Role And Immune Mechanism Of PD-1/PD-L1 Signaling Pathway In Abortion Of Autoimmune Thyroiditis

Background:Autoimmune thyroiditis(AIT)with euthyroid is associated with miscarriage.But the exact mechanism remains unclear.There are many reasons for abortion,in which the imbalance of maternal-fetal interface and the invasion of endometrial disorders by placental trophoblast cells during pregnancy are important causes of abortion.Studies have shown that the programmed cell death-1(PD-1)/programmed cell death-matching 1(PD-L1)pathway is essential for normal pregnancy.PD-1(CD279),a member of the immunoglobulin superfamily,is widely expressed.PD-L1(B7-H1 or CD274)is one of the ligands of PD-1 and is widely expressed in immunologically exempt sites such as the placenta.In one aspect,the PD-1/PD-L1 signaling pathway maintains immune tolerance and stability in the body by maintaining a balance between regulatory T cells and effector T cells(Treg/Teff).On the other hand,PD-L1 confers cell invasiveness through the MER/ERK signaling pathway axis.Aim:To study whether autoimmune thyroiditis is due to the decrease of PD-1/PD-L1 content,on the one hand,the inhibition of phosphatidylinositol 3-kinase(PI3K)signaling pathway is weakened,which affects the maternal-fetal immune balance,and on the other hand,the ERK phosphoric acid.Decreased levels affect the invasiveness of embryonic trophoblast cells and lead to miscarriage.Method:(1)CBA/J female mice were immunized with m Tg to induce AIT mice positive for normal Tg Ab,and then caged with Balb/c male mice.(2)Group the mice.(I)control group;(II)m Tg(+)group;(III)m Tg(+)+ PD-L1-Fc(+);(IV)m Tg(+)+ PI3 K inhibitor(+).Animals were sacrificed at 13.5 days(GD13.5)and serum,thyroid and placenta were taken for follow-up examination.(3)Detection of PD-1,PD-L1,p-PI3 K,p-PTEN,p-AKT,p-m TOR,Foxp3,RORγt protein expression in placenta by immunohistochemistry and Western blot.(4)The expression levels of PD-L1,p-ERK,MMP-2 and MMP-9 in placenta were detected by immunohistochemistry and Western blot.(5)Using RNA interference(RNAi)technology to block the expression of PD-L1 protein in human chorionic trophoblast cells of HTR-8/SVneo(HTR8).(6)The expression levels of PD-L1,p-ERK,MMP-2 and MMP-9 in HTR8 cells were detected by immunohistochemistry and Western blot.(7)Transwell method to measure the invasive ability of HTR8 cells.Result:(1)The concentration of Tg Ab in the peripheral blood of each group of mice was observed by ELASA.The serum Tg Ab level in the m Tg group was significantly higher than that in the Con group(P<0.05).The PD-L1-Fc group was lower than the m Tg group(P<0.05).HE staining showed a large amount of lymphocytic infiltration in the thyroid gland in the m Tg group compared with Con,while the thyroid follicles were dilated and destroyed,and the placental stenosis and blood cell distribution were reduced.However,the above phenomenon was improved in the PD-L1-Fc group.(2)Immunohistochemistry and Western blot analysis of placental tissue showed that P13 K,Akt,m-TOR phosphorylation levels and IL-6 and RORγt protein expression levels were increased in the m Tg group compared with the Con group;PD-1,PD-L1,TGF-β and Foxp3 protein expression levels and PTEN phosphorylation were decreased.However,injection of PD-L1-Fc or PI3 K inhibitor reversed these changes,phosphorylation of PI3 K,Akt,m TOR and proteins of IL-6 and RORγt in the PD-L1-Fc and PI3 K inhibitor groups compared to the m Tg group.PD-1,PD-L1,TGF-β and Foxp3 protein expression levels and PTEN phosphorylation were increased.(3)The results of immunohistochemistry and Western blot analysis showed that the expression of PD-L1,phosphorylated ERK,MMP-2 and MMP-9 in AIT pregnant mice was compared with the control group.The content is significantly reduced.(4)Immunohistochemistry and Western blot detection of HTR-8/SVneo(HTR8)human chorionic trophoblast cells showed that PD-L1,phosphorylated ERK in cells interfered with PD-L1 si RNA compared with NC cells The expression of MMP-2 and MMP-9 was significantly reduced.(5)Transwell results showed that the invasion ability of HTR-8/SVneo cells interfered with PD-L1 si RNA was significantly lower than that of NC group cells.Conclusion:(1)Autoimmune thyroiditis due to the decrease of PD-1/PD-L1 content,the inhibition of phosphatidylinositol 3-kinase(PI3K)signaling pathway is weakened,which affects maternal-fetal immune balance,but treatment with PD-L1-Fc The autoimmune thyroid model has a protective effect,suggesting that the use of PD-L1-Fc may be a potential therapeutic target for the treatment of autoimmune thyroiditis.(2)Autoimmune thyroiditis affects the phosphorylation of ERK,MMP-2 and MMP-9 in cells by reducing the expression of PD-L1 in placental trophoblast cells,attenuates the invasiveness of embryonic trophoblast cells and ultimately induces abortion.