| Objective:Programmed death protein(PD-1)is a negative costimulatory receptor of the immunoglobulin gene superfamily.It can mediate the immune escape function of tumor cells and pathogens by binding to its ligand PD-L1/PD-L2.As the main cell population at the maternal-fetal interface,uNK cells can regulate endometrial remodeling and angiogenesis during pregnancy,and play a key role in the growth and development of embryos.Before and after acute inflammation during pregnancy,the expression of PD-1/PD-L1 pathway at the maternal-fetal interface will fluctuate,and the subpopulations of uNK cells also have significant changes.However,the number and subpopulations of uNK cells in the pregnancy model of acute Toxoplasma infection in the first trimester The fluctuation of and its interaction with PD-1 is still unclear.Therefore,this article discusses the establishment of a Toxoplasma gondii infection model in early pregnancy:The dynamic expression of PD-1 in Toxoplasma gondii-infected mice in the first trimester,and the mutual regulation of IFN-γ in a mouse model of Toxoplasma gondii infection.And its effect on uNK differentiation.Method:1.Establishment of an acute Toxoplasma infection model in the first trimester:Twenty 0-day pregnant female mice were randomly divided into 4 groups,respectively named 12 days pregnant normal(12 dpn)group,12 days pregnant infected(12 dpi)group,18 days pregnant(18 dpn)group and 18 days infected(18 dpi)group.On the 6th day of pregnancy,150 Toxoplasma Pru strain tachyzoites were injected intraperitoneally into the two infected pregnant mice,and the two control pregnant mice were injected the same amount of PBS.Four groups of mice were sacrificed on the 12th and 18th day of pregnancy,respectively,and the weight of the placenta and fetus were weighed and counted to preliminarily measure the impact of Toxoplasma infection on embryonic development.2.Pathological examination of uterus and placenta of pregnant mice:the placenta and uterine tissues of pregnant mice were taken to observe histopathological changes.3.Detection and analysis of the expression of PD-1 and other related cytokines:Real-time fluorescent quantitative polymerase chain reaction(qPCR)was used to detect and analyze the mRNA expression levels of PD-1,PD-L1,NKG2A,NKG2D,DAP 10,Toxoplasma gondii surface antigen SAG-1 and cytokine IFN-γ in placental and uterine tissues.The correlation between PD-1 andIFN-γ expression.4.Immunohistochemistry:immunohistochemical staining(IHC)was used to detect the localization of PD-1 in uterus and placenta tissues;5.Flow Cytometry:flow cytometry was used to detect toxic subgroups of NK cells in placenta and liver tissues in infection The dynamic changes before and after,as well as the expression of PD-1 and IFN-γ on the surface of NK cells,preliminarily measure the effect of PD-1 on the differentiation of uNK cells and the regulation of the expression of IFN-γ molecules.Result:1.Toxoplasma gondii infection in the first trimester leads to adverse pregnancy outcomes:The pregnant rats without Toxoplasma infection were in good mental state,while the pregnant rats in the 12 dpi and 18 dpi infection groups showed typical symptoms of toxoplasma infection,bulging hairs,bowed backs,and listlessness.The pregnant rats in individual infection groups showed vaginas.Hemorrhage,miscarriage,and premature delivery.The general observation of the embryos of pregnant mice found that the embryos of the control group were well developed,the placenta had sufficient blood supply,and the placenta was uniform in size.In the infected group,there were stillbirths,teratomas,and absorptive fetuses.In this case,the weight of pregnant mice and embryos and the abortion rate of pregnant mice were significantly lower than those of the normal group during the same period(t=5.52,t=11.44,t=12.63,t=11.67,P<0.01).2.Toxoplasma infection causes pathological changes in the uterus and placenta of pregnant