| Objective:To investigate the potential role of Tim-3 on decidual macrophage in the abnormal pregnant outcomes induced by T.gondii infection.Methods:In vitro:Human decidual tissue samples were taken and separated.Single cell suspension was obtained.Human decidual macrophages were purified by magnetic bead separation and then divided into three groups:uninfected group,infected group and anti-Tim-3 neutralized infected group.The infected group and anti-Tim-3 neutralized infected group were cultured respectively with T.gondii RH strain tachyzoites(cell counts:tachyzoites counts =1:2)for 24h.The expressions of Tim-3,M1 and M2 membrane functional molecules CD80,CD86,CD 163,CD209,CD206 on decidual macrophages,intracellur cytokine TNF-a,IL-10 secreted by decidual macrophages were detected by flow cytometry.The synthesis of arginase metabolic enzymes inducible nitric oxide synthase(iNOS)and type-I arginase(Arg-I)in decidual macrophages were analyzed by Western Blot.The level of TNF-a and IL-10 in the supertant of decidual macrophages were also detected by ELISA.In vivo:Infected C57/BL6 pregnant mice model and Tim-3-/-infected pregnant mice model were successfully established in our study.The pregnant mice were divided into uninfected group,infected group and Tim-3-/－ infected group randomly.400 T.gondii tachyzoites(RH strain)were injected intraperitoneally to pregnant mice of infected group and Tim-3-/-infected group on gestation day(GD)8.The mice were executed on GD14,then the pregnancy outcomes were recorded including mental state,absorbed fetus and stillbirth,the weight of placenta and fetues,and so on.Single cell suspension was obtained by single cell preparation instrument.The expressions of Tim-3,M1 and M2 membrane functional molecules CD80,CD86,CD206 on F4/80+ decidual macrophages,arginase metablic enzymes inducible nitric oxide synthase(iNOS)and type-I arginase(Arg-I)synthesized by F4/80+ decidual macrophages,intracellur cytokines TNF-a and IL-10 secreted by F4/80+ decidual macrophage were detected by flow cytometry.Results:In vitro:With flow cytometry,Tim-3 expression levels on human decidual macrophages were significantly decreased after T.gondii infection(p<0.05),and only a few Tim-3 could be detected in anti-Tim-3 neutralized infected group.M1 membrane functional molecules CD80,CD86 were significantly up-regulated after T.gondii infection compared to uninfected group(p<0.05).M2 membrane functional molecules CD 163,CD209,CD206 expressions on human decidual macrophages were significantly down-regulated by T.gondii infection(p<0.05).Arginine catabolism enzyme iNOS was no expression in uninfected human decidual macrophages by Western Blot analysis,but could be obviously induced expression after T.gondii infection.Arg-I synthesis in decidual macrophages was significantly down-regulated by T.gondii infection.TNF-a secretion in human decidual macrophages was obviously up-regulated after T.gondii infection by flow cytometry and ELISA.IL-10 secretion in human decidual macrophages was also up-regulated after T.gondii infection by flow cytometry and ELISA.In addition,the ratio of TNF-a/IL-10 was increased in infected cells compared to uninfected cells.In order to explore the effect of Tim-3 on decidual macrophages in above functional molecules,related functional molecules were further detected in anti-Tim-3 neutralized infected group.The results showed that CD80,CD86 were further obviously up-regulated,CD 163,CD209,CD206 were reduced compared to infected group(p<0.05).Western Blot analysis showed iNOS was further up-regulated and Arg-I was further downregulated in anti-Tim-3 neutralized infected group compared to infected group(p<0.05).Flow cytometry analysis showed TNF-a secretion in human decidual macrophages was reduced,and IL-10 was also reduced in anti-Tim-3neutralized infected group compared to infected group,but TNF-a/IL-10 ratios were greater in anti-Tim-3 neutralized infected cells than in infected cells(p<0.05).ELISA analysis showed TNF-a secretion in human decidual macrophages was up-regulated,and IL-10 was also up-regulated in anti-Tim-3 neutralized infected group compared to infected group,but TNF-a/IL-10 ratios were greater in anti-Tim-3 neutralized infected cells than in infected cells(p<0.05).In vivo:Pregnant mice in infected group showed obvious abnormal pregnancy outcomes,they were shambling,moving less and erect fur,absorbed fetus and stillbirth were more serious in infected group,abnormal fetal ratios of infected group were higher than those of uninfected group,placentas and fetuses weight were significantly less(p<0.05).Tim-3 on mice decidual macrophages with T.gondii infection was obviously down-regulated compared to uninfected group(p<0.05),also a little of Tim-3 on decidual macrophages in Tim-3-/-infected mice could detected with flow cytometry.CD80 and CD86 were significantly increased and CD206 was significantly decreased after T.gondii infection by flow cytometry analysis(p<0.05).iNOS in mice decidual macrophages-was showed an obviously expression and Arg-I was obviously decreased after T.gondii infection by flow cytometry analysis(p<0.05).TNF-a in mice decidual macrophages was showed a obvious up-regulated expression and IL-10 was reduced after T.gondii infection,The TNF-a/IL-10 ratio was increased in infected decidual macrophages compared to uninfected decidual macrophages(p<0.05).In order to explore the effect of Tim-3 on decidual macrophages in above functional molecules,related functional molecules were further detected in Tim-3-/-infected group.The results showed that CD80 and CD86 were further obviously up-regulated and CD206 was further reduced(p<0.05).iNOS was further up-regulated and Arg-I was further down-regulated inTim-3-/-infected group compared to infected group.TNF-a was further up-regulated and IL-10 was significantly increased in decidual macrophages of Tim-3-/-infected mice compared to infected group by flow cytometry,the TNF-a/IL-10 ratio was increased in Tim-3-/－infected decidual macrophages compared to infected decidual macrophages(p<0.05).Conclusions:Taken together,T.gondii infection could down-regulate the expression of inhibitory receptor Tim-3 on decidual macrophages.The down-regulation of Tim-3 could up-regulate M1 functional moleculars(membrane molecules CD80,CD86,arginine metabolic enzymes iNOS,cytokine TNF-a)which went against maternal-fetal tolerance.While the down-regulation of Tim-3 could down-regulate M2 functional molecules(membrane molecules CD 163,CD209,CD206,arginine metabolic enzymes Arg-I)which in favor of maternal-fetal tolerance.Decidual macrophages shift toward Ml decidual macrophages and away M2 decidual macrophages.All above results led to the disruption of decidual macrophages function and polarization.finally contributed to abnormal pregnancy outcomes caused by T.gondii infection.So the down-modulation of Tim-3 on decidual macrophages by T.gondii infection may be an important molecular mechanism of abnormal pregnancy through affecting decidual macrophage functions.The results of this study provide not only a deeper understanding of the immune mechanisms operational during abnormal pregnancy,induced by T.gondii infection,but also identify potential avenues for therapeutic and preventive treatment of congenital toxoplasmosis. |
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