| Background and purpose:The body’s immune response plays a pivotal role in the process ofHelicobacter.pylori(H.pylori) infection. As a part of the immune system, macrophageacts as a significant role in the innate immune response, what’s more, it has greatimpact on adaptive immunity. TLR4is a kind of pattern recognition receptor, andby recognizing pathogen-associated molecule patterns (PAMPs), it can activatemacrophages to secrete plenty of cytokines which initiate and regulate body’simmune response. Tim-3is an important member of TIM family, a recentlydiscovered transmembrane protein family. Studies showed that Tim-3was a moleculemarker differentiating Th1from Th2cells and Tim-3can downregulate Th1cellfunction when activated. Lots of researches also have shown Tim-3was expressed byinnate immune cells such as macrophages and can impact macrophage functionthrough interacting with TLR4signaling pathway. Until now, it is unclear how H.pylori impacts Tim-3and TLR4signaling in macrophages. By establishingmacrophages overexpressing Tim-3, we attempt to clarify the impact of H.pylori onTim-3and TLR4signaling pathway and the relationship between Tim-3and TLR4signaling pathway.These results may provide important experimental evidence forcontrol and prevention of H.pylori infection and its related diseases by targetingTim-3.Method:⑴The impact of H.pylori on macrophage proliferationH.pylori SS1(SS1) was used to infect RAW264.7at different multiplicityof infection (MOI), and the research groups were as following：Control group: cells without infectionGroup A：MOI is equal to25Group B：MOI is equal to50Group C：MOI is equal to100Group D：MOI is equal to200 Group E：MOI is equal to400Group F：MOI is equal to800The cell proliferation rates for each group were detected at3,6,12,24and48hours after coculture respectively.⑵T he impact ofH.pylori on Tim-3, TLR4, MyD88expression inmacrophagesSS1was used to infect RAW264.7at different MOI, and the researchgroups were as following：Control group：cells without infectionGroup A：MOI is equal to25Group B：MOI is equal to50Group C：MOI is equal to100Group D：MOI is equal to200The cells were collected after12hours of coculture⑶Construction and identification of Tim-3recombinant eukaryotic expressionplasmid①Splenic mononuclear cells of mice were isolated, and specific primers forTim-3gene were designed.Using the primers and proper procedure, amplification ofTim-3gene was performed.②T im-3amplification product was inserted into pLVX-IRES-ZsGreen1vectorto construct recombinant plasmid.Amplifying and extracting the recombinant plasmidwere performed, and then Tim-3recombinant plasmid was identified byrestriction enzyme digestion and sequencing.⑷Eukaryotic expression of Tim-3recombinant plasmidUsing lipofection transfection technology to transferpLVX-IRES-ZsGreen-Tim-3into mouse macrophages, we observed whether therewas fluorescence or not and the level of fluorescence intensity. Cells were collectedto test the expression of Tim-3mRNA and protein.⑸T he impact ofH.pylori infection on Tim-3and TLR4signaling pathway andsecretion of inflammatory cytokines in mouse macrophages overexpressing Tim-3①T im-3transfection ②experimental groupingSS1with100MOI was used to infect RAW264.7, and the researchgroups were as following：Control group1：cells+transfection reagentControl group2：cells+transfection reagent+Tim-3plasmidControl group3：cells+transfection reagent+plasmid without Tim-3Experimental group1：cells+transfection reagent+H.pyloriExperimental group2：cells+transfection reagent+H.pylori+Tim-3plasmidExperimental group3：cells+transfection reagent+H.pylori+plasmid without Tim-3Cells and supernatant were collected after12hours of infection.⑸Detection methods：①MTT was used to detect the impact of H.pylori on macrophages proliferation.②R T-PCR was used for detecting levels of Tim-3, TLR4and MyD88mRNAin macrophages of each group.③Western Blot was applied to examine Tim-3, TLR4, MyD88and NF-κBp65expression in macrophages of each group.④The concentrations of TNF-α, IL-6, IFN-γ and IL-10in supernatant weredetermined by ELISA.Results：⑴The impact of H.pylori on macrophages proliferationThere was no statistical differences in cell proliferation rates among everygroups in3hours and12hours. There was significant statistical differences amongevery groups in6hours,12hours and48hours（all P value＜0.01）. Cell proliferationrates in each group gradually increased with the value of MOI in6hours And groupsD, E and F were significantly higher than the control group（P＜0.05，P＜0.01）,while groups E and F were significantly higher than group A（P＜0.05，P＜0.01）.Moreover, group F was significantly higher than groups B, C and Drespectively（P≤0.01）. Cell proliferation rates in each group gradually increased withthe value of MOI in12hours, and groups C, D, E and F were all significantly higher than the control group and group A（P＜0.05，P＜0.01）.Groups D, E and Fwere all significantly higher than group B（P＜0.01）, and group F was significantlyhigher than group C and D（P＜0.05，P＜0.01）. Cell proliferation in each groupgradually decreased with the value of MOI Groups B, C, D, E and F were allsignificantly lower than the control group（P＜0.05，P＜0.01）.Groups C, D, E and Fwere all significantly lower than groups A and B respectively（P＜0.05，P＜0.01）,and groups D, E and F were all significantly lower than group C（P＜0.05，P＜0.01）,and groups E and F were all significantly lower than group D（P＜0.05，P＜0.01）.