Rationale: Emerging evidence demonstrates that insufficient migration and invasion of trophoblasts play critical roles in the pathogenesis of recurrent spontaneous abortion (RSA). Cell-to-cell communication at the maternal-fetal interface is essential to maintain the invasion and migration of trophoblasts. M1 macrophages, important immune cellular components at the maternal-fetal interface, have been reported to be elevated in decidua tissues from patients with RSA. Recent studies indicate that M1 macrophages modulate trophoblast biological behaviors; however, the underlying mechanisms remain poorly understood. Methods: Extracellular vesicles (EVs) were isolated from the supernatant of M1 macrophages inducted from THP-1 cells (M1-EVs) by ultracentrifugation, identified by transmission electron microscopy, nanoparticle tracking analysis, and western blotting, and their miRNA profile was characterized by miRNA sequencing. Scratch wound healing and transwell assays were used to investigate the effect of M1-EVs on trophoblast migration and invasion. RT-PCR, western blotting, and luciferase reporter assays were conducted to uncover the underlying mechanism. Finally, animal experiments were employed to explore the effect of M1-EVs on embryo absorption in mice. Results: M1 macrophages suppressed trophoblast EMT to reduce their migration and invasion abilities in vitro by secreting EVs. Through miRNA sequencing, miR-146a-5p and miR-146b-5p were identified as the most upregulated miRNAs in trophoblasts treated with M1-EVs. Further functional experiments showed that M1-EVs inhibited trophoblast migration and invasion by transferring miR-146a-5p and miR-146b-5p. Mechanistically, EV miR-146a-5p and miR-146b-5p inhibited EMT of trophoblasts by directly suppressing TNF receptor-associated factor 6 (TRAF6) expression at the post-transcriptional level. Furthermore, M1-EVs aggravated embryo absorption in mice. Clinically, expression of miR-146a-5p, miR-146b-5p, and TRAF6 were aberrant in placental villous tissues from patients with RSA, and negative correlations were found between miR-146a-5p/miR-146b-5p and TRAF6 expression levels. Conclusions: Our findings indicate that miR-146a-5p and miR-146b-5p derived from EVs play important roles in intercellular communication between M1 macrophages and trophoblasts, illuminating a novel mechanism in M1 macrophage regulation of trophoblasts and their role in RSA.

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Insufficient invasion ability of trophoblasts might be associated with the development of recurrent miscarriage (RM). Ubiquitin-specific protease 25 (USP25) can regulate the processes of invasion and migration in different types of cancer cells. However, the effect of USP25 on trophoblasts and its roles in the development of RM are unknown. In this study, we first analyzed the USP25 expression in placental villous tissues from RM patients, and then assessed the roles of USP25 in epithelial-to-mesenchymal transition (EMT), invasion and migration of trophoblasts. Furthermore, bioinformatics prediction and luciferase reporter assay were used to explore the mechanism of microRNA on USP25 expression, and regulation of USP25 expression in trophoblasts was assessed following transfection with microRNA mimics or inhibitor. The results showed that the expression of USP25 in the placental villous tissues was downregulated in RM patients.Knockdown of USP25 suppressed the EMT process, the invasion and migration capability of trophoblast cells, while overexpression of USP25 exhibited opposite results.Mechanistically, miR-27a-3p could regulate USP25 expression by binding to the 3′-untranslated region of USP25 in trophoblasts. Quantitative real-time polymerase chain reaction results found the expression of miR-27a-3p were negatively related to USP25 in RM patients. MiR-27a-3p mimics inhibited but miR-27a-3p inhibitor enhanced the migration and invasion capability of trophoblasts. Furthermore, sh-USP25 counteracted the promotion of invasion and migration mediated by the miR-27a-3p inhibitor.Taken together, these data indicate that USP25 downregulation by miR-27a-3p contributes to the EMT process, thereby inhibiting the migration and invasion of trophoblast cells, and these findings might provide potential biomarkers for RM. K E Y W O R D Sinvasion, migration, miRNA-27a-3p, recurrent miscarriage, trophoblasts, USP25

Trophoblasts are important parts of the placenta and exert vital roles in the maternal‐foetal crosstalk, and sufficient trophoblasts migration and invasion is critical for embryo implantation and normal pregnancy. Macrophages, as the major components of decidual microenvironment at maternal‐foetal interface, can interact with trophoblasts to participate in the regulation of normal pregnancy. Previously, our group have demonstrated that trophoblasts could induce macrophages polarization to M2 subtype by secreting interleukin‐6 (IL‐6); however, the understanding of macrophages regulating the migration and invasion of trophoblasts is limited. In the present study, we used the co‐cultured model to further investigate the effects of macrophages on trophoblasts migration and invasion. Our results showed that co‐culture with macrophages promoted epithelial‐to‐mesenchymal transition (EMT) of trophoblasts, thereby enhancing their migrative and invasive abilities. Further experiments revealed that M2 macrophage‐derived G‐CSF was a key factor, which promoted the EMT, migration and invasion of trophoblasts via activating PI3K/Akt/Erk1/2 signalling pathway. Clinically, G‐CSF was highly expressed in placental villous tissues of normal pregnancy patients compared to patients with recurrent spontaneous abortion, and its expression level was significantly correlation with EMT markers. Taken together, these findings indicate the important role of M2 macrophages in regulating trophoblasts EMT, migration and invasion, contributing to a new insight in concerning the crosstalk between macrophages and trophoblasts in the establishment and maintenance of normal pregnancy.

BackgroundTissue expansion is a procedure that promotes skin regeneration by mechanical stretch. During the stress and relaxation cycle, the skin undergoes a repeated microtrauma which triggers an immune response leading to the recruitment of macrophages to repair the damaged tissue. Macrophages have been found to be necessary for tissue repair and wound healing, but their effects on skin regeneration during mechanical stretch remain unclear.MethodsThe dynamic changes of macrophages in the rat skin tissues undergoing expansion were quantitatively determined by immunohistochemistry staining. The area of the expanded skin, skin thickness, dermal collagen density, cell proliferation and tissue vascularization were examined to determine the effects of macrophages on the expanding skin. The phenotypes of macrophages and the growth factors related to skin regeneration were also examined to evaluate the underlying mechanisms for the involvement of macrophages in skin regeneration. As a comparison, the tissue samples of expanding skin in which the macrophages were depleted by topically utilizing clodronate liposomes were also evaluated.ResultsThe number of skin macrophages in skin maintained in the high level during the skin expansion compared to non-expanded skin. We found that a switch from an M1- to M2-dominant response during tissue expansion. After the macrophages were depleted, the skin regeneration was inhibited, as evidenced by a smaller expansion area, thinner skin layers and decreased cell proliferation rate, collagen synthesis and, skin vascularization. The secretion of epidermal growth factor (EGF), fibroblast growth factor (FGF), and vascular endothelial growth factor (VEGF) were decreased when macrophages were depleted.ConclusionsOur findings suggest that macrophages are necessary for skin regeneration during tissue expansion. Modulating inflammation may provide a key therapeutic strategy to promote skin growth under mechanical strain.Electronic supplementary materialThe online version of this article (10.1186/s12967-019-1780-z) contains supplementary material, which is available to authorized users.