The Mechanism Of M1 Macrophages-derived Exosoms In Recurrent Miscarriage

Macrophages are the second largest type of leukocytes at the maternal-fetal interface.Macrophages are divided into M1 and M2 subtypes according to their activation status.The polarization balance of M1 and M2 macrophages is related to various processes of normal pregnancy,and the abnormal polarization of macrophages is related to insufficient uterine vascular remodeling and poor trophoblast invasion,which may eventually lead to recurrent miscarriage(RM),preeclampsia and premature birth.Extravillous trophoblast cells are one of the important components of the placenta,which play vital roles in embryo implantation and pregnancy.Insufficient invasion may affect the establishment of maternal-fetal junctions,leading to miscarriage.Macrophages play important roles in the development of pregnancy through intercellular interactions or interactive dialogue with trophoblasts.In addition to cytokines,exosomes may also play an intermediary role in this process.The purpose of this study is to determine the distribution of macrophage subtypes(M1/M2)at the maternal-fetal interface,and the mechanism by which abnormal macrophages affect trophoblast function,thereby participating in the pathogenesis of recurrent miscarriage(RM).This topic mainly includes three parts,as the following:Part I.Distribution of decidual M1 and M2 macrophages in RM patientsObjective:To investigate the distribution of macrophages in decidual tissue of RM patients,and to clarify the relationship between abnormal macrophage polarization and the pathogenesis of RM.Methods:The decidual tissues of patients with artificial abortion and RM were collected.The number of decidual macrophages and related markers in two groups were detected by RT-PCR and immunohistochemical staining.Results:The results of immunohistochemical staining showed that CD68~+and CD86~+cells in the decidual tissue of RM group were significantly higher,while CD163~+cells were significantly lower than those in the control group.RT-PCR results showed that i NOS and TNF-αin RM group were significantly higher,while CD206 and TGF-βwere lower.Conclusions:The distribution of macrophages in the villous tissue of RM patients was abnormal,M1 macrophages increased and M2 macrophages decreased.Part II.The Mechanism of M1 Macrophages-derived Exosomal mi R-146a/b-5p Inhibit Invasion and Migration of TrophoblastsObjective: To explore the molecular mechanism of M1 macrophages-derived exosomes affecting the epithelial mesenchymal transformation(EMT)process,invasion and migration of trophoblasts.Methods: M1 culture system and co-culture model were constructed in vitro,and M1 macrophages were co-cultured with trophoblast cell lines(HTR-8/JEG3).RT-PCR and Western blot were used to detect related markers of epithelial-mesenchymal transition(E-cadherin,N-cadherin and Vimentin);scratch and invasion assay were applied to detect the migration and invasion ability in the co-culture group and control group.The exosomes of the supernatants of M1 macrophages collected by ultracentrifugation were used to treat HTR-8.RNA Sequencing was used to detect the distribution of mi RNAs in two groups and exosomes of M1 macrophages.RT-PCR was used to detect the mi RNAs with the largest difference in two groups and the most mi RNAs in the exosomes,identifying the mi RNAs with the most significant differences.RT-PCR and Western blot were used to detect changes in EMT-related indicators of HTR-8/JEG3 after transfection of mi RNA mimics or inhibitors.Scratch and invasion assay were used to detect the migration and invasion ability.Bioinformatics was used to predict possible target genes of mi RNAs,and dual luciferase reporter genes were used to verify the target genes.The si RNA and adenovirus vectors were constructed for the target gene,and applied to transfect HTR-8/JEG3.RT-PCR and Western blot were used to detect changes in EMT-related indicators,while scratch and invasion assay were used to detect the migration and invasion ability.Results: M1-derived exosomes significantly inhibit the EMT process of trophoblasts,inhibiting the invasion and migration abilities.RNA Sequencing and RT-PCR results confirmed that mi R-146a-5p and mi R-146b-5p were significantly up-regulated in trophoblasts after co-cultured with M1-Exos.Further mechanistic studies have shown that M1-Exos can target tumor necrosis factor receptor-associated factor 6(TRAF6)at the post-transcriptional level by transmitting mi R-146a-5p and mi R-146b-5p,thereby inhibiting the EMT process,invasion and migration of trophoblasts.Response experiments confirmed that overexpression of TRAF6 can reverse mi R-146a-5p or mi R-146b-5p mimics’ inhibitory effect on the migration and invasion of trophoblasts.Conclusions: M1 macrophages-derived exosomes target mi R-146a-5p and mi R-146b-5p to suppress the expression of TRAF6 in trophoblasts and inhibit the EMT process,thereby inhibiting their invasion and migration capacity.Part III.Analysis of mi R-146a-5p,mi R-146b-5p and TRAF6 expression levels and correlation in villous tissue of RM patients.Objective: To analyze the expression of mi R-146a-5p,mi R-146b-5p and TRAF6 in villous tissue of two groups,and their correlations in RM patients.Methods: The villus tissues of patients with artificial abortion with normal pregnancy and RM were collected.RT-PCR was used to detect the expression of mi R-146a-5p,mi R-146b-5p and TRAF6.Western blot were used to detect the protein expression of TRAF6 in tissue samples.Serial tissue sections were used to detect the expression of mi R-146a-5p and mi R-146b-5p in the decidual tissues by in situ hybridization,and the expression of TRAF6 was stained by immunohistochemistry.Results: Compared with the control group,the expression of mi R-146a-5p and mi R-146b-5p was abnormally increased in the villus tissue of RM patients,while the expression of TRAF6 m RNA and protein was significantly reduced.Pearson correlation analysis showed that the expression of mi R-146a-5p and mi R-146b-5p in RM villous tissue was significantly negatively correlated with the expression of TRAF6.Conclusions: The expression levels of mi R-146a-5p and mi R-146b-5p in the villus tissue of RM patients were significantly up-regulated,while the expression of TRAF6 was significantly decreased,and the expression of mi R-146a-5p and mi R-146b-5p was significantly negatively correlated with TRAF6.