⑵The impact of H.pylori on levels of Tim-3, TLR4and MyD88mRNA inmacrophagesH.pylori can elevate the level of Tim-3, TLR4and MyD88mRNA inmacrophages after12hours of coculture（P＜0.01）.Levels of Tim-3, TLR4andMyD88mRNA increased with the value of MOI within certain limits（group A to C）,and Tim-3mRNA expression in each experimental group was significantly higherthan the control group（P＜0.01）, however, there were no significant differenceamong these experimental groups（P＞0.05）. TLR4mRNA expression in eachexperimental group was significantly higher than that in control group（P＜0.05，P＜0.01）, and group C was significantly higher than groupsAand B (P＜0.01).GroupD was significantly higher than groups A and B (P＜0.01，P＜0.05),while there wasno significant difference between groups A and B (P＞0.05).There was also nosignificant difference between groups C and D (P＞0.05). MyD88mRNA expressionin groups B, C and D were significantly higher than that in control group (P＜0.01，P＜0.05), and groups C and D were significantly higher than groups A and B (P＜0.01，P＜0.05), but there was no significant difference between groups A and thecontrol group, groups B and A as well as groups C and D (P＞0.05).⑶Construction and identification of Tim-3recombinant eukaryotic expressionplasmidpLVX-IRES-ZsGreen-Tim-3recombinant expression plasmid was successfullyconstructed. After digested by restriction enzyme, two bands in the agarose gel wereobserved. The molecular weight was consistent with expected size. Sequencinganalysis showed the inserted segment had100%similarity to Tim-3gene sequence provided by Pubmed gene library (GenBank Accession: AF450241.1).⑷Expression of Tim-3recombinant eukaryotic expression plasmidAfter Tim-3recombinant plasmid was transferred into mouse macrophages,green fluorescence could be observed under fluorescence microscope, and theexpression of Tim-3mRNA and protein in macrophages was significantly higher thanthat in the groups untransfected(P<0.05), which meaned transfection was verysuccessful.⑸The impact of H.pylori infection on Tim-3expression of mousemacrophages overexpressing Tim-3The effect of H.pylori infection on Tim-3expression in macrophagesoverexpressing Tim-3Whether transfected with Tim-3plasmid or not, the expressionof Tim-3mRNAand protein in mouse macrophages was significantly higher in the groups infectedwith H.pylori than that in the control group（sP＜0.01，P＜0.05）.In the control groups,the expression of Tim-3mRNA in macrophages transfected with Tim-3plasmid wassignificantly higher than that in the groups untransfected or transfected with plasmidwithout Tim-3（P＜0.01）.In the groups infected by H.pylori, the expression levels ofTim-3mRNA and protein in mouse macrophages transfected with Tim-3plasmid wassignificantly higher than that in the groups untransfected and transfected with plasmidwithout Tim-3（P＜0.01）.Furthermore, whether infected with H.pylori or not, therewas no significant difference between groups untransfected and groups transfectedwith plasmid without Tim-3（P＞0.05）.⑹T he impact ofH.pylori infection on TLR4signaling pathway in macrophagesoverexpressing Tim-3①Changes of TLR4expressionIn the groups untransfected and transfected with plasmid without Tim-3, theexpressionof TLR4mRNA and protein in mouse macrophages infected by H.pyloriwere significantly higher than that in the control groups（P＜0.01，P＜0.05）.In thegroups overexpressing Tim-3, the expression of TLR4mRNA was significantlyhigher in the groups infected with H.pylori than that in the control groups（P＜0.01）,and there was no significant difference in the expressionof TLR4mRNA and protein among the groups without H.pylori infection（P＞0.05）.In the groups infectedwith H.pylori, the expression of TLR4mRNA was significantly lower in the groupsoverexpressing Tim-3than that in the groups untransfected or transfected withplasmid without Tim-3（P＜0.01）, and there was no significant difference betweenthe groups untransfected and transfected with plasmid without Tim-3（P＞0.05）.②Changes of MyD88expressionIn the groups untransfected and transfected with plasmid without Tim-3, theexpression of MyD88mRNA and protein in mouse macrophages infected withH.pylori was significantly higher than that in the control groups（P＜0.01，P＜0.05）.In the groups overexpressing Tim-3, the expression of MyD88mRNA wassignificantly higher in the groups infected with H.pylori than that in the controlgroups（P＜0.01），and there was no significant difference in the expression ofMyD88mRNA and protein among the groups without H.pylori infection（P＞0.05）.In the groups infected by H.pylori, the expression of MyD88mRNA wassignificantly lower in the groups overexpressing Tim-3than that in the groupsuntransfected and transfected with plasmid without Tim-3（P＜0.05）, and there wasno significant difference between the groups untransfected and transfected withplasmid without Tim-3（P＞0.05）.