The Study On The Mechanism Of MiR-93 Targeting PD-L1 To Improve Immune Tolerance In Dairy Cows During Pregnancy

Reproductive disorder is one of the key factors restricting the reproductive efficiency in dairy cattle,which mainly occurs in the early stage of maternal pregnancy identification,causing substantial economic losses to the dairy industry.Cow endometrium needs to maintain a certain intensity of immune tolerance environment in the early gestation period to protect the development of a "semi-allogeneic fetus" in utero.Failure to enhance immune tolerance will lead to embryo death and pregnancy establishment failure.Therefore,it is of great significance to explore the mechanism of uterine immune tolerance in the early pregnancy of dairy cows.As the most abundant endometrial immune cells in cows during peri-implantation,macrophages’ specific functional phenotype and local immune homeostasis are closely related to maternal-fetal immune tolerance.However,the mechanism of their action on the maternal-fetal interface of dairy cows remains unclear.Previous studies have shown that human decidual macrophages during pregnancy are regulated by programmed cell death receptor 1(PD-1)and programmed cell death ligand 1(PD-L1)to help establish maternal-fetal immune tolerance.IFN-τ is associated with the establishment of pregnancy of dairy cows.The previous sequencing results of our laboratory found that the expression of miR-93 decreased significantly after IFN-τ stimulation.However,the role of miR-93 in immune tolerance and the possible interaction mechanism between Mir-93 and PD-L1 remain unclear.In this study,the endometrial immune microenvironment and macrophage polarization of cows in the early gestation period were investigated by taking the uterine tissue of cows in the early gestation period and non-gestation period as the research object;Then,the co-culture model of bovine endometrial luminal epithelial cells(b EECs)and bovine macrophages(Bo Mac)was established to reveal the targeting relationship between miR-93 and PD-L1 and further explore the molecular mechanism of miR-93 induced by IFN-τ in regulating Bo Mac polarization by targeting PD-1/AKT/m TOR pathway during pregnancy immunosuppression,which provides a theoretical basis for clarifying the endometrial immune tolerance mechanism in early pregnancy dairy cows.The main research results and conclusions are as follows:1.The endometrium of cows presented a certain level of immunosuppression in early pregnancy.Immunohistochemical results showed that the proportion of macrophages in endometrium of dairy cows increased significantly in early pregnancy,while M1 macrophages decreased and M2 macrophages increased.The proportion of cells with positive expression of PD-L1 and PD-1 increased significantly after pregnancy.The results of WB and RT-q PCR further confirmed that PD-L1 was highly expressed in the endometrium of cows in early pregnancy.Meanwhile,RT-q PCR results showed that,compared with non-pregnant cows,the expression of pro-inflammatory Th1 cytokine IFN-γdecreased and the expression of anti-inflammatory Th2 cytokine IL-4 increased in endometrium of cows in early pregnancy.These results suggest that macrophage recruitment and M2-type bias,high expression of PD-L1 and PD-1,and local immunosuppressive environment in endometrium of cows in early pregnancy.2.MiR-93 targeting PD-L1 regulates immunosuppression induced by IFN-τ.The immunosuppression model was constructed using IFN-τ in b EECs.Firstly,CCK-8 test detected that b EECs viability was not affected by IFN-τ stimulation.RT-q PCR results showed that the expressions of PD-L1 and anti-inflammatory cytokines increased in a dose-dependent manner when the concentration of IFN-τ was 10,20 and 50 ng/m L,and the optimal intervention condition of IFN-τ was 50 ng/m L for 24 h.According to the previous sequencing results of our laboratory,it was found that miR-93 was significantly down-regulated after IFN-τ treatment in b EECs.Combined with the significant decrease of miR-93 expression in the endometrial tissues of dairy cows during early pregnancy,the results suggest that IFN-τ induced immunosuppression model has been successfully constructed,and miR-93 may play an important role in the early gestation period.Through the analysis of biological information software,it was found that the target bases of miR-93 were paired with PD-L1,and PD-L1 was preliminarily identified as the target gene of miR-93.It showed that miR-93 significantly inhibited the activity of PD-L13’-UTR luciferase by dual luciferase assay,and overexpression of miR-93 significantly reduced the translation levels of PD-L1 in b EECs,confirming that PD-L1 was the target gene of miR-93,and miR-93 negatively regulated the expression of PD-L1.3.miR-93 promotes M2-type polarization of Bo Mac through PD-1/AKT/m TOR.b EECs and Bo Mac contacting co-culture models were constructed and transfected with miR-93 inhibitor.Flow cytometry showed that the proportion of M2-type macrophages increased significantly,while that of M1-type macrophages decreased after transfection with an miR-93 inhibitor.RT-q PCR results showed that the expression of M2 cytokines such as IL-10 and CCL17 increased significantly.WB experiment demonstrated that after the inhibition of miR-93,the expressions of PD-1 and the major proteins of AKT/m TOR pathway in Bo Mac were increased,which proved that the inhibition of miR-93 activated the PD-1/AKT/m TOR pathway and promoted the M2-type polarization of Bo Mac.4.PD-L1 was blocked to inhibit the process of miR-93 regulating Bo Mac polarization.Atezolizumab was used to block the function of PD-L1.RT-q PCR showed that the expression of PD-L1 were markedly decreased after Atezolizumab treatment in b EECs,and the expression level of PD-1 was also significantly decreased in Bo Mac.Atezolizumab was added to the co-culture system and transfected with an miR-93 inhibitor.Flow cytometry showed that the proportion of M2-type cells decreased significantly,and that of M1-type cells increased.RT-q PCR proved that the expression of M2 cytokines such as CCL17 and CCL24 was also significantly inhibited after added PD-L1 monoclonal antibody,and the expression of M1 cytokines such as CXCL9 increased.In addition,the expressions of PD-1,p-AKT,and p-m TOR were down-regulated in the PD-1/AKT/m TOR pathway.In conclusion,blocking of PD-L1 and the downstream PD-1/AKT/m TOR pathway inhibits the M2-type polarization of Bo Mac,thus damaging the immunosuppressive environment.5.AgomiR-93 promotes embryo loss in mice.A mouse pregnancy model was established and agomiR-93 was injected into the uterus of healthy pregnant mice,and significant loss of embryos was observed.Immunofluorescence staining showed that a decrease in the proportion of M2-type macrophages and a significant decrease in PD-L1 expression compared with healthy pregnant mice.RT-q PCR detected decreased expression of M2 cytokines IL-10 and Arg1 in agomiR-93 injected group.Meanwhile,the protein levels of PD-1/AKT/m TOR pathway was inhibited after the activation of miR-93,which showed same results of in vitro experiments.Therefore,it was further verified in mice that AgomiR-93 negatively regulates the post-transcriptional translation level of PD-L1,inhibits the activity of PD-1/AKT/m TOR signaling pathway,and thus negatively regulates the M2-type polarization and immune tolerance environment.The results showed that IFN-τ inhibited miR-93,regulated target gene PD-L1 and then activated PD-1/AKT/m TOR pathway,thus promoting the polarization of macrophages towards M2-type and regulating the balance of local immune microenvironment in uterus.This may be one of the main reasons for the immunotolerance of cow endometrium in early pregnancy,laying the foundation for embryo implantation and resistance to pathogen invasion.