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

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Escherichia coli maltose-binding protein (MBP) directly induces mouse Th1 activation through upregulating TLR2 and downregulating TLR4 expressions Fang Wang 1 , Weihua Ni 1 , Guomu Liu, Juan Wang, Fei Xie, Hongyan Yuan, Yingying Guo, RuiPing Zhai, Tanxiu Chen, Qiongshu Li, Guixiang Tai ∗ Department of Immunology, Norman Bethune College of Medicine, Jilin University, Changchun 130021, China

a r t i c l e

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Article history: Received 8 November 2014 Received in revised form 16 December 2014 Accepted 23 December 2014 Available online xxx Keywords: Maltose-binding protein, MBP CD4+ T cell Th1 TLR2 TLR4

a b s t r a c t 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-␥ 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 I␬B phosphorylation. Notably, the addition of anti-TLR2 antibody abrogated the MBP-induced CD4+ T cells proliferation, IFN-␥ 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 I␬B 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. © 2015 Elsevier GmbH. All rights reserved.

Introduction Maltose-binding protein (MBP) is a component of the maltose transport system of Escherichia coli (Riggs 2000). It is believed that MBP has minimal effects on the bioactivity of its fusion protein, so it has been widely used for the purification of recombinant proteins as a tagged protein (Butt et al. 1989). However, MBP was recently used as a chaperone component in various subunit vaccines against pathogenic bacteria (Kang et al. 2005; Lee et al. 1999). Our previous studies found that the combination of MBP and Bacillus Calmette–Guerin (BCG) induces synergistic antitumor activity in Lewis lung carcinoma-bearing mice; these synergistic antitumor effects might be mediated mainly through the activation of T helper 1 (Th1) cells, NK cells and macrophages (Zhang et al. 2011). The findings above confirmed the potent immune-enhancing

∗ Corresponding author at: Department of Immunology, Norman Bethune College of Medicine, Jilin University, 126 Xinmin Street, Changchun 130021, China. Tel.: +86 043185619476; fax: +86 04319403. E-mail address: [email protected] (G. Tai). 1 These authors contributed equally to this work.

activities of MBP. Further understanding of how MBP activates multiple immune cells may help to explain the adjuvanticity of MBP. An additional study found that MBP directly promotes U937 cell viability, as well as activating mouse peritoneal macrophages and inducing M1 polarization (Zhao et al. 2011; Ni et al. 2014). However, the effects of MBP on T cells are largely unexplored. Toll-like receptor 2 (TLR2) and Toll-like receptor 4 (TLR4) functionally express on mouse CD4+ T cells and directly regulate these cells’ functions (Raveendra et al. 2011; Kawai and Akira 2010). Several studies have shown that TLR2 agonist, P3CSK4 can induce IFN-␥ secretion of mouse CD4+ T cells in vitro combined with TCR stimulation (Scott et al. 2014), whereas TLR4 agonist, low-level of LPS can skew mouse CD4+ T cells to T helper 2 (Th2) response (Stephanie et al. 2002). Our previous studies implied that MBP can bind to human monocytes and modulate their viability through TLR2 (Zhao et al. 2009); MBP additionally activates mouse peritoneal macrophages and induces M1 polarization via TLR2 and TLR4 (Ni et al. 2014). Fernandez also found that MBP induces human dendritic cells (DCs) maturation and the proinflammatory cytokines production via TLR4 (Fernandez et al. 2007). In the present study, we sought to investigate the regulatory effect of MBP on mouse CD4+ T cells in vitro. To further explore the possible mechanism,

http://dx.doi.org/10.1016/j.imbio.2014.12.016 0171-2985/© 2015 Elsevier GmbH. All rights reserved.

