Escherichia Coli Maltose Binding Protein Combined With BCG Synergistically Induces Th1Activation Via The Cross-talk Of TLR2/TLR4/TLR9

Maltose-binding protein (MBP), a component of the maltose transport system ofEscherichia coli, has been thought to have minimal effects on the bioactivity of itsfusion protein. However, our previous studies found that MBP increased theproliferation of splenic mononuclear cell, induced the activation of Th1cells, NKcells and macrophages. These findings above confirmed the potentimmune-enhancing activities of MBP. Additionally, we found that MBP significantlyincreased IFN-γ production of lymphocytes immuned with the combination of MBPand BCG could induced Th1activation synergistically, which may explain why MBPcombined with BCG induces synergistic antitumor activity in Lewis lungcarcinoma-bearing mice. However, the effects of MBP combined with BCG on mouseCD4+T cells in vitro are largely unexplored and the mechanism still has not been wellcharacterized. Our group found that MBP could induce U937cells proliferation viaTLR2，induce macrophages polarized to M1subtype via TLR2and TLR4. Fernandezreported that MBP induced DC maturation and IL-12production via TLR4. It isreported that BCG can induce mouse DC maturation and increase T helper1(Th1)cytokine secretion via TLR2, TLR4and TLR9. It has been reported that TLRssignaling pathway includes MyD88-dependent and–independent pathway(TRIF-dependent pathway)，which play a synergistic effect or an antergic effect on regulatingimmune response. Therefore, we supposed that the combination of MBP and BCGinduced Th1activation was associated with the cross-talk of TLRs signaling pathway.In this paper, we further explored the mechanism of MBP combined withBCG-induced Th1activation. The effect of MBP and BCG on mouse spleen lymphocytes proliferation wasanalyzed by CFSE assay. The results showed that the combination of MBP and BCGcould synergistically induce the proliferation of lymphocytes.2. The combination of MBP and BCG synergistically increased the percentage ofCD4+T cellTo further analyze the subpopulation, the percentage of CD3+CD4+, CD3+CD8+，CD19+cells were detected by flow cytometer responded to MBP, BCG andMBP+BCG. The results showed that MBP，BCG and MBP+BCG reduced thepercentage of CD19+cells, with no influence of the percentage of CD3+CD8+cells.The percentage of CD3+CD4+cells of MBP or BCG group was higher than that ofcontrol group, while the MBP+BCG group exhibited the higest percentage of CD3+CD4+cells among all the groups. These results indicated that the combination ofMBP and BCG could synergistically increased the percentage of CD4+T cell3. The combination of MBP and BCG synergistically increased the IFN-γproduction of CD4+T cellsTo investigate whether MBP combined with BCG induced Th1activation, thesplenic mononuclear cells was stimulated with MBP, BCG, MBP+BCG for48h andthe IFN-γ and IL-4production was detected by ELISA. The results showed that theIL-4production level was low and changed little responded to MBP and BCG. Bycontrast, there was a higher IFN-γ production level of both MBP and BCG-treatedcells, and the IFN-γ production level of the combination-treated cells was highest. Theresults of intracellular cytokine staining assay showed that either MBP or BCG couldincrease the percentage of CD4+IFN-γ+cells, which was highest in MBP+BCG group.The percentage of CD4+IL-4+, CD8+IFN-γ+and CD8+IL-4+cells changed littleresponded to MBP, BCG or the combination, suggesting that the combination of MBPand BCG synergistically induced the IFN-γ production of CD4+T cells.4. The combination of MBP and BCG upregulated the expression of TLR2andTLR4on CD4+T cellsThe expression of TLR2and TLR4on CD4+T cells was analyzed by flowcytometer. The results exhibited that the combination of MBP and BCGsynergistically upregulated the expression of TLR2on CD4+T cells. The expressionof TLR4on CD4+T cells was downregulated by MBP, but upregulated by MBPcombined with BCG. MBP or BCG has no influence on the expression of TLR2, TLR4on CD8+T cells.二、The mechanism of MBPcombined with BCG-induced Th1activation1. The combination of MBP and BCG synergistically induced Th1activationTo analyze the direct effect of MBP combined with BCG on mouse CD4+Tcells, we purified mouse CD4+T cells by magnetic beads and activated the cells usedanti-CD3/CD28antibodies. The ELISA assay result showed that both MBP and BCGalone resulted in an increase of IFN-γ production, and the combination of MBP andBCG exhibited a synergistic effect. Additionally, MBP or BCG alone or thecombination had no influence on IL-4secretion. The result of qRT-PCR showed asimilar result. The results above indicated that the stimulation of MBP combined withBCG could synergistically induce Th1response.2. The combination of MBP and BCG synergistically activates theMyD88-dependent and TRIF-dependent pathway of TLR2, TLR4and TLR9signaling pathwaysTo determine the contribution of TLRs on MBP combined with BCG-inducedTh1activation, we analyzed the molecules related to TLR signaling pathway. Theresults showed that MBP upregulated the expressions of TLR2, MyD88, TRIF andTRAF3, but downregulated TLR4expression, indicating that MBP may favorMyD88-dependent pathway and TRIF-dependent TRAF3pathway, inhibitTRIF-dependent TRAF6pathway via TLR2and TLR4. BCG upregulated theexpressions of TLR2, TLR4, TLR9, MyD88, TRIF and TRAF6, but downregulatedTRAF3expression. The combination of MBP and BCG exhibited a synergistic effect.The results indicated that the combination of MBP combined with BCG activatedMyD88-dependent pathway and TRIF-dependent TRAF6pathway, significantlyinhibited TRIF-dependent TRAF3pathway. The