The dysfunction of NK cells in women with endometriosis (EMS) contributes to the immune escape of menstrual endometrial fragments refluxed into the peritoneal cavity. The reciprocal communications between endometrial stromal cells (ESCs) and lymphocytes facilitate the development of EMS. However, the mechanism of these communications on cytotoxicity of natural killer (NK) cells in endometriotic milieus is still largely unknown. To imitate the local immune microenvironment, the co-culture systems of ESCs from patients with EMS and monocyte-derived macrophages or of ESCs, macrophages and NK cells were constructed. The cytokine levels in the co-culture unit were evaluated by ELISA. The expression of functional molecules in NK cells was detected by flow cytometry (FCM). The NK cell behaviors in vitro were analyzed by cell counting kit-8 and cytotoxic activation assays. After incubation with ESCs and macrophages, the expression of CD16, NKG2D, perforin and IFN-γ, viability and cytotoxicity of NK cells were significantly downregulated. The secretion of interleukin (IL)-1β, IL-10 and transforming growth factor (TGF)-β in the co-culture system of ESCs and macrophages was increased. Exposure with anti-IL-10 receptor β neutralizing antibody (αhIL-10Rβ) or αTGF-β could partly reverse these effects of ESCs and macrophages on NK cells in vitro. These results suggest that the interaction between macrophages and ESCs downregulates cytotoxicity of NK cells possibly by stimulating the secretion of IL-10 and TGF-β, and may further trigger the immune escape of ectopic fragments and promote the occurrence and the development of EMS.
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Hui-Li Yang, Wen-Jie Zhou, Kai-Kai Chang, Jie Mei, Li-Qing Huang, Ming-Yan Wang, Yi Meng, Si-Yao Ha, Da-Jin Li, and Ming-Qing Li
Xuan-Tong Liu, Hui-Ting Sun, Zhong-Fang Zhang, Ru-Xia Shi, Li-Bing Liu, Jia-Jun Yu, Wen-Jie Zhou, Chun-Jie Gu, Shao-Liang Yang, Yu-Kai Liu, Hui-Li Yang, Feng-Xuan Xu, and Ming-Qing Li
It has been reported that the impaired cytotoxicity of natural killer (NK) cells and abnormal cytokines that are changed by the interaction between ectopic endometrial cells and immune cells is indispensable for the initiation and development of endometriosis (EMS). However, the mechanism of NK cells dysfunction in EMS remains largely unclear. Here, we found that NK cells in peritoneal fluid from women with EMS highly expressed indoleamine 2,3-dioxygenase (IDO). Furthermore, IDO+NK cells possessed lower NKp46 and NKG2D but higher IL-10 than that of IDO-NK. Co-culture with endometrial stromal cells (nESCs) from healthy control or ectopic ESCs (eESCs) from women with EMS led to a significant increase in the IDO level in NK cells from peripheral blood, particularly eESCs, and an anti-TGF-β neutralizing antibody suppressed these effects in vitro. NK cells co-cultured with ESC more preferentially inhibited the viability of nESCs than eESCs did, and pretreating with 1-methyl-tryptophan (1-MT), an IDO inhibitor, reversed the inhibitory effect of NK cells on eESC viability. These data suggest that ESCs induce IDO+NK cells differentiation partly by TGF-β and that IDO further restricts the cytotoxicity of NK cells in response to eESCs, which provides a potential therapeutic strategy for EMS patients, particularly those with a high number of impaired cytotoxic IDO+NK cells.
Jia-Wei Shi, Hui-Li Yang, Zhen-Zhen Lai, Hui-Hui Shen, Xue-Yun Qin, Xue-Min Qiu, Yan Wang, Jiang-Nan Wu, and Ming-Qing Li
The survival and development of a semi-allogeneic fetus during pregnancy require the involvement of decidual stromal cells (DSCs), a series of cytokines and immune cells. Insulin-like growth factor 1 (IGF1) is a low molecular weight peptide hormone with similar metabolic activity and structural characteristics of proinsulin, which exerts its biological effects by binding with its receptor. Emerging evidence has shown that IGF1 is expressed at the maternal–fetal interface, but its special role in establishment and maintenance of pregnancy is largely unknown. Here, we found that the expression of IGF1 in the decidua was significantly higher than that in the endometrium. Additionally, decidua from women with normal pregnancy had high levels of IGF1 compared with that from women with unexplained recurrent spontaneous miscarriage. Estrogen and progesterone led to the increase of IGF1 in DSCs through upregulating the expression of WISP2. Recombinant IGF1 or DSCs-derived IGF1 increased the survival, reduced the apoptosis of DSCs, and downregulated the cytotoxicity of decidual NK cells (dNK) through interaction with IGF1R. These data suggest that estrogen and progesterone stimulate the growth of DSCs and impair the cytotoxicity of dNK possibly by the WISP2/IGF1 signaling pathway.
Pei-Li Wu, Jing-Wen Zhu, Cheng Zeng, Xin Li, Qing Xue, and Hui-Xia Yang
Insufficient trophoblast invasion at the maternal–fetal interface contributes to abortion-prone pregnancy. Our study shows that decreased levels of IGFBP7 in unexplained recurrent spontaneous abortion (URSA) trophoblast cells inhibit MMP2 and Slug expression as well as trophoblast invasion, suggesting that IGFBP7 should be considered a potential therapeutic protein target in URSA.