Please cite this article in press as: Wang, F., et al., Escherichia coli maltose-binding protein (MBP) directly induces mouse Th1 activation through upregulating TLR2 and downregulating TLR4 expressions. Immunobiology (2015), http://dx.doi.org/10.1016/j.imbio.2014.12.016


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TLR2 and TLR4 expressions and phosphorylation of nuclear factor␬B (NF-␬B) signaling pathway-related molecules were probed. Materials and methods Reagents MBP used in the present study was obtained from E. coli strain carried the MBP expression vector pMAL-c2 (New England Biolabs, USA), which consists of MBP preceded by methionine, with the final four amino acids replaced by 23 residues encoded by the polylinker of pMAL-c2. Endotoxin was removed with 100 kDa ultrafiltration tube and the endotoxin level was lower than 10 endotoxin units (EU)/ml. The CD4+ T cell mouse lymphocytes enrichment kit, APC-antiCD3 antibody, PE/Cy7-anti-CD4 antibody, PE-anti-CD4 antibody, PE-anti-CD8 antibody, PE/Cy7-anti-CD8 antibody and PE-antiCD19 antibody were purchased from Becton Dickinson. CFSE and TRIzol were obtained from Invitrogen. Anti-mouse CD3 monoclonal antibody was purchased from Sungene Biotech. FITC-anti-IFN-␥ or IL-4, anti-CD28, anti-TLR2 or TLR4 antibody, mouse IgG antibody, fixation buffer and permeabilization buffer were obtained from eBioscience. Anti-MyD88, anti-␣-tubulin, anti-I␬B antibody and polymyxin B were all purchased from Cell signaling Technology. Cell culture medium and reagents were purchased from Gibco. The mouse lymphocyte separation medium and WST1 were obtained from Dakewe Biotech. Cell preparation and culture Splenic mononuclear cells from C57BL/6 mice were prepared with mouse lymphocyte separation medium by density gradient

centrifugation. CD4+ T cells were purified from C57BL/6 mouse spleens by negative selection using the CD4+ T cell mouse lymphocytes enrichment kit according to the manufacturer’s protocol. After isolation, the purity of CD4+ T cell population was >98% assessed by flow cytometry (Becton Dickinson, USA) (data not shown). Cell viability was evaluated over 90% by Trypan blue staining. All of the cells were cultured with Iscove Modified Dulbecco Medium (IMDM) supplemented with 10% fetal bovine serum (FBS), 100 ␮g/ml streptomycin and 100 U/ml penicillin in humidified atmosphere of 5% CO2 at 37 ◦ C.

WST1 assay Splenic mononuclear cells were stimulated with different concentrations of MBP (2.5 ␮g/ml, 5 ␮g/ml, 10 ␮g/ml and 20 ␮g/ml) or LPS (2 ␮g/ml) for 48 h with or without polymyxin B (2 ␮g/ml). Anti-mouse CD3 monoclonal antibody (5 ␮g/ml) was coated in 96-well plate at 4 ◦ C in 100 ␮l PBS overnight. The purified CD4+ T cells were cultured with anti-CD28 antibody (1 ␮g/ml) for 48 h in the pre-coated wells. The cells were treated with 10 ␮g/ml antiTLR2, anti-TLR4 or mouse IgG for 2 h and then stimulated with 10 ␮g/ml MBP for 48 h. WST1 was added to each well at the final concentration of 10% (v/v) and cultured in dark at 37 ◦ C for 1 h. The absorbance was detected at 450 nm with a microplate reader (BioTek, USA). The results were showed as relative cell viability. Relative cell viability was calculated as A450 (MBP-stimulated group) − A450 (control group)/A450 (control group).

Fig. 1. The effect of MBP on CD4+ T cells proliferation in splenic mononuclear cells. (A) Splenic mononuclear cell proliferation induced by different concentrations of MBP was detected by WST1 assay. Cells were treated with 0, 2.5, 5, 10 and 20 ␮g/ml MBP for 48 h. Relative cell viability is calculated as A450 (MBP-stimulated group) − A450 (control group)/A450 (control group). (B) The proliferation of splenic mononuclear cell stimulated with MBP (10 ␮g/ml) for 48 h was detested by CFSE labeling. Histogram plots showed the percentage of cells labeled with CFSE. (C) The effect of endotoxin on MBP-induced cell proliferation was detected by WST1 assay. Splenic mononuclear cell was treated with 10 ␮g/ml MBP, 2 ␮g/ml LPS and 2 ␮g/ml polymyxin B for 48 h. Cell proliferation was detected by WST1 assay. (D and E) Splenic mononuclear cell subpopulations were analyzed by flow cytometry. Splenic mononuclear cells were treated with 10 ␮g/ml MBP for 48 h, and stained with anti-CD19, anti-CD3, anti-CD4 and anti-CD8 antibodies. The percentages of CD4+ and CD8+ cells were gated on CD3+ cells. The data shown were the averages of three experiments ± standard errors of the means. *P < 0.05 compared with control group.