results above indicated that TRAF6and TRAF3play condratict roel in Th1activation.3. TLR2partly favored Th1activation induced by MBP combined with BCG viaMyD88-dependent pathwayTo further explore the roles of TLR2in MBP, BCG and MBP combined withBCG-induced Th1activation, the TLR2expression of purified CD4+T cells wereblocked with anti-TLR2antibody before stimulating with MBP/BCG alone or thecombination. We detected the IFN-γ production level by ELISA and found that TLR2antibody partly decreased IFN-γ secretion of each group. The level of MBP combined 一. The effect of MBP combined with BCG on lymphocytics1. The combination of MBP and BCG synergistically induced mouse spleenlymphocytes proliferationwith BCG-induced IFN-γ secretion was decreased more significantly, but still higherthan that of control group. Next, we detected the molecules related to TLR2signalingby Western blot and qRT-PCR. The results showed that TLR2antibody significantlydownregulated MyD88expression. The MyD88expression induced by MBPcombined with BCG was decreased more significantly, even lower than that of controlgroup. TLR2antibody had no influence of TRIF, TRAF3expression responded toMBP, BCG and the combination. The results above indicated that TLR2signalingpartly favored MBP， BCG and the combination-induced Th1activation viaMyD88-dependent pathway.4. TLR4partly favored MBP combined with BCG-induced Th1activation viaTRAF6upregulation and TRAF3downregulation of TRIF pathwayTo further explore the roles of TLR4in MBP, BCG and MBP combined withBCG-induced Th1activation, the TLR4expression of purified CD4+T cells wereblocked with anti-TLR4antibody before stimulating with MBP/BCG alone or thecombination.The result of ELISA assay showed that TLR4antibody increasedMBP-induced IFN-γ production. IFN-γ production of the cells stimulated with thecombination was reduced more significantly than that of cells stimulated with BCGresponded to TLR4antibody, but still higher than that of control group.Next, we detected the molecules related to TLR4signaling by Western blot andqRT-PCR. The results showed that TLR4antibody markly downregulatedMBP-induced TRIF and TRAF3expression, upregulated MyD88expression, had noinfluence on TRAF6expression, indicating that TLR4inhibited MBP-induced Th1activation via TRIF/TRAF3pathway. TLR4antibody markly downregulated MBPcombined with BCG-induced TRIF and TRAF6expression, upregulated MyD88andTRAF3expression, indicating that the TRIF pathway of TLR4induced Th1activationvia TRAF6upregulation and TRAF3downregulation. Additionally, the upregulationof MyD88expression induced by TLR4antibody indicated that TLR4may have across-talk with TLR2or TLR9signaling pathway.5. TLR9significantly favored Th1activation via the activation ofMyD88-dependent pathway and inhibition of TRIF/TRAF3To further explore the roles of TLR9in MBP, BCG and MBP combined withBCG-induced Th1activation, the TLR9expression of purified CD4+T cells wereblocked with TLR9inhibitor before stimulating with MBP/BCG alone or the combination.The result of ELISA assay showed that TLR9inhibitor significantly reducedBCG and MBP+BCG-induced IFN-γ production. The level of MBP combined withBCG-induced IFN-γ secretion was decreased more significantly, which was nearlyequal to that of control group. Next, we detected the molecules related to TLR9signaling by Western blot and qRT-PCR. The results showed that TLR9inhibitorreduced MyD88, TRAF6, TRIF expression and IκB phosphorylation inducd by BCGand MBP+BCG. The MyD88, TRAF expression and IκB phosphorylation ofMBP+BCG group was nearly equal to that of control group. TLR9inhibitor marklyunregulated TRAF3expression of BCG and the combination-treated cells. The resultsabove indicated that TLR9played a key role in MBP, BCG and thecombination-induced Th1activation through the activation of MyD88/TRAF6andTRIF/TRAF6pathway and the inhibition of TRIF/TRAF3pathway. The resultssuggested that the balance of TRAF6andTRAF3regulated Th1activation. It has beenreported that MyD88is the only way for TLR9to activate TRAF6. The resultsshowed that TLR9inhibitor inhibited TRIF/TRAF3pathway, suggesting that theremay be a cross-talk between TLR9and TLR4signaling induced by BCG and thecombination.6. The cross-talk of TLR2/4/9in Th1activation induced by MBP combined withBCGTo explore the cross-talk of TLR2/4/9responded to MBP, BCG or thecombination, TLR2/4/9expression was blocked and the expressions of TLR2/4/9were detected by qRT-PCR. The result showed that TLR2blockage had no influenceof TLR4and TLR9expression responded to MBP, BCG or the combination.Conversely, TLR4antibody treatment significantly increased TLR2expression, withno influence of TLR9expression. When TLR9was blocked, there was a reduction inboth TLR2and TLR4expression. Next, TLR2/4/9agonist was used to confirm theresult above. The result showed that Pam3CSK4, LPS and CPG-ODN increasedIFN-γ production, the combination of Pam3CSK4, LPS and CPG-ODN exhibited asynergistic effect. Additionally, the expression of TLR4and TLR9were notinfluenced by Pam3CSK4. The expression of TLR2and TLR4were increased byCPG-ODN. The results above indicated that in the MBP combined with BCG-inducedTh1activation, TLR4inhibited TLR2-meditated signaling; TLR9favored TLR2and TLR4meditated signaling, suggesting that there was a cross-talk among TLR2，TLR4and TLR9，and TLR9played a key role in this cross-talk.Collectively, our research found that MBP combined with BCG couldsynergistically induced Th1activation and TLR2/TLR4/TLR9activation. Here, wefirstly reveal the role of TLR2/4/9cross-talk-mediated TRAF6upregulation versusTRAF3downregulation in regulating Th1polarization induced by MBP combinedwith BCG.