The Combination Of Escherichia Coli Maltose Binding Protein And BCG-induced Dendritic Cell Maturation Promote Th1 Activation Through TLR2/TLR4

MBP（Escherichia coli maltose-binding protein）encoded by the mal E gene is responsible for the capture and transport of maltodextrins in E.coli（Escherichia coli）.Previously,MBP was deemed to have minimum effects on bioactivity and is frequently used as a protein tag.Recent studies have characterized the immunological attributes of MBP.Our previous experiments indicated that MBP induced the activation of Th1 cells,NK cells and macrophages,indicating that MBP has potent immune-enhancing activities.Furthermore,we found that MUC1 and BCG show better antitumor effects after being fused to MBP and the combination of MBP and BCG can induce Th1 activation synergistically and significantly increase the IFN-γproduction of lymphocytes.Furthermore,we found that MUC1 and BCG show better antitumor effects after being fused to MBP and the combination of MBP and BCG can induce Th1 activation synergistically and significantly increase the IFN-γproduction of lymphocytes.Our latest studies showed that Th1 polarization and TLR2/TLR4/TLR9 activation were synergistically induced by the combination of MBP and BCG and were the first to reveal that the cross-talk between TLR signaling pathways was associated with the activation of Th1 cells by the combination of MBP and BCG.DCs are generally viewed as the most potent professional APCs（antigen presenting cells）.Various microbial components induce the activation of DCs（dendritic cells）through TLR（Toll-like receptor）signaling,which plays a critical role in the generation of protective immune responses.However,very little is known about the function and maturation of DCs that are induced by the combined effects of MBP and BCG and promote Th1 type immunity.To clarify the molecular mechanism of MBP or the combination of MBP and BCG and its potential use as a TLR2/TLR4 agonist in DC-based immune therapies,we mainly investigated the synergistic effect of the combination of MBP and BCG on the maturation and function of DCs.Furthermore,our findings highlight MBP as a TLR2/TLR4 agonist that favors DCinduced Th1 polarization indirectly.The combination of MBP and BCG-induced DC maturation promote Th1 activation in vitro1 Isolation of dendritic cells and CD4⁺T cellsThe spleen samples were took out from normal mice,and the single-cell suspensions were prepared by enzymatic disaggregation with Collagenase D.The mononuclear cells were isolated from spleen with mouse percoll.Then one part of mononuclear cells were prepared to isolate the dendritic cell after washing with PBS,the other part of mononuclear cells were prepared to isolate CD4⁺T cells.The mononuclear cells were added with anti-CD11 C micro Beads and labeled with specific anti-CD11 C micro Beads is loaded onto a MACS column using positive selection.The procedure of isolation of CD4⁺T cells from spleens was performed using negative selection.Then the purities of dendritic cells and CD4⁺T cells were analyzed by Flow cytometry,respectively.2 The combination of MBP and BCG induced the duration and activation of DCs in vitroTo determine if the combination of MBP and BCG had an effect on the activation and maturation of DCs,we characterized the expression of co-stimulatory molecules and MHC II molecules on DCs from normal mice following stimulation with MBP,BCG or the combination of MBP and BCG.DCs stimulated with MBP showed greater CD80 and CD86 expression than DCs in the control group.Similarly,BCG increased the percentage of CD11C+ cells expressing CD80 and CD86.In particular,the combination of MBP and BCG synergistically increased the percentage of CD11C+ cells expressing CD80 and CD86,compared to the DCs in the control group.In addition,MBP or BCG increased the expression of MHC class II on the surface of DCs relative to the DCs in the control group,and the combination of MBP and BCG significantly increased the percentage of cells expressing the surface molecule MHC class II.In addition,we detect the level of IL-12p70 