The Effects And Mechanisms Of Escherichia Coli Maltose-binding Protein Switching Tumor-associated Macrophages From M2to M1

Maltose-binding protein (MBP) is a critical component of themaltose/maltodextrin transport system in Escherichia coli, which is responsible forthe uptake and catabolism of maltodextrins. It has been widely used for thepurification of recombinant pathogenic bacteria and virus subunit vacccines as atagged protein in modern research of molecular biology. Recent study shows thatMBP can induce dendritic cell maturation and produce proinflammatory cytokinesthrough Toll-like receptor4, and recombinant protein-MBP vaccines can enhance theimmunogenicity of the recombinant proteins. In our previous study, we found thatMBP can enhance the viability of U937cells through TLR2, and MBP cannon-specifically promote mice spleen lymphocyte proliferation, induces T helpertype1cell activation and activate peritoneal macrophages obtained from mouse;Moreover, MBP can enhance the immunogenicity of the Mucin1-MBP recombinantprotein vaccine, and induce effective antitumor cellular immune response; and wealso found that the combination of MBP and Bacillus Calmette-Guerin (BCG)induces synergistic antitumor activity in Lewis lung carcinoma or B16melanoma-bearing mice. The findings above confirmed the potentimmune-enhancing activities of MBP, however, the mechanisms remains unknown.In the present study, we sought to determine if MBP can activate macrophagesdirectly and influence macrophage polarization, and further to explore themoderating effects of MBP in the polarity process of tumor cell supernatant-inducedtumor-associated macrophages. To date, only a few agents have been reported todrive macrophages toword M1polarization. Therefore, it is essential to develop new adjuvants and immunostimulants for inducing the M1polarization of macrophages,which can be used in the treatment of oncological diseases. In the present study, wemainly study the moderating effects of MBP in the polarization process oftumor-associated macrophages, and the possible mechanism of MBP in switchingtumor-associated macrophages from M2to M1. This research includes the followingaspects:1. Preparation of MBPThe pMAL plasmid was transfected into E. coli DH5α. The MBP wasexpressed upon induction with IPTG. MBP was purified by Amylose resin affinitychromatography, and the protein was desalted by Sephadex G-25. MBP was thenpassed through a polymyxin B-agarose column to get rid of endotoxins, and filteredby Millipore ultrafiltration device. MBP was tested for endotoxin remnants using alimulus amebocyte lysate-based assay.2. MBP induced RAW264.7cells polarized to M1macrophagesThe effects of MBP in activation and polarization of RAW264.7cells wereinvestigated in the study. RAW264.7cells were stimulated with MBP in vitro. Theproduction of NO was detected by Griess reagents and the secretion of cytokines asIL-1β, IL-6, IL-10, and IL-12p70were analyzed by commercial ELISA kit. Theexpression of iNOS on RAW264.7cells was analyzed by flow cytometry. The resultsshowed that MBP significantly increased NO production in RAW264.7cells in a dose andtime-dependent manner (12-48h), when stimulated with MBP (0.1-10μg/ml), theproduction of nitric oxide (NO) was increased in RAW264.7cells (P <0.01). Thesecretion of IL-1β, IL-6and IL-12p70was significantly increased in MBP-inducedRAW264.7cells, but the secretion of IL-10was not increased (P>0.05). Theexpressions of CD80, MHC class II and inducible nitric oxide synthase (iNOS) weresignificantly increased in MBP-treated RAW264.7cells. CD80and MHC class IIwere the molecular markers of the activation of macrophages, and iNOS was themarker of M1macrophages. The results indicated the activation and polarization of RAW264.7cells into M1macrophages induced by MBP.3. The molecular mechanisms of M1type polarization of RAW264.7cellsinduced by MBPThe expressions of TLR2and TLR4on RAW264.7cells were analyzed by flowcytometry. TLR2and TLR4play critical roles in the activation of immunoeffectorcells induced by MBP. The results showed that both TLR2and TLR4were upregulatedwhen RAW264.7cells were treated with MBP, which indicated that MBP may promote theactivation and polarization of RAW264.7cells via TLR2and TLR4pathways. Furtherstudy showed that MBP stimulation upregulated the expression of TLR2and TLR4on RAW264.7cells, which was accompanied by subsequent phosphorylation ofIκB-α and p38MAPK, moreover, the results showed that MyD88was upregulated inMBP-treated RAW264.7cells, which indicated the possible involvement of MyD88in theprocess of MBP stimulation. Pretreatment with anti-TLR2or anti-TLR4antibodieslargely inhibited the phosphorylation of IκB-α and p38MAPK, suggesting that theMBP-induced macrophage activation and polarization were mediated by TLR2andTLR4signaling pathways.4. Tumor-associated macrophages in vitro cell model was induced by tumorcell supernatantTumor cell supernatants (TCS) were obtained from the cell culture supernatantsof murine breast cancer4T1cells. RAW264.7cells were co-cultured with theconditioned TCS for48h. The specific molecular markers of M1and M2macrophages were analyzed by immunofluorescence and flow cytometry, and thespecific cytokines IL-10and IL-12p70were tested by ELISA kits. CD206wasselected as the specific marker for M2macrophages, iNOS and CD16/32were takenfor the specific marker for M2macrophages. LPS-induced M1macrophages andIL-4-induced M2macrophages were used as positive control. The results showed thatTCS-induced RAW264.7cells express CD206molecules, and secrete high level ofIL-10, that means TCS induced RAW264.7cells polarizing to M2macrophages.5. MBP modulated TAM polarizing from M2to M1RAW264.7cells were cultured in RPMI-1640medium contained40% conditioned tumor cell supernatants in the absence or presence of5μg/ml MBP. Thespecific molecular markers of M1and M2macrophages were analyzed byimmunofluorescence and flow cytometry, and the specific cytokines IL-10andIL-12p70were tested by ELISA kits. CD206was selected as the specific marker forM2macrophages, iNOS and CD16/32were taken for the specific marker for M2macrophages. The results showed that compared with TCS-induced RAW264.7cells,the expressions of CD16/32and iNOS were increased, CD206was decreased, thesecretion of IL-12was increased, and IL-10was decreased in MBP+TCS-treatedRAW264.7cells. The results suggested TAM induced by conditioned tumor cellsupernatants can be converted to M1by MBP stimulation.Our study provides a new insight into a mechanism by which MBP can directlyinduce macrophages activation and M1type polarization. MBP may have the effectsto enhance antitumor immune responses through switching TAM polarization fromM2to M1. Our study warrants the potential application of MBP as an immuneadjuvant in tumor immune therapies.