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Fig. 2. The analysis of MBP-induced cytokines production in splenic mononuclear cells. (A) IFN-␥ and IL-4 production were assessed by ELISA. Splenic mononuclear cells (1 × 106 ) were treated with 10 ␮g/ml MBP, 2 ␮g/ml LPS and polymyxin B. Cell supernatants were harvested after 48 h and cytokine productions were assessed. The data shown were the averages of three experiments ± standard errors of the means. *P < 0.05 compared with control group; (B) IFN-␥ and IL-4 production were assessed by intracellular cytokine staining. Cells were treated with 10 ␮g/ml MBP for 22 h and cytokine secretions were blocked by Brefeldin A for an additional 26 h. Numbers represent percentages of CD4+ and CD8+ cells that produce IFN-␥ or IL-4 gated on CD3+ cells. The data was representative of three independent experiments.

CFSE labeling and proliferation assay Splenic mononuclear cells were labeled with 2 ␮M CFSE in PBS contained 5% FBS at 37 ◦ C for 15 min. The CFSE labeling was quenched with the addition of FBS to a final concentration of 30% (v/v). Labeled cells were washed three times with IMDM, and then cultured in 96-well plate at 1 × 106 cells/well with 10 ␮g/ml MBP for 48 h. The cell proliferation was evaluated by FACS analysis.

Flow cytometry assay Splenic mononuclear cells (1 × 106 cells) were stimulated with MBP (10 ␮g/ml) for 48 h and washed twice with FACS solution (PBS containing 2% FCS and 0.1% NaN3 ). The cells were stained with APC-anti-CD3 antibody, PE/Cy7-anti-CD4 antibody, PE-anti-CD8 antibody, PE-anti-CD19 antibody, FITC-anti-TLR2 and anti-TLR4 antibodies on ice for 60 min darkly, and then washed twice again. The samples were analyzed on a FACS Calibur flow cytometer.

to detect IFN-␥ and IL-4 level by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s protocol. The purified CD4+ T cells were treated as described in Section ‘WST1 assay’. The IFN-␥ level was assayed by ELISA. Intracellular cytokine staining assay Splenic mononuclear cells (1 × 106 ) were stimulated with MBP (10 ␮g/ml) for 48 h. After incubating for 22 h, brefeldin A (1:10,000) was added to prevent cytokines secretion. The cells were collected, washed twice and stained with APC-anti-CD3 antibody, PE-antiCD4 antibody, PE/Cy7-anti-CD8 antibody on ice for 60 min. Then the cells were fixed with 1% fixation buffer for 30 min, permeabilized with permeabilization buffer for 30 min, and finally stained with FITC-labeled anti-IFN-␥ or anti-IL-4 antibody for 60 min on ice. All of the procedures were performed in the dark. The stained cells were washed and analyzed on a FACS Calibur flow cytometer. qRT-PCR assay

ELISA analysis Splenic mononuclear cells (1 × 106 ) were stimulated with MBP (10 ␮g/ml) and LPS (2 ␮g/ml) for 48 h in the presence or absence of polymyxin B (2 ␮g/ml), then the cell supernatants were harvested

Anti-mouse CD3 monoclonal antibody (5 ␮g/ml) was coated in 96-well plate at 4 ◦ C in 100 ␮l PBS overnight. The purified CD4+ T cells were cultured for 48 h in the pre-coated wells and then stimulated with MBP (10 ␮g/ml) for 2 h. RNA was isolated using TRIzol. Total RNA was converted to cDNA using M-MLV reverse


