 antibody followed by treatment with MBP were detected via RT‐PCR and western blotting, respectively. MBP decreased the production of IFN‐&ggr; in CD4+T cells in the presence of anti‐TLR2, accompanied by the down‐regulated expression of MyD88 and TRAF6. However, MBP increased the production of IFN‐&ggr; in CD4+T cells in the presence of anti‐TLR4 antibody accompanied by the up‐regulated expression of MyD88 and the down‐regulated expression of TRIF, TRAF6 and TRAF3. The results suggested that the MyD88‐dependent pathway of TLR2 and TRIF‐dependent pathway are involved in the mechanism of Th1 activation induced by MBP. Our study has contributed to the clarification of the molecular mechanism of MBP‐induced activation of CD4+T cells. HighlightsMBP elevated mRNA levels of IFN‐&ggr; in activated CD4+T cells and promoted production of IFN‐&ggr; in activated CD4+T cellsDownstream molecules of TLR2/TLR4 signaling may be involved in MBP‐induced activation of CD4+T cells3 MyD88‐dependent pathway of TLR2 and TRIF‐dependent pathway of TLR4 are involved in MBP‐induced activation of Th1

The combination of maltose-binding protein and BCG-induced Th1 activation is involved in TLR2/9-mediated upregulation of MyD88-TRAF6 and TLR4-mediated downregulation of TRIF-TRAF3

Our previous study demonstrated that maltose-binding protein (MBP) activated Th1 through the TLR2-mediated MyD88-dependent pathway and the TLR4-mediated TRIF-dependent pathway. The combination of MBP and BCG synergistically induced Th1 activation, and the TLR2/9-mediated MyD88-dependent pathway is involved in this process. To further explore this mechanism, we stimulated purified mouse CD4+ T cells with MBP and BCG in vitro. The results demonstrated that MBP combined with BCG synergistically increased IFN-γ production and TLR2/4/9 expression, suggesting the involvement of TLR2/4/9 in the combination-induced Th1 activation. Next, TLRs 2/4/9 were blocked to analyze the effects of TLRs on Th1 activation. The results demonstrated that MBP induced a low level of Th1 activation by upregulating TLR2-mediated MyD88-TRAF6 and TLR4-mediated TRIF-TRAF3 expression, whereas MBP combined with BCG induced synergistic Th1 activation, which was not only triggered by strong upregulation of TLR2/9-mediated MyD88-TRAF6 expression but also by shifting TLR4-mediated TRIF-TRAF3 into the TRIF-TRAF6 pathway. Moreover, we observed that a TLR4 antibody upregulated MyD88 expression and a TLR9 inhibitor downregulated TRIF expression, indicating that there was cross-talk between TLRs 2/4/9 in MBP combined with BCG-induced Th1 activation. Our findings may expand the knowledge regarding TLR cross-talk involved in regulating the Th1 response.