The Role Of Tim-3 Signaling Pathway Regulating Macrophage Polarization In The Pathogenesis Of H.Pylori Infection

Background and objective:Amost more than half of the world's population is infected with H.pylori.Once the bacteria colonized in gastric mucosa,the infection will be lifelong,moreover,the infection may cause chronic gastritis,peptic ulcer,and even gastric cancer.The invasion of H.pylori can cause infiltration of a large number of inflammatory cells in the gastric mucosa intrinsic layer,inducing a strong inflammatory reaction in the gastric mucosa.Macrophages can secrete inflammatory factors at the first time after Helicobacter pylori invade,initiate immune response,and ultimately achieve the killing effect on pathogens,in which macrophage polarization is an important mechanism for its function.Tim3,one of the important member of TIM family,has an important role in immune regulation and tumor response.Previous research findings of our research group showed that Tim3 signaling pathway and TLR4signaling pathway can regulate each other in H.pylori infected mouse and vaccine immunized mouse,and compared with wild macrophages,the actvation of TLR4signaling pathway and the secretion of inflammatory cytpkines were lower than H.pylori infected Tim3 overexpressing macrophages significantly,indicatin that Tim3can affect the function of macrophages,but whether it is through the regulation of macrophage polarization to play this role is not yet clear.So,the paper will explore this conduct.Method:1.Groups:?1?Wild RAW264.7 cell;?2?Wild RAW264.7 cell+H.pylori;?3?Tim3^Low RAW264.7 cell;?4?Tim3^Low RAW264.7+H.pylori;?5?Tim3^Highigh RAW264.7cell;?6?Tim3^Highigh RAW264.7 cell+H.pylori.2.Co-culture of Helicobacter pylori and cell:After cultured 12h in six well plate and the cells are well attached,add suspension of H.pylori into six well plate according to the designed program.The cells then are incubated for 2 hours in the CO₂ incubator,after that,we collect the cells and culture supernatant for the next experiment.3.Observation indicators and methods:?1?Real-time PCR mesure the relative mRNA expression of NOS2?IL-10 in the H.pylori infected macrophages.?2?Test the expression of F4/80?CD16/CD32?CD206 of macrophages infected by H.pylori by flow cytometry.?3?Flow cytometry mesure the percentage of engulfed fitc-dextran by H.pylori infected macropages to evaluate the phagocytic function of macrophages.?4?Mesure the expression of TLR4?MyD88?p-NF-?B of H.pylori infected macrophages by western blot.?5?Mesure the expressiong of TNF-??TGF-?in the cell culture supernatant.Result:1.The effect of H.pylori infection on macrophage polarization and the phagocytic function of macrophages1.1.The expression of macrophage polarization-related cytokines on H.pylori infected macrophagesNOS2 is the marker of M1 macrophage,and IL-10 presents the M2 macrophage.And the NOS2 expression of the H.pylori-infected wild type macrophages gradually increased with time,compared with control group,the increase has statistical significance?p<0.05?;while IL-10 expression was higher than controlled group too,only 2 hours group higher than control group significantly?p<0.05?,but the expression reduced by time.1.2.Detection of TNF-?and TGF-?in macrophage culture supernatants after H.pylori infection by ELISAData showed that M1 type cytokine TNF-?was significantly increased after stimulation?p<0.01?,while M2 The type of cytokine TGF-?did not change significantly after H.pylori stimulation?p>0.05?1.3.Detection of macrophage M1 and M2 cell ratio after H.pylori infection by flow cytometryThe proportion of M2 macrophages increased significantly in wild-type RAW264.7 cells after the infection of H.pylori?p<0.01?,while the proportion of M1macrophages decreased?p<0.01?.1.4.The detection of macrophage phagocytosis after H.pylori infection by flowcytometryAfter stimulating wild-type RAW264.7 cells with H.pylori,the phagocytosis