The Effects Of Endometrial Epithelial Cell-derived Exosomes On The Recruitment And Activation Of Inflammatory Lymphocytes In Dairy Cows Uterus

Objectives:The endometrium is the protective layer of the uterus.Endometritis mainly manifests inflammatory changes in the endometrial cells.When the disease occurs,lymphocytes will aggregate towards the inffected area,and its related subgroups will play their respective functions to regulate the body’s immunity.Therefore,lymphocytes gather to the site of uterine inflammation and exert their immune function by suppressing the spread of inflammation.Regulatory T cells maintain immune tolerance.Changes in the number of regulatory T cells can directly affect the immune function of T cells,and then affect the immune function of the body.Exosomes are tiny vesicles released by cells with genetic information from the host cell.The purpose of this test was to investigate the role of endometrial epithelial-cell-derived exosomes in regulating the activity of T lymphocytes in the dairy cows uterus with endometritis.It has important reference value for preventing endometritis and improving immune efficiency.Methods:Cytological staining was used to identify the neutrophilic granulocyte in healthy and diseased uterine fluid.Lymphocytes Extract Kit was used to extract lymphocytes in the uterus and analyze the subtypes of T lymphocyte.After that,the expressions of FOXP3 and IL-17 in T lymphocytes were detected by qRT-PCR and western blot.Exosomes was extracted by Lymphocytes Extract Kit from normal culture and LPS-treated endometrial epithelial cells and electron microscope was used to identify exosomal morphology and western blot was used to identify the protein expression of CD9.The bovine Tregs were sorted by flow cytometry,and then exosomes derived from normal culture and LPS-treated endometrial epithelial cells were added.After that,The differences in bovine Treg-related factors CTLA4,PD-1,and Galectin-1 were determined by real-time PCR.For further study,mouse regulatory T cells was induced with IL-2 and exosomes were added.The expression changes of CTLA4,PD-1,Galectin-1,FOXP3 and IL-17 were detected by Western blot and immunofluorescence methods.Results:① Clusters of neutrophils were found in the uterine fluid of diseased cows by cytology.No neutrophil infiltration in the uterine fluid of healthy cows;②The number of T lymphocytes in the diseased group increased while the number of subgroups also changed,and the proportion of Tc cells increased significantly compared to the healthy group;③The relative expression of FOXP3 protein in the healthy group increased significantly(p<0.01);The expression of IL-17 in the healthy group was significantly lower than that in the diseased uterus(p<0.01);④ The shape and size of the exosomes isolated and the protein expression of related marker CD9 identified by Transmission Electron Microscope and Western Bolt respectively can prove that the isolation was successful;⑤ The expression levels of PD 1,CTLA4,and Galectin-1 mRNA in the Treg cells of co-cultured with LPS-treated were significantly decreased compared to the untreated group(p<0.01);⑦ The relative expression of PD1,CTLA4,Galectin-1,and FOXP3 proteins in mouse Treg cells co-cultured with the exosomes derived from LPS-treated endometrial epithelial cells were significantly decreased compared to the control group.(p<0.01),while the relative expression of IL-17 was significantly increased in vitro inflammation model group(p<0.05).Conclusion:① The number of normal T lymphocyte subpopulations in the dairy cows uterus was disturbed under the condition of endometritis,especially the ratio of Treg to cytotoxic T cells;②The activation of local Treg in the uterus can be regulated by endometrial epithelial cells through secreted exosomes,and eventually affect the local immune microenvironment in the dairy cows uterus under the condition of endometritis.The changes of uterine immune microenvironment may be an inherent cause of lower pregnancy rates in dairy cows with endometritis.