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Fig. 3. The analysis of TLR2 and TLR4 expression on MBP-treated T cells. (A) TLR2 and TLR4 expressions on CD3+ T cells were analyzed by flow cytometry. Splenic mononuclear cell was treated with 10 ␮g/ml MBP for 48 h. TLR2 and TLR4 expressions were examined. (B and C) TLR2/4 expressions on CD4+ T cells were detected by qRT-PCR and Western blotting. CD4+ T cells were purified from C57BL/6 mouse spleens by negative selection using magnetic beads and activated with anti-CD3 antibody, then stimulated with MBP (10 ␮g/ml) for 2 h. The TLR2/4 mRNA and protein level were analyzed. Relative protein level was calculated as the ratio of TLR2 or TLR4/a-tubulin. The data shown were the averages of three experiments ± standard errors of the means. *P < 0.05 compared with activated CD4+ T cells. Table 1 Primer sequences and reaction parameters used for qRT-PCR analysis. Gene

Primer

␤-Actin

Forward: 5 -TTCTTTGCAGCTCCTTGG-3 Reverse: 5 -TTCTGACCCATTCCCACC-3 Forward: 5 -TTGCGTTACATCTTGGAACTG-3 Reverse: 5 -ACTACGTCTGACTCCGAGGG-3 Forward: 5 -CTTCATTCAAGACCAAGCCTTTC-3 Reverse: 5 -AACCGATGGACGTGTAAACCAG-3

TLR2 TLR4

transcriptase and oligo (dT) primers (Promega Corporation, USA) according to the manufacturer’s protocol. ␤-Actin was used as internal control gene. The primer sequences and reaction parameters are shown in Table 1. The expressions of TLR2 and TLR4 were normalized to that of the internal control gene. Calculations were performed using the 2−CT method, and the results were presented as the fold change in gene expression relative to expression in the control sample. For the control sample, 2−CT = 1. Western blotting analysis The purified CD4+ T cells were activated with only anti-CD3 antibody or anti-CD3 and anti-CD28 antibodies as Section ‘qRT-PCR assay’ or ‘WST1 assay’ described. The cells were pretreated with or without 10 ␮g/ml anti-TLR2 or anti-TLR4 antibody for 2 h, and then stimulated with 10 ␮g/ml MBP for 2 h. Cell lysate was prepared with RIPA Lysis Buffer in the presence of protease inhibitor and phosphatase inhibitor. Protein concentration was obtained by bicinchoninic acid protein detection system. Proteins were separated by SDS-PAGE and subjected to western blotting. The following

antibodies were used: anti-␣-tubulin, anti-TLR2, anti-TLR4, antiMyD88, anti-I␬B and anti-p-I␬B antibodies. Statistical analysis All statistical analyses were performed with SPSS 13.0 software. The data are expressed as mean ± standard deviation (SD). P < 0.05 was considered as statistically significant. Results MBP mainly promotes the proliferation of CD4+ T cells in splenic mononuclear cells To analyze the effect of MBP on mouse splenic mononuclear cells in vitro, splenic mononuclear cells were stimulated with different concentration of MBP for 48 h and cell proliferation was detected by WST1 assay. As Fig. 1A shown, cell proliferation was significantly increased by 10 ␮g/ml and 20 ␮g/ml MBP. There was little difference of cell proliferation capacity between the two groups, so 10 ␮g/ml was selected as the suitable concentration in this paper. Next, splenic mononuclear cells were labeled with CFSE and then stimulated with 10 ␮g/ml MBP for 48 h to detect cell proliferation. The result (Fig. 1B) was consistent with that of Fig. 1A. To further discount potential endotoxin contamination in MBP, splenic mononuclear cells were treated with MBP or LPS in the presence or absence of LPS binding antibiotic polymyxin B and the cell proliferation was measured by WST1 assay. As Fig. 1C shown, polymyxin B abrogated the cell proliferation induced by 2 ␮g/ml




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Fig. 4. The effects of TLR2 and TLR4 on MBP-induced CD4+ T cell proliferation and IFN-␥ production. CD4+ T cells were purified from C57BL/6 mouse spleens by negative selection using magnetic beads and activated with anti-CD3/28 antibody. Activated CD4+ T cells were pre-treated with anti-TLR2/4 antibody (10 ␮g/ml) for 2 h and then stimulated with MBP (10 ␮g/ml) for 48 h. IgG was used as isotype control. (A) The proliferation of CD4+ T cells was detected by WST1 assay. (B) IFN-␥ production of CD4+ T cells was detected by ELISA. The data shown were the averages of three experiments ± standard errors of the means. *P < 0.05 compared with activated CD4+ T cells. # P < 0.05 compared with activated CD4+ T cells treated with MBP and IgG.