mice:Histopathological observations showed that the decidua and junctional areas of the placental tissues of the pregnant mice in the infection group were loosely connected and the cell arrangement was disordered.Congestion,cell edema,fibrinoid necrosis and a large number of inflammatory cell infiltration occurred in the placental labyrinth area.In the infected group,the uterine mucosal columnar epithelial cells were severely damaged,the red blood cells in the blood vessels were reduced,and there was inflammatory cell infiltration.3.Detection and analysis of the expression of PD-1 and other related factors:(1)Toxoplasma infection causes the expression of PD-1,PD-L1 and IFN-γmolecules to increase at 12 days of gestation and decrease at 18 days of gestation:After qPCR detection,PD-1,(F=22.48,51.23,P<0.05)PD-L1(F=9.61,47.49,P<0.05)in the placenta and uterine tissues of mice in the 12dpn,12dpi,18dpn and 18dpi groups There are differences in the expression of IFN-γ(F=16.08,21.52,P<0.05).Among them,the 12dpi group mouse placenta(P<0.05)and uterine tissue(P<0.05)in PD-1,PD-L1 And IFN-γ expression level was higher than that of 12dpn group;compared with 18dpn group,the expression levels of PD-1 and PD-L1 in mouse placenta tissue of 18dpi group decreased(P<0.05)while the expression level of IFN-γ Increased(P<0.05);compared with the 12dpi group,the expression levels of PD-1,PD-L1 and IFN-γ in the placenta and uterus of pregnant rats in the 18dpi group decreased(all P<0.05).The above results indicate that Toxoplasma gondii infection The expression trend of PD-1,PD-L1 and IFN-γ molecules rises at 12 days gestation and decreases at 18 days gestation.(2)Toxoplasma infection increases the expression of Toxoplasma-specific tachyzoite protein SAG-1:After qPCR detection,there were differences in the expression of SAG-1(F=28.66,238.90,P<0.05)in mouse placenta and uterine tissues in the 12dpn,12dpi,18dpn groups and 18dpi groups.The expression of SAG-1 was not detected in the 18dpn group,but the high expression of SAG-1 was detected in the 12dpi and 18dpi groups,and compared with the 12dpi group,the expression of SAG-1(all P<0.05)in the 18dpi group decreased,Indicating that Toxoplasma gondii infection increased the expression of the Toxoplasma-specific tachyzoite protein SAG-1,and the increase was most obvious at the 12th day of gestation.(3)Toxoplasma infection makes the expression of NKG2A/2D in the placenta and uterus unbalanced:In normal pregnancy,the NKG2A receptor,which is beneficial to embryonic development,is highly expressed at the maternal-fetal interface,while the toxicity-related receptor NKG2D exhibits a low expression level.Toxoplasma infection makes the expression of NKG2A/2D in the placenta and uterus unbalanced.The qPCR test found that there were differences in the expression of NKG2A/2D in the placenta and uterus tissues of the mice in the 12dpn,12dpi,18dpn and 18dpi groups(F=27.59,24.26,P<0.05).Compared with the 12dpn,the NKG2D of the 12dpi group(P<0.05))And DAP-10(P<0.01)increased significantly,but compared with 12dpi,the expression of NKG2D(P<0.05)and DAP-10(P<0.01)in the 18dpi group was down-regulated,and the expression of NKG2A in placental tissues The amount showed a downward trend(P<0.05).Compared with the control group,the expression of NKG2A in the 12 dpi and 18 dpi groups increased(P<0.05).4.Immunohistochemistry:The results of IHC staining showed that PD-1 was expressed on the inflammatory cells infiltrated in the placenta tissue of pregnant mice in the 12 dpi group,but not the mice in the 18 dpi group;PD-1 was strongly positively expressed in the uterine epithelium of pregnant mice in the 12 dpi group,and weakly positive in the uterine epithelium of the mice in the 18 dpi group.The results of correlation analysis showed that the placenta(rs=0.99,P<0.01),uterus(rs=0.97,P<0.01)of mice in the 12dpi group,the placenta(rs=0.82,P<0.05),and uterus(rs=0.81,P<0.05)of mice