protein in supernatant of DCs from WT mice treatment with MBP,BCG and the combination of MBP and BCG.Both MBP and BCG increased the production of IL-12p70 protein compared with control group.The combination of MBP and BCG synergistically increased the level ofIL-12p70 protein.These results demonstrated that the combination of MBP and BCG induced a more mature phenotype.Taken together,MBP and BCG synergistically induced the activation and maturation of DCs.3 MBP and BCG synergistically induced Th1 activation in vitroTo investigate whether DCs treated with the combination of MBP and BCG affect the proliferation of CD4⁺T cells,the proliferation of pure CD4⁺T cells co-cultured with DCs from normal mice was detected with CCK8 kits.CD4⁺T cells co-cultured with DCs derived from WT mice were incubated with MBP（10 μg/ml）,BCG（20 μg/ml）and the combination of MBP（10 μg/ml）and BCG（20 μg/ml）for 48 hours.MBP,BCG and the combination of MBP and BCG increased the proliferation of CD4⁺T cells co-cultured with DCs relative to that of the control group.Moreover,the results showed that the combination of MBP and BCG synergistically increased the proliferation of CD4⁺T cells relative to that in the MBP group and the BCG group.To further study the effect of the combination of MBP and BCG on the activation of CD4⁺T cells co-cultured with DCs,the secretion of IFN-γ and IL-4 was examined using ELISPOT.Co-culturing CD4+ T cells with DCs stimulated with the combination of MBP and BCG significantly increased the production of IFN-γ but decreased the levels of IL-4 production.The mechanism of MBP combined with BCG-induced DC maturation promote Th1 activation in MBP elevated TLR2/TLR4 surface expression on DCs in vitroIn this study,we investigated the influence of MBP and BCG on TLR2/TLR4 expression in DCs from normal mice.The surface expression of TLR2 and TLR4 on DCs from normal mice stimulated with MBP,BCG and MBP+BCG for 48 h was analyzed using flow cytometry.The data demonstrated that MBP significantly increased the percentage of CD11C+ cells expressing surface TLR2 in the control group.Similarly,MBP increased the expression of the TLR4 surface molecule on CD11C+ cells compared to the control group.BCG increased the surface TLR2 and TLR4 expression in DCs relative to the control group.2 TLR2 signaling is also required for the MBP plus BCG-induced maturation of DCsTo elucidate the functional significance of TLR2 expression on DCs,DCs isolated from TLR2^-/-mice were stimulated with MBP,BCG or the combination ofMBP and BCG for 48 hours.The expression of the maturation markers CD80,CD86 and MHC class II on the DCs were examined with flow cytometry.DCs lacking TLR2 expressed lower levels of co-stimulatory molecules on their surface compared to DCs from normal mice.These results indicated that these cells had a more immature state.However,no changes were observed in MHC class II expression on DCs from TLR2^-/-mice.Moreover,MBP,BCG and the combination of MBP and BCG up-regulated the expression of CD80,CD86 and MHC class II in DCs from normal mice,whereas these treatments failed to induce the up-regulation of co-stimulatory molecules and MHC class II molecules in DCs from TLR2^-/-mice.These results showed that the combination of MBP and BCG up-regulates the expression of the surface markers CD80,CD86 and MHC class II on DCs via TLR2.3 TLR4 signaling is required for the MBP plus BCG-induced maturation of DCsTo further investigate the significance of TLR4 in the MBP plus BCG-induced up-regulation of markers of a mature phenotype on DCs,DCs derived from TLR4^-/-mice were stimulated with MBP,BCG or the combination of MBP and BCG for 48 hours.The expression of CD80,CD86 and MHC class II on the surface of DCs was analyzed using flow cytometry.TLR4 deficiency decreased the expression of the surface markers CD80 and CD86 on DCs;that is,the expression of the TLR4 molecule on the surface of DCs is very important to the maturation of DCs.No changes in the expression of MHC class II were observed in DCs from TLR4^-/-mice.Moreover,MBP,BCG and the combination of MBP and BCG failed