LPS. As expected, the addition of polymyxin B did not influence MBP-induced cell proliferation, indicating that the bioactivity of MBP was not involved in endotoxin. Subpopulation analysis indicated that MBP significantly increased the percentage of CD3+ T cell, while decreased the B cell (CD19+ cells) percentage (Fig. 1D). Furthermore, we investigated the change in CD4+ T cell and CD8+ T cell after MBP treatment gated on CD3+ T cell. As shown in Fig. 1E, MBP stimulation induced CD3+ CD4+ T cell proliferation, and decreased the CD3+ CD8+ cell percentage. These results indicated that MBP mainly promoted CD4+ T cell proliferation.

absence of polymyxin B and harvested the supernatants to detect IFN-␥ and IL-4 production by ELISA. As shown in Fig. 2A, MBP increased IFN-␥ production of splenic mononuclear cells, but the IL-4 level was very low and changed little. The addition of polymlxin B did not affect the MBP-induced IFN-␥ production. The results of intracellular cytokine staining assay showed that CD4+ T cells, rather than CD8+ T cells, responded to MBP by producing more IFN-␥. On the other hand, the levels of IL-4 produced in both CD4+ T cells and CD8+ T cells were decreased responding to MBP (Fig. 2B). These results indicated that MBP could induce Th1 activation.

MBP induces Th1 activation in splenic mononuclear cells

MBP regulates TLR2 and TLR4 expression of purified CD4+ T cells

To investigate whether MBP could induce Th1 activation in splenic mononuclear cells, we stimulated splenic mononuclear cells with 10 ␮g/ml MBP or 2 ␮g/ml LPS for 48 h in the presence or

Our previous study found that MBP induced mouse M1 polarization via TLR2/4 (Ni et al. 2014). Fernandez also pointed out that MBP induced human DCs maturation via TLR4 (Fernandez et al.

Fig. 5. The analysis of TLR-signaling pathway in CD4+ T cells. CD4+ T cells were purified from C57BL/6 mouse spleens by negative selection using magnetic beads and activated with anti-CD3/28 antibody. (A) The cells were pre-treated with 10 ␮g/ml anti-TLR2 antibody for 2 h. (B) The cells were pre-treated with 10 ␮g/ml anti-TLR4 antibody for 2 h. Then cells were stimulated with 10 ␮g/ml MBP for 2 h. MyD88 expression and I␬B phosphorylation were determined by Western blotting. Relative protein level was calculated as the ratio of MyD88/␣-tubulin or p-I␬B/I␬B. The data shown were the averages of three experiments ± standard errors of the means. *P < 0.05 compared with activated CD4+ T cells; # P < 0.05 compared with activated CD4+ T cells treated with MBP.




