The Mechanism Of Tim-3 On Decidual NK Cells In Abnormal Pregnancy Outcomes Induced By T.gondii Infection

Objective:To explore the exact role and the specific signaling pathway of Tim-3 on decidual NK cells(dNK cells)in adverse pregnancy outcomes induced by T.gondii infection.Methods:In vitro:The primary human decidual cells were obtained aseptically by the combination of enzymatic digestion and mechanical method through the collection of decidual tissue from normal early pregnancy(40-60 days of pregnancy)induced abortion.Human CD3-CD56+dNK cells were collected by magnetic bead separation and then divided into uninfected dNK cells,infected dNK cells and anti-Tim-3 neutralized infected dNK cells.The anti-Tim-3 neutralized infected group was firstly blocked with anti-Tim-3 functional antibody.The infection of T.gondii RH strain tachyzoites was by co-culturing primary human dNK cells and T.gondii RH strain tachyzoites,with a ratio of 1:2(NK cells:tachyzoites counts=1:2)for 36 h.The normal NK cells were treated same volume of culture medium.Then the expressions of Tim-3,inhibitory receptor KIR2DL4 and activating receptor NKG2D on dNK cells were detected by flow cytometry.Cytotoxic granules GranzymeA,GranzymeB and Perforin as well as intracellular cytokine IL-10 and IFN-y of CD3-CD56hright dNK cells were detected by both flow cytometry and Western blot.We further explored the PI3K-AKT-mTOR and Jak-Stat signaling pathway.In vivo:C57BL/6 wild-type(WT)mice were research objects.We further introduced C57BL/6 background Tim-3 knockout mice.Female mice were mated with male mice to obtain pregnant mice models and WT pregnant mice were divided into 2 groups as uninfected and infected group.Mice in infected group and all Tim-3-/-pregnant mice were infected by T.gondii.The infection of T.gondii is done by intraperitoneal injection of 400 T.gondii tachyzoites(RH strain)on gd7.The normal pregnant mice were treated with same volume of PBS as control.After 4 days of injection,we observed the physical of all mice by activity abilities and fur.Mice above were then put to death by cervical dislocation and fetuses as well as placentas were observed and weighted.The record of the rate of abnormal fetuses including absorbed and stillbirth embryos.We obtained single cell suspension by cutting and grinding placentas and decidual.The expressions of Tim-3,inhibitory receptor NKG2A and activating receptor NKG2D,cytotoxic granules such as GranzymeA,GranzymeB and Perforin and intracellur cytokines IL-10,IFN-y secreted by CD3-CD122+dNK were analyzed by flow cytometry.Results:In vitro:Data of flow cytometric analysis showed Tim-3 expression levels on human dNK cells were significantly declined after T.gondii infection,while a little of Tim-3 expression was still detected in anti-Tim-3 neutralized infected group.Activating receptor NKG2D on dNK cells were significantly up-regulated after T.gondii infection compared to uninfected group,and inhibitory receptor KIR2DL4 slightly increased.While the ratio of NKG2D/KIR2DL4 up-regulated after infection and the negative tendency of Tim-3 expression and the ratio NKG2D/KIR2DL4 were observed.Cytotoxic granules GranzymeA,GranzymeB and Perforin were relatively low in uninfected human dNK cells by flow cytometry analysis and Western blot analysis,but could be obviously enhanced by T,gondii infection.IL-10 and IFN-? secretion in human dNK cells also increased after T.gondii infection by flow cytometry and Western blot.And the ratio of IFN-?/IL-10 in human dNK cells showed a relevant inverse association with its Tim-3 expression.In order to further explore the effect of Tim-3 on dNK cells on above receptors,cytotoxic granules and cytokines.We then established and investigated the anti-Tim-3 neutralized infected group.The results showed that activating receptor NKG2D on dNK cells were further increased in anti-Tim-3 neutralized infected group compared to infected group,with a slightly upregulation of inhibitory receptor KIR2DL4.And the ratio NKG2D/KIR2DL4 is even higher than that of infected group.Flow cytometry and Western blot analysis showed GranzymeA,GranzymeB and Perforin further up-regulated in anti-Tim-3 neutralized infected group compared to infected group.Besides,flow cytometry and Western blot analysis showed although IFN-? and IL-10 secretion in human dNK cells was both up-regulated after infection,but the ratio IFN-?/IL-10 is higher caused by T.gondii.In anti-Tim-3 neutralized infected group,we observed even higher IFN-?/IL-10 ratio compared to infected group by flow cytometry and Western blot as a result of significant up-regulation of IFN-?.Pathway analysis showed that as the decrease of Tim-3,the PI3K-AKT-mTOR and Jak-Stat pathway activated and participated in modulating dNK cells functional molecules.In vivo:Infected WT mice displayed apparent severe pregnancy outcomes with a state of spiritual malaise,extrados,erected fur and inflammatory hyperemia of placentas.The fetuses of infected WT group displayed abnormal bleeding manifestations and a higher rate of abnormal fetuses than the uninfected group as well as the decrease in fetal and placental weights.Compared to infected WT pregnant mouse,T.gondii infected Tim-3-/-pregnant mouse had sluggish response,trembled,with significant bleeding of placenta.The worse abnormal pregnancy outcomes in infected Tim-3-/-pregnant mouse were based on the the decrease in fetal and placental weights and higher rate of abnormal fetuses in comparison with infected WT group.Flow cytometry results showed Tim-3 expression on dNK cells in infected group was much lower compared to uninfected group,and little of Tim-3 on dNK cells could be detected in Tim-3-/-infected mice.For murine dNK cells,flow cytometric analysis indicated a significant up-regulation of GranzymeA,GranzymeB and Perforin in WT infected dNK cells,and further increased in infected Tim-3-/-dNK,correlated with high cytotoxicity of dNK in Tim-3-/-mouse.As the expression of Tim-3 reduced,both IL-10 and IFN-y expression were significantly elevated in T.gondii infected mouse and then decrease in Tim-3-/-infected mouse.While IFN-y/IL-10 ratio was higher in infected mouse than the uninfected group.Interestingly,IFN-y/IL-10 ratio of Tim-3-/-mice further increased and was higher than infected WT mice.Conclusions:Based on our research,the expression of inhibitory receptor Tim-3 on dNK cells could be influenced by T.gondii infection and declined after infection.The decrease of Tim-3 could up-regulate activating and inhibitory receptors on dNK cells and change the balance of NKG2D/KIR2DL4 and NKG2D/NKG2A.And Tim-3 expression of dNK cells is associated with cytotoxic granules which went against maternal-fetal tolerance and were up-regulated after infection.Besides the down-regulation of Tim-3 could also lead to Thl bias in cytokines secretion with a high ratio of IFN-y/IL-10.All above results in a enhanced cytotoxicity of dNK cells.The dysfunction of dNK cells eventually lead to severe pregnancy outcomes due to T.gondii infection.In all,the decrease of Tim-3 on dNK cells might be an new and vital molecular mechanism affecting dNK cells functions and finally contribute to adverse pregnancy outcomes by T.gondii infection.Our results further shed new light to the specific mechanism of T.gondii infection caused abnormal pregnancy.And the research will provide a new strategy of treatment to prevent and cure pregnancy failure caused by T.gondii infection.