in the 18dpi group between IFN-y and PD-1 mRNA expression levels were positively correlated.5.Flow Cytometry:(1)Toxoplasma infection increases the proportion of NK cells related to toxicity in the placenta and liver tissues:The ratio of CD3-NK1.1+NK cells to lymphocytes in placenta and liver tissue changed between the four groups(F=228.6,F=73.56,P<0.0001):compared with 12 dpn in placenta and liver tissue Compared with the 12 dpi group,the ratio of CD3-NK1.1+NK cells in mice in the 12 dpi group was significantly increased(P<0.001,P<0.0001),and the ratio of CD3-NK1.1+NK cells between the 18 dpn and 18 dpi groups did not Significant difference,but the proportion of NK cells in the 18 dpn group was significantly up-regulated compared with 12 dpn(all P<0.0001),and the proportion of NK cells in the 18 dpi group was also increased compared with the 12 dpi group(P<0.01);placental and liver tissues were detected The ratio of DX5+CD3-NK1.1+NK cells/CD3-NK1.1+NK cells in the middle,the result shows the ratio of DX5+CD3-NK1.1+NK cells/CD3-NK1.1+NK cells among the four groups Significant statistical difference(F=91.18,F=150.60,P<0.0001):Compared with 12dpn,the proportion of DX5+CD3-NK1.1+NK in the 12dpi group increased significantly(P<0.05,P<0.0001),placenta The proportion of DX5+CD3-NK1.1+NK in the 18dpi group was also increased compared with that in the 18dpn group(P<0.05),and the proportion of DX5+CD3-NK1.1+NK in the 18dpn and 18dpi group in the placenta and liver tissue was higher than that in the 18dpi group.Compared with the 12dpi group,they all decreased significantly(P<0.01);(2)Toxoplasma infection increases the expression of PD-1 on the surface of toxic NK cell subsets in the second trimester of the placenta and liver tissues:Further observe the ratio of CD3-NK1.1+DX5+PD-1+cells in the placenta and liver tissue,and find that the ratio of CD3-NK1.1+DX5+PD-1+between the placenta and liver tissue among the four groups has statistics Differences in science(F=39.87,F=14.51,P<0.001):The proportion of CD3-NK1.1+DX5+PD-1+NK cells between the placenta and liver tissues of pregnant rats in the 12dpi group was significantly higher than that in the 12dpn group(P<0.0001,P<0.001),the proportion of CD3-NK1.1+DX5+PD-1+NK cells in the placental tissue in the 18dpi group was lower than that in the 18dpn group(P<0.05),but in the liver tissue in the 18dpi group The ratio of CD3-NK1.1+DX5+PD-1+NK cells was not significantly different from the 18dpn group.(3)Toxoplasma infection increases the secretion of IFN-y of toxic NK cells:Observing the ratio of CD3-NK1.1+DX5+IFN-y+NK cells in the placenta tissue,it was found that the ratio of CD3-NK1.1+DX5+IFN-γ+NK cells was statistically different between the four groups(F=54.22,P<0.0001):The proportion of CD3-NK1.1+DX5+IFN-y+NK cells in the 12dpi group was up-regulated compared with the 12dpn group(P<0.0001),and the CD3-NK1.1+DX5+IFN-γ+in the 18dpi group Compared with the 18dpn group,the ratio of NK cells was up-regulated(P<0.01),but the 18dpi group was significantly down-regulated compared with the 12dpi group(P<0.01).Conclusion:1.After Toxoplasma gondii infection occurs in the first trimester,the expression level of PD-1/PD-L1 pathway at the maternal-fetal interface increases first and then decreases,and it is positively correlated with the main inflammatory factor IFN-γ,suggesting that after Toxoplasma infection The mutual regulation of PD-1 and IFN-γ.2.After Toxoplasma gondii infection occurs in the first trimester,the ratio of NK cells related to maternal liver and maternal-fetal interface toxicity is increased,and the PD-1/PD-L1 factor on the surface of the toxic subtype uNK cell and IFN-γ factor are simultaneously increased,suggesting PD-1 It can regulate the differentiation of toxic subtypes of NK cells,thereby participating in the process of resisting Toxoplasma infection during pregnancy. |
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