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2007). Thus, we hypothesized that the regulation of mouse CD4+ T cells by MBP was associated with the expression of TLR2 and TLR4. Splenic mononuclear cells were stained with anti-CD3, anti-TLR2 or anti-TLR4 antibody to analyze the TLR2/4 expression on CD3+ T cells by flow cytometer assay. After stimulation with MBP, a significant increase in the percentages of CD3+ T cells expressing TLR2 was observed compared with that of control, but the percentage expressing TLR4 was decreased (Fig. 3A). Subsequently, we purified CD4+ T cells from mouse splenic mononuclear cells using magnetic beads and detected the TLR2/4 mRNA levels by qRT-PCR. TLR2/4 mRNA expressions in CD4+ T cells activated with anti-CD3 antibody were higher than in naive CD4+ T cells. MBP significantly increased TLR2 mRNA expression level in activated CD4+ T cells but decreased TLR4 mRNA expression compared to that of control (Fig. 3B). We confirmed TLR2 and TLR4 protein expression on purified CD4+ T cells by western blotting (Fig. 3C). TLR2 and TLR4 play different roles in Th1 activation directly induced by MBP To examine the direct effects of MBP on purified CD4+ T cells, cell proliferation and IFN-␥ secretion were detected by WST1 and ELISA, respectively. Naive CD4+ T cells were stimulated through TCR with plate-bound anti-CD3 and soluble anti-CD28 antibodies alone or combined with MBP. As expected, naive CD4+ T cells were responsive to TCR stimulation, with higher level of cell proliferation and IFN-␥ production. MBP significantly increased the proliferation and IFN-␥ secretion of activated CD4+ T cells (Fig. 4). The results strongly suggested that MBP could directly induced Th1 activation. To further explore the roles of TLR2 and TLR4 in MBP-induced Th1 activation, the TLR2/4 expression of purified CD4+ T cells were blocked with anti-TLR2/4 antibody before stimulating with MBP. MBP-induced proliferation and IFN-␥ secretion of activated CD4+ T cells were largely abrogated when TLR2 was blocked but significantly increased when TLR4 was blocked (Fig. 4). These results were not isotype dependent, as both blocking antibodies were mouse IgG. These differences in the abilities of TLR2 and TLR4 to induce proliferation and cytokine production suggested that TLR2 signal favored Th1 differentiation, whereas TLR4 was responsible for counterbalancing this effect by decreasing IFN-␥ production. MBP-induced MyD88 and IB activations are associated with TLR2 and TLR4 All TLRs signals, with the exception of TLR3, are activated through MyD88, which results in NF-␬B activation (Kawai and Akira 2011). Signaling through NF-␬B pathway is followed by increased level of I␬B phosphorylation, which causes the release and nuclear translocation of NF-␬B (Koyama et al. 2010). Therefore, we explored the effects of MBP on activating TLR2 and TLR4 by measuring MyD88 expression and I␬B phosphorylation. As shown in Fig. 5, MBP stimulation induced strong MyD88 expression and I␬B phosphorylation. Pretreatment of activated CD4+ T cells with either anti-TLR2 or anti-TLR4 antibody both reduced I␬B phosphorylation. Surprisingly, the expression of MyD88 was down-regulated in TLR2-blocked cells, but up-regulated in TLR4-blocked cells. The results above indicated that TLR2 favored Th1 activation via MyD88-dependent pathway, whereas TLR4 inhibited Th1 activation through MyD88-independent pathway. Discussion Our previous studies found that the combination of MBP and BCG induces synergistic antitumor activity in Lewis lung carcinoma-bearing mice, which might be mediated through the