of FITC-dextran was significantly enhanced?p<0.01?.2.The infection of Tim3 on the macrophages polarization and the phagocytic function of macrophages2.1.The building of Tim3^LowRAW264.7 cell line and Tim3^HighRAW264.7 cell lineData showed that Tim3^LowRAW264.7 cells Tim3 mRNA expression was significantly lower than the control cells?p<0.01?,Tim3^HighRAW264.7 cells Tim3mRNA expression was significantly higher than control cells?p<0.01?.2.2.The expression of macrophage polarization-related cytokings on infected Tim3^LowRAW264.7 cell and Tim3^HighRAW264.7 cellAfter stimulated by H.pylori,the expression of NOS2?M1 marker?in wild-type RAW264.7 cells,Tim^LowRAW264.7 cells and Tim3^HighRAW264.7 cells was higher than that of unstimulated cells.With or without H.pylori stimulation,the NOS2expression in Tim^LowRAW264.7 cells and Tim3^HighRAW264.7 cells was significantly higher than that in wild-type RAW264.7 cells?p<0.01?;and NOS2 in Tim3^HighRAW264.7 cells after H.pylori stimulation was significantly higher than that of Tim3^LowRAW264.7 cells?p<0.05?.IL-10?M2 marker?expression was significantly higher in wild-type RAW264.7cells,Tim^LowRAW264.7 cells,and Tim3^HighRAW264.7 cells which were infected with H.pylori than in uninfected cells?p<0.01?.In the absence of H.pylori stimulation,NOS2 expression was significantly higher in Tim^LowRAW264.7 cells and Tim3^HighRAW264.7 cells than in wild-type RAW264.7 cells?p<0.05?;compared to wild-type RAW264.7 cells after H.pylori stimulation,the expression of IL-10 in Tim3^LowRAW264.7 cells was significantly increased?p<0.05?,IL-10 expression in Tim3^HighRAW264.7 cells was also increased,but there was no statistical difference?p>0.05?;and after H.pylori stimulation,The IL-10 expression of Tim3^LowRAW264.7cells was significantly higher than that of Tim3^HighRAW264.7 cells?p<0.05?.2.3.Detection of TNF-?and TGF-?in culture supernatant of Tim3^Low and Tim3^HighRAW264.7 cells after H.pylori infection by ELISAThe results showed that the levels of the M1 cytokine TNF-?in the culture supernatant of wild-type and Tim3^Low RAW264.7 cells after H.pylori stimulation were higher than control group.In the unstimulated group,there was no significant change in the level of M1 cytokine TNF-?after stimulation with H.pylori in Tim3^HighRAW264.7 cells?p>0.05?;in the absence of H.pylori stimulation,TNF-?in the culture supernatant of Tim3^HighRAW264.7 cells was significantly higher than that of wild-type and Tim3^LowRAW264.7 cells?p<0.01?.In the presence of H.pylori stimulation,the levels of TNF-?in the culture supernatant of wild-type RAW264.7and Tim3^HighRAW264.7 cells were significantly higher than that of Tim3^LowRAW264.7.?p<0.05?,buttherewasnodifferencebetween Tim3^HighRAW264.7 and wild-type RAW264.7?p>0.05?.However,a M2 marker,TGF-?,did not change after stimulation with H.pylori?p>0.05?,regardless of up-or down-regulation of Tim3 expression?p>0.05?.2.4.The ratio of M1 and M2 macrophages in H.pylori-infected Tim3^Low and Tim3^HighRAW264.7 cellThe proportion of M1 macrophages in wild type and Tim3^HighRAW264.7 cells stimulated by H.pylori was significantly lower than that of the group without H.pylori stimulation?p<0.01?.Although there was a decrease in Tim3^Low group,there was no statistical difference?p>0.05?.In the absence of H.pylori stimulation,the ratio of M1 macrophages in Tim3^LowRAW264.7 was lower than that of the wild-type RAW264.7 group,but there was no statistical difference?p>0.05?,while the proportion of M1 macrophage in the Tim3^HighRAW264.7 group was significantly higher than that of the wild-type RAW264.7 and Tim3^LowRAW264.7 groups?p<0.05?;After H.pylori stimulation,the proportion of M1 macrophages in Tim3^LowRAW264.7and Tim3^HighRAW264.7 was significantly higher than that of the wild-type RAW264.7 group?p<0.05?.The proportion of M2 macrophages stimulated by H.pylori in three gropus was significantly higher than that in the unstimulated