to increase the expression of CD80,CD86 and MHC class II on the surface of splenic DCs from TLR4^-/-mice.These results show that the combination of MBP and BCG up-regulates the expression of the surface markers CD80,CD86 and MHC class II in DCs via TLR4.4 The effects of TLR2/TLR4 on the proliferation and activation of CD4⁺T cells induced by the combination of MBP and BCG in vitroTo detect the indirect effect of TLR2/TLR4 expression in DCs on the proliferation of CD4⁺T cells,the proliferation of CD4⁺T cells from WT mice co-cultured with TLR2^-/-DCs or with TLR4^-/-DCs was assessed using a CCK8 assay following treatment with MBP,BCG or the combination of MBP and BCG.DCs lacking TLR2 or TLR4 failed to increase the proliferation of CD4⁺T cells from WT mice after stimulation with MBP,BCG or the combination of MBP and BCG.Theseresults indicated that TLR2/TLR4 expression on DCs is very important to the proliferation of CD4⁺T cells from WT mice stimulated with MBP,BCG or the combination of MBP and BCG.Moreover,to further detect the indirect effect of the expression of TLR2/TLR4 by DCs on the polarization of Th cells,ELISPOT method was used to detect IFN-γand IL-4 production by CD4⁺T cells from WT mice co-cultured with DCs from TLR2^-/-mice or TLR4^-/-mice that were treated with the combination of MBP and BCG.The results showed that the combination of MBP and BCG failed to increase the level of IFN-γ produced by CD4+ T cells from WT mice co-cultured with TLR2^-/-DCs or TLR4^-/-DCs.These results indicated that TLR2/TLR4 expression in DCs plays an important role in polarizing the Th response toward Th1.The increase in IL-4 production in co-cultures of normal CD4⁺T cells with TLR2^-/-DCs indicated that the combination of MBP and BCG has an indirect influence on the activation of CD4⁺T cells.Moreover,the data about IL-10 protein show that the same tendency as compared with the results of IL-4.The combination of MBP and BCG-induced DC maturation promote Th1 activation in vivo1 The combination of MBP and BCG induced the up-regulation of TLR2/TLR4 expression on DCs from WT mice in vivoTo detect the effect of the combination of MBP and BCG on the expression of the surface molecules TLR2/TLR4 on DCs in vivo,the expression of TLR2 and TLR4 on DCs from WT mice immunized with MBP,BCG or the combination of MBP and BCG was analyzed using flow cytometry.Compared to the control group,MBP,BCG or MBP plus BCG significantly elevated TLR2 expression on the DCs.The expression of TLR2 on DCs from the BCG group was higher than that in the MBP group and the MBP plus BCG group.Compared with the control group,MBP,BCG or MBP plus BCG significantly increased the expression of TLR4 on DCs.The level of TLR4 on the DCs in the BCG group was significantly higher than that in the MBP group and in the MBP plus BCG group.2 The combination of MBP and BCG induced DC maturation via TLR2/TLR4 in vivoTo examine more directly whether the combination of MBP and BCG is very important in influencing the expression of co-stimulatory molecules on DCs throughthe TLR2/TLR4 pathways,an in vivo study further detected the influence of the combination of MBP and BCG on the expression of co-stimulatory molecules in DCs from WT mice,TLR2^-/-mice and TLR4^-/-mice.A notable increase in CD80 and CD86 expression in DCs from WT mice was detected in the MBP,BCG and MBP plus BCG groups relative to that in the control group.A similar trend in MHC-II expression on DCs was observed,although the difference did not reach statistical significance.These results are in accordance with the in vitro experiments.To test the contribution of the TLR4 molecule in the MBP plus BCG-induced functional ability of DCs,the expression of the CD80,CD86 and MHC-II molecules on the surface of DCs from TLR4^-/-mice immunized with MBP,BCG or the combination of MBP and BCG was examined with flow cytometry.TLR4 deficiency significantly decreased the expression of CD80,CD86 and MHC-II on DCs.There was no obvious change in CD80,CD86 and MHC-II expression in DCs from TLR4^-/-mice immunized with MBP,BCG or the combination of MBP