activation of Th1 cells, NK cells and macrophages (Zhang et al. 2011). We also found that MBP directly promotes U937 cell viability, as well as activating mouse peritoneal macrophages and inducing M1 polarization (Zhao et al. 2011; Ni et al. 2014). Further understanding of how MBP activates multiple immune cells may help to explain the adjuvanticity of MBP. In this paper, we primarily explored the effects of MBP on mouse T cells in vitro. The results showed that MBP induced the proliferation and IFN-␥ production of CD4+ T cells in mouse splenic mononuclear cells. Subsequently, to detect whether MBP could directly induce Th1 activation in the absence of APCs, we purified CD4+ T cells from mouse splenic mononuclear cell using magnetic beads. Results showed that MBP provided a proliferation stimulius to antiCD3/CD28 antibody-activated CD4+ T cells. Activated CD4+ T cells produced higher level of IFN-␥ responded to MBP, which implied that MBP had a direct effect on Th1 activation. It has been confirmed that TLR2 and TLR4 express on CD4+ T cells and play an important role in regulating the effector functions of CD4+ T cells (Socorro et al. 2011; Angela et al. 2011). TLR2 engagement directly regulates Th1 activation in vitro (Andre et al. 2005; Takayuki et al. 2007). The role of TLR4 on mouse CD4+ T cell differentiation is controversial. In some reports TLR4 agonist favors Th1 response (Joseph et al. 2012; Mark et al. 2013), but in others promotes Th2 response (González-Navajas et al. 2010). Our previous studies showed that MBP modulated U937 cells viability through TLR2 and activated mouse peritoneal macrophages to M1 type via TLR2/4 (Zhao et al. 2011; Ni et al. 2014). Therefore, we supposed that the effects of MBP on CD4+ T cells may be associated with TLR2 and TLR4. Here, we detected TLR2 and TLR4 expression on CD4+ T cells and found that MBP increased both the mRNA and protein levels of TLR2 in CD4+ T cells, whereas the TLR4 expression level was decreased. Additionally, the proliferation and IFN-␥ production of MBP-treated CD4+ T cells was abrogated when TLR2 was blocked but increased when TLR4 was blocked. These results were consistent with the reports of Scott et al. (2014) and Phipps et al. (2009) who confirmed that TLR2 induced Th1 response while TLR4 was responsible for Th2 response. TLR2 can induce Th1 activation through MyD88-dependent pathway (Wang et al. 2007; Fang et al. 2011), which may be attributed to the involvement of the MyD88 molecule in IL-1␤ and IL-18 receptor signaling (Adachi et al. 1998). These findings are in agreement with our results showing that the blockage of TLR2 decreased MBP-induced MyD88 expression and I␬B phosphorylation. TLR4 is known to activate both the MyD88 and TRIF pathway (Nicholas et al. 2011). There was report that MyD88 actively regulate the Th1 response, whereas TRIF can decrease IFN-␥ secretion and increase IL-4 production to counteract this effect (Dalia et al. 2011). Our results supported this idea, showing that the blockage of TLR4 signaling leaded to an increase of IFN-␥ production and MyD88 expression. The crosstalk of TLR2 and TLR4 in MBP-induced Th1 activation is currently being studied in our laboratory. Our latest results showed that besides the MyD88-dependent pathway, the TRIF pathway was activated in MBP-induced Th1 activation and TLR4 blockage increased TLR2 expression level, which may help to explain the clear increase in MyD88 expression when TLR4 was blocked (data not shown). Our previous study found that subcutaneous immunization with MBP induced weak IFN-␥ production in vivo. However, in this paper we found that MBP significantly increased IFN-␥ level in vitro. One possible explanation is that MBP can directly interact with TLR2 expressed on CD4+ T cells and induce Th1 activation in vitro, which results in much stronger IFN-␥ production. Another possible reason is the critical role of DCs in directing the differentiation of CD4+ T cells (Boonstra et al. 2003). In this regard, we suppose that the two different administration routes caused distinct phenotype of DCs induced by MBP. All these factors may be responsible for the




low level of IFN-␥ secretion in vivo. This interesting possibility is currently under investigation in our laboratory. In summary, our study revealed that MBP directly promoted activated CD4+ T cell proliferation and induced Th1 activation in vitro. This study lays the foundation for the development of MBP as an immunomodulatory agent. TLR2 favored the MBP-induced Th1 activation via the MyD88-dependent pathway, whereas TLR4 inhibited the MBP-induced Th1 activation via the MyD88independent pathway. This cross-talk between TLR2 and TLR4 provides a new way to understand the complicated mechanisms of TLRs-triggered immune response. Conflict of interest The authors declare no conflicts of interest. Acknowledgment This work was supported by the National Natural Science Foundation of China (nos. 31170875 and 31300740). References Adachi, O., Kawai, T., Takeda, K., Matsumoto, M., Tsutsui, H., Sakagami, M., Nakanishi, K., Akira, S., 1998. Targeted disruption of the MyD88 gene results in loss of IL-1and IL-18-mediated function. Immunity 9, 143–150. Andre, B., Charles, A.S., Carl, G.F., Cynthia, L., Allen, C., Alan, S., 2005. TLR9 regulates Th1 responses and cooperates with TLR2 in mediating optimal resistance to Mycobacterium tuberculosis. J. Exp. Med. 202, 1715–1724. Angela, L., Michael, T.W., Paul, J.U., 2011. CpG and non-CpG oligodeoxynucleotides directly costimulate mouse and human CD4+ T cells through a TLR9- and MyD88independent mechanism. J. Immunol. 187, 3033–3043. Boonstra, A., Asselin, P.C., Gilliet, M., 2003. Flexibility of mouse classical and plasmacytoidderived dendritic cells in directing T helper type 1 and 2 cell development: dependency on antigen dose and differential toll-like receptor ligation. J. Exp. Med. 197, 101–109. Butt, T.R., Jonnalagadda, S., Monia, B.P., Sternberg, E.J., Marsh, J.A., Stadel, J.M., Ecker, D.J., Crooke, S.T., 1989. Ubiquitin fusion augments the yield of cloned geneproducts in Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 86, 2540–2544. Dalia, E.G., Suzanne, M.M., Jannet, K., 2011. TLR4 signaling via MyD88 and TRIF differentially shape the CD4+ T cell response to Porphyromonas gingivalis hemagglutinin B. J. Immunol. 3, 5772–5783. Fang, H.L., Wu, Y.F., Huang, X.H., Wang, W.Y., Bing Ang Cao, X.T., Tao, W., 2011. Tolllike receptor 4 (TLR4) is essential for Hsp70-like protein 1 (HSP70L1) to activate dendritic cells and induce Th1 response. J. Biol. Chem. 7, 30393–30400. Fernandez, S., Palmer, D.R., Simmons, M., Sun, P., Bisbing, J., McClain, S., Mani, S., Burgess, T., Gunther, V., Sun, W., 2007. Potential role for Toll-like receptor 4 in mediating Escherichia coli maltosebinding protein activation of dendritic cells. Infect. Immun. 75, 1359–1363. González-Navajas, J.M., Fine, S., Law, J., Datta, S.K., Nguyen, K.P., Yu, M., Corr, M., Katakura, K., Eckman, L., Lee, J., Raz, E., 2010. TLR4 signaling in effector CD4+T cells regulates TCR activation and experimental colitis in mice. J. Clin. Invest. 120, 570–581.