group?p<0.01?;in the absence of H.pylori stimulation,the proportion of M2 macrophages in theTim3^HighRAW264.7group was significantly higher than that of the Tim3^LowRAW264.7 and wild type RAW264.7 groups?p<0.01?,but there was no difference in the ratio of the M2macrophages between the Tim3^LowRAW264.7 group and the wild type RAW264.7group?p>0.05?;After H.pylori stimulation,the proportion of M2 macrophages in the Tim3^HighRAW264.7 and Tim3^LowRAW264.7 groups was significantly higher than that in the wild-type RAW264.7 group?p<0.01?,and the proportion of M2 macrophages in the Tim3^LowRAW264.7 group was significant higher than Tim3^HighRAW264.7group?p<0.05?.2.5.Detection of phagocytic function about infected Tim3^Low and Tim3^Highigh RAW264.7 by flow cytometryThe results showed that the phagocytosis of three kinds of cells stimulated by H.pylori was significantly stronger than unstimulated group?p<0.05?;with or without H.pylori stimulation,the phagocytosis of Tim3^HighRAW264.7 group was stronger than that in the Tim3^LowRAW264.7 and wild-type RAW264.7 groups?p<0.05?.There was no difference in phagocytosis between Tim3^LowRAW264.7 and wild-type RAW264.7?p>0.05?.3.The regulation of Tim3 on macrophage polarization in H.pylori infection3.1.The expression of TLR4 in H.pylori infected wild-type?Tim3^Low and Tim3^High RAW264.7 cellBoth wild-type RAW264.7 cells and Tim3^HighRAW264.7 cells showed increased TLR4 protein expression after H.pylori stimulation,and wild type RAW264.7 cells showed significantly higher expression of TLR4 after H.pylori stimulation?p<0.05?.The expression of TLR4 after stimulation with H.pylori in Tim3^HighRAW264.7 cells was increased,but the difference was not statistically significant?p>0.05?.However,TLR4 protein expression did not change after stimulation with H.pylori in Tim3^LowRAW264.7 cells?p>0.05?.TLR4 expression in Tim3^LowRAW264.7 group after H.pylori stimulation was significantly lower than that of Tim3^High RAW264.7?p<0.05?.3.2.The expression of MyD88 in H.pylori infected wild-type?Tim3^Low and Tim3^High RAW264.7 cellThe expression of MyD88 protein in wild-type RAW264.7 and Tim3^HighRAW264.7 cells after H.pylori stimulation was significantly higher than that in the unstimulated group?p<0.05?,while the increase of infected Tim3^LowRAW264.7has no Statistical significance.After H.pylori stimulation,MyD88 in Tim3^LowRAW264.7 cells was significantly lower than wild-type RAW264.7 cells and Tim3^HighRAW264.7 cells?p<0.01?,and there was no difference between wild-type MyD88 cells and Tim3^LowRAW264.7 cells?p>0.05?.3.3.The expression of p-NF-?B in H.pylori infected wild-type?Tim3^Low and Tim3^High RAW264.7 cellAfter H.pylori stimulation,the p-NF-?B protein level of three groups was higher than that of the unstimulated group,among them,the wild-type group was higher than controlled group significantly?p<0.05?.Stimulated with H.pylori,p-NF-?B protein in Tim3^LowRAW264.7 Cell was lower than wild-type RAW264.7cells?p>0.05?,and lower than Tim3^LowRAW264.7 cells significantly?p<0.05?,but the difference has no Statistical significance?p>0.05?.Conclusion:1.Early in the H.pylori infection,macrophages appeared as mixed M1 and M2increase,mainly based on M2 increase,besides the M2 can convert to M1 at the late infection,therefor,macrophage polarization is a dynamic process in H.pylori infection.2.Overall,Tim3 promotes the polarization of macrophages to M1,their intensity is different at base line and after H.pylori stimulated,the expression of the polarization makers are different too.3.Down-regulation of Tim3 could inhibit the activation of TLR4 signaling pathway in macrophages after H.pylori stimulation,which may be one of the mechanisms that promote the polarization of H.pylori-stimulated macrophages to M2 after Tim3 down-regulation.4.Helicobacter pylori infection enhances phagocytosis of macrophages,while Tim3 can enhance phagocytosis of macrophages furtherly.