and BCG.The expression of CD80 and CD86 on DCs from TLR4^-/-mice immunized with MBP,BCG or the combination of MBP and BCG was significantly higher than that in TLR2^-/-mice.These data suggested that the up-regulation of co-stimulatory molecules in DCs induced by the combination of MBP and BCG may be more dependent on the TLR2 molecule than TLR4 molecule.To confirm whether TLR2 molecules are involved in the functional ability of DCs induced by the combination of MBP and BCG,the expression of co-stimulatory molecules on DCs derived from TLR2^-/-mice immunized with MBP,BCG or the combination of MBP and BCG were analyzed by flow cytometry.TLR2 deficiency decreased significantly the expression of CD80 and CD86 on the surface of DCs.The CD80 and CD86 expression in DCs from TLR2^-/-mice immunized with MBP,BCG or the combination of MBP and BCG also decreased significantly relative to the other groups derived from WT mice.There was no obvious change in MHC II expression in DCs from TLR2^-/-mice immunized with MBP,BCG or the combination of MBP and BCG.This suggested that TLR2 molecules are very important in the expression of CD80 and CD86 on the surface of DCs.These results indicated that the combination of MBP and BCG significantly increased the expression of CD80,CD86 and MHC-II on the surface of DCs mainly through the TLR2 molecule.3 The combination of MBP and BCG promoted the activation of CD4⁺T cell via TLR2/TLR4 in vivoTo confirm the functional involvement of the combination of MBP and BCG in the proliferation of CD4⁺T cells,we examined the proliferation of CD4⁺T cells from WT mice,TLR2^-/-mice and TLR4^-/-mice immunized with MBP,BCG or the combination of MBP and BCG.MBP or BCG significantly increased the proliferation of CD4⁺T cells from normal mice,and the combination of MBP and BCG synergistically increased the proliferation of CD4⁺T cells.These data indicated that the combination of MBP and BCG promotes the proliferation of CD4⁺T cells in vivo.To observe whether TLR2/TLR4 influence the proliferation of CD4⁺T cells,TLR2^-/-mice or TLR4^-/-mice were immunized with MBP,BCG or the combination of MBP and BCG.Compared to normal mice,MBP,BCG or the combination of MBP and BCG failed to increase the proliferation of CD4⁺T cells from TLR2^-/-mice or TLR4^-/-mice in vivo.In this study,these results suggested that the proliferation of CD4+ T cells induced by the combination of MBP and BCG may be dependent on TLR2/TLR4 in vivo.To further confirm the effect of the combination of MBP and BCG on the activation of Th1 in vivo,we examined the level of IFN-γ m RNA in CD4⁺T cells from mice in different groups.CD4⁺T cells from normal mice immunized with BCG expressed significantly higher levels of IFN-γ m RNA than the cells in the control group or MBP group.The combination of MBP and BCG significantly increased IFN-γ m RNA levels in CD4⁺T cells.The combination of MBP and BCG failed to increase the level of IFN-γ m RNA in CD4⁺T cells from TLR4^-/-mice in vivo.In addition,TLR4 deficiency decreased the m RNA level of IFN-γ in CD4⁺T cells following immunization with BCG.Likewise,TLR2 deficiency in CD4⁺T cells significantly decreased the level of IFN-γ m RNA produced following immunization with MBP and BCG.However,no change was observed in the IFN-γ m RNA level in CD4⁺T cells from TLR2^-/-mice immunized with BCG.This indicated that the combination of MBP and BCG has a direct effect on the polarization of Th cells toward a Th1 phenotype mainly through TLR2 but also partly through TLR4 expressed in CD4⁺T cells in vivo.To sum up,the present study mainly explored the expression of co-stimulatory molecules and the activation of DCs induced by the combination of MBP and BCG in vitro and in vivo.The results suggested that the expression of TLR2/TLR4 may be one of the potent mechanisms involved in altering DC maturation and in priming a Th1 response after stimulation with the combination of MBP and BCG.We conclude that the combination of MBP and BCG has the potential to be used as a TLR2/TLR4 agonist for strengthening immunity against life-threatening diseases.