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Cathelicidins Inhibit Escherichia coli-Induced TLR2 and TLR4 Activation in a Viability-Dependent Manner.

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

Mycobacterium tuberculosis Rv2882c Protein Induces Activation of Macrophages through TLR4 and Exhibits Vaccine Potential.

4 in vivo and in vitro.

Correlation of TLR2 and TLR4 expressions in peripheral blood mononuclear cells to Th1- and Th2-type immune responses in children with henoch-schönlein purpura.

SecB-independent export of Escherichia coli ribose-binding protein (RBP): some comparisons with export of maltose-binding protein (MBP) and studies with RBP-MBP hybrid proteins.

Akt and upregulating p38 MAPK signaling pathways.

Cucurbitacin E Induces Autophagy via Downregulating mTORC1 Signaling and Upregulating AMPK Activity.

Protein-bound polysaccharide-K induces IL-1β via TLR2 and NLRP3 inflammasome activation.

Escherichia coli outer membrane protein F (OmpF): an immunogenic protein induces cross-reactive antibodies against Escherichia coli and Shigella.

β2-AR activation induces chemoresistance by modulating p53 acetylation through upregulating Sirt1 in cervical cancer cells.

Luteolin induces apoptosis in BE colorectal cancer cells by downregulating calpain, UHRF1, and DNMT1 expressions.

Escherichia coli sec mutants accumulate a processed immature form of maltose-binding protein (MBP), a late-phase intermediate in MBP export.

miRNA143 Induces K562 Cell Apoptosis Through Downregulating BCR-ABL.

Microglia Induce Neurotoxic IL-17+ γδ T Cells Dependent on TLR2, TLR4, and TLR9 Activation.

Evaluating Effect of Mesenchymal Stem Cells on Expression of TLR2 and TLR4 in Mouse DCs.

TLR4, rather than TLR2, regulates wound healing through TGF-β and CCL5 expression.

TLR2 and TLR4 polymorphisms influence mRNA and protein expression in colorectal cancer.

The general protein-export pathway is directly required for extracellular pullulanase secretion in Escherichia coli K12.

TLR2 and TLR4 in healthy children: age and gender differences.

TLR4 Signaling Enhances TGF-β Response Through Downregulating BAMBI During Prostatic Hyperplasia.

Relationship of TLR2, TLR4 and tissue remodeling in chronic rhinosinusitis.

TLR2, TLR4 and CD86 gene polymorphisms in recurrent aphthous stomatitis.

Leishmania mexicana lipophosphoglycan activates ERK and p38 MAP kinase and induces production of proinflammatory cytokines in human macrophages through TLR2 and TLR4.