③Changes of pNF-κB p65expressionIn the groups without H.pylori infection, there was no significant difference inthe expression of pNF-κB p65regardless of Tim-3transfection（P＞0.05）.In thegroups infected with H.pylori, the expression of pNF-κB p65was slightly lower inthe groups overexpressing Tim-3than that in the groups untransfected and transfectedwith plasmid without Tim-3,however it was not of statistical difference（P＞0.05）,and the expression of pNF-κB p65in the groups untransfected and transfected withplasmid without Tim-3was significantly higher in the groups infected with H.pylorithan that in the groups without infection（P＜0.05）.Moreover, there was nosignificant difference in the expression of pNF-κB p65of mouse macrophagesoverexpressing Tim-3regardless of H.pylori infection（P＞0.05）.⑺The impact of H.pylori infection on the secretion of inflammatory cytokines inmouse macrophages overexpressing Tim-3. ①Impact on TNF-αIn the groups infected with H.pylori, TNF-α secretion in the groupsoverexpressing Tim-3was significantly lower than that in the groups untransfectedand transfected with plasmid without Tim-3（P＜0.01）.In the groups withoutinfection, there was no significant difference in TNF-α secretion regardless of Tim-3transfection（P＞0.05），and in the groups untransfected and transfected with plasmidwithout Tim-3, TNF-α secretion was significantly higher in the groups infected withH.pylori than that in the groups without infection （P＜0.01）.In the groupsoverexpressing Tim-3, there was no significant difference in TNF-α secretionregardless of H.pylori infection （P＞0.05）, and there was no significantdifference in TNF-α secretion between groups untransfected and transfected withplasmid without Tim-3regardless of H.pylori infection（P＞0.05）。②Impact on IL-6In the groups infected with H.pylori, IL-6secretion in the groups overexpressingTim-3was significantly lower than that in the groups untransfected and transfectedwith plasmid without Tim-3（P＜0.01）.In the groups without infection, there was nosignificant difference in IL-6secretion regardless of Tim-3transfection（P＞0.05）.And in the groups untransfected and transfected with plasmid without Tim-3, IL-6secretion was significantly higher in the groups infected by H.pylori than that in thegroups without infection（P＜0.01）. In the groups overexpressing Tim-3, there wasno significant difference in IL-6secretion regardless of H.pylori infection（P＞0.05）, and there was no significant difference in IL-6secretion between groupsuntransfected and transfected with plasmid without Tim-3regardless of H.pyloriinfection（P＞0.05）。③Impact on IFN-γIn the groups infected by H.pylori, IFN-γsecretion in the groups overexpressingTim-3was significantly lower than that in the groups untransfected and transfectedwith plasmid without Tim-3（P＜0.01，P＜0.05）. In the groups without infection,there was no significant difference in IFN-γsecretion regardless of Tim-3transfection（P＞0.05）And in the groups untransfected and transfected with plasmid withoutTim-3, IFN-γsecretion was significantly higher in the groups infected by H.pylori than that in the groups without infection（P＜0.01，P＜0.05）. In the groupsoverexpressing Tim-3, there was no significant difference in IFN-γsecretion betweengroups with and without H.pylori infection（P＞0.05）, and there was also nosignificant difference in IFN-γ secretion between groups untransfected andtransfected with plasmid without Tim-3regardless of H.pylori infection（P＞0.05）。④Impact on IL-10In the groups infected with H.pylori, IL-10secretion was significantly lower inthe groups overexpressing Tim-3than that in the groups untransfected and transfectedwith plasmid without Tim-3（P＜0.01，P＜0.05）. In the groups without infection,Tim-3transfection had no significant impact on IL-10secretion（P＞0.05）.Regardlessof Tim-3transfection, IL-10secretion was significantly lower in groups withinfection than that in groups without infection（P＜0.01，P＜0.05）. Regardless ofinfection, there was no significant difference in IL-10secretion between groupsuntransfected and transfected with plasmid without Tim-3（P＞0.05）.Conclusion：1.H.pylori SS1can enhance the proliferation of RAW264.7in a concentration ofH.pylori–dependent way.2.RAW264.7constitutively expresses TLR4and Tim-3, and H.pylori stimulationcan elevate TLR4signaling pathway and the expression of Tim-3. This kind ofstimulation could increase proinflammatory cytokines secretion and decreaseanti-inflammatory cytokine secretion which was dependent on the concentration ofH.pylori.3.Overexpression of Tim-3in RAW264.7has no significant impact on TLR4signaling pathway and inflammatory cytokines secretion.However, under thecircumstances of H.pylori infection, overexpression of Tim-3could downregulateTLR4signaling pathway and inflammatory cytokines secretion. |
PDF Full Text Request