Insufficient trophoblast invasion at the maternal–fetal interface contributes to abortion-prone pregnancy. Cyclosporine A (CsA) can exert therapeutic effects on URSA by promoting trophoblast invasion. A previous study showed decreased expression of insulin-like growth factor-binding protein 7 (IGFBP7) in the sera of recurrent spontaneous abortion patients. However, the role of IGFBP7 in URSA remains unknown. The aim of this study was to determine whether IGFBP7 modulates trophoblast invasion in URSA and the underlying molecular mechanisms. We found that IGFBP7 was expressed at lower levels in villous specimens from URSA patients. Manipulating IGFBP7 expression significantly affected the MMP2 and Slug expression in HTR-8/SVneo cells as well as trophoblast invasion in vitro. Inactivation of IGF-1R by IGFBP7 was observed, and IGF-1R inhibition increased the IGFBP7-induced MMP2 and Slug expression in HTR-8/SVneo cells. Moreover, the level of c-Jun was significantly upregulated in the URSA group. Silencing IGFBP7 increased the binding of downstream c-Jun to the MMP2 and Slug promoter regions in HTR-8/SVneo cells, thus suppressing transcription. In addition, increased expression of IGFBP7 in HTR-8/SVneo cells was observed upon CsA treatment. Knockdown of IGFBP7 inhibited the CsA-enhanced MMP2 and Slug expression in HTR-8/SVneo cells. Our results suggest that in normal pregnancy, IGFBP7 induces MMP2 and Slug expression via the IGF-1R-mediated c-Jun signaling pathway, thereby promoting trophoblast invasion. IGFBP7 depletion in URSA inhibits MMP2 and Slug expression as well as trophoblast invasion. Moreover, IGFBP7 participates in CsA-induced trophoblast invasion, suggesting that IGFBP7 is a potential therapeutic target for URSA.
Tao Yu, Shuai Lin, Rui Xu, Tian-Xi Du, Yang Li, Hui Gao, Hong-Lu Diao, and Xiu-Hong Zhang
Embryo implantation is a crucial step for the successful establishment of mammalian pregnancy. Cyclophilin A (CYPA) is a ubiquitously expressed intracellular protein and is secreted in response to inflammatory stimuli to regulate diverse cellular functions. However, there are currently no reports about the role of CYPA in embryo implantation. Here, we examine the expression pattern of CYPA during mouse early pregnancy and explore the potential role of CYPA during implantation. CYPA is expressed in the subluminal stroma surrounding the implanting blastocyst on day 5 of pregnancy, but not at inter-implantation sites. In ovariectomized mice, estrogen and progesterone significantly stimulate CYPA expression. When pregnant mice are injected intraperitoneally with CYPA inhibitor, the numbers of implantation sites are significantly reduced. Using an in vitro stromal cell culture system, Ppia siRNA knockdown of CYPA and CYPA-specific inhibitor treatment partially inhibits levels of CD147, MMP3 and MMP9. Decreased CYPA expression also significantly inhibits Stat3 activity and expands estrogen responsiveness. Taken together, CYPA may play an important role during mouse embryo implantation.
Wen-Lin Chang, Qing Yang, Hui Zhang, Hai-Yan Lin, Zhi Zhou, Xiaoyin Lu, Cheng Zhu, Li-Qun Xue, and Hongmei Wang
Placenta-specific protein 1 (PLAC1), a placenta-specific gene, is known to be involved in the development of placenta in both humans and mice. However, the precise role of PLAC1 in placental trophoblast function remains unclear. In this study, the localization of PLAC1 in human placental tissues and its physiological significance in trophoblast invasion and migration are investigated by technical studies including real-time RT-PCR, in situ hybridization, immunohistochemistry, and functional studies by utilizing cell invasion and migration assays in the trophoblast cell line HTR8/SVneo as well as the primary inducing extravillous trophoblasts (EVTs). The results show that PLAC1 is mainly detected in the trophoblast columns and syncytiotrophoblast of the first-trimester human placental villi, as well as in the EVTs that invade into the maternal decidua. Knockdown of PLAC1 by RNA interference significantly suppresses the invasion and migration of HTR8/SVneo cells and shortens the distance of the outgrowth of the induced EVTs from the cytotrophoblast column of the explants. All the above data suggests that PLAC1 plays an important role in human placental trophoblast invasion and migration.
Hui Li, Huan Wang, Jianmin Xu, Xinxin Zeng, Yingpu Sun, and Qingling Yang
Oocyte quality and its NAD+ level decrease with time during in vitro culture. This study shows that nicotinamide riboside (NR) supplementation improves early embryonic development potential in post-ovulatory oocytes by decreasing the reactive oxygen species (ROS) levels and reducing DNA damage and apoptosis which could potentially increase the success rate of assisted reproductive technology (ART).
The quality of post-ovulatory oocytes deteriorates over time, impacting the outcome of early embryonic development during human ART. We and other groups have found that NAD+, a prominent redox cofactor and enzyme substrate, decreases in both aging ovaries and oocytes. In this study, we found that the NAD+ levels decreased in the post-ovulatory mouse oocytes during in vitro culture and this decrease was partly prevented by NR supplementation. NR treatmenty restored MII oocyte quality and enhanced the early embryonic development potential of post-ovulatory oocytes via alleviating mitochondrial dysfunction and maintaining normal spindle/chromosome structure. Also, treatment with NR decreased ROS levels and reduced DNA damage and apoptosis in post-ovulatory oocytes. Taken together, our findings indicated that NR supplementation increases the oocyte quality and early embryonic development potential in post-ovulatory oocytes which could potentially increase the success rate of ART.
Xue-Min Qiu, Zhen-Zhen Lai, Si-Yao Ha, Hui-Li Yang, Li-Bing Liu, Yan Wang, Jia-Wei Shi, Lu-Yu Ruan, Jiang-Feng Ye, Jiang-Nan Wu, Qiang Fu, Xiao-Fang Yi, Kai-Kai Chang, and Ming-Qing Li
Immune cells and cytokines have important roles in the pathogenesis of endometriosis. However, the production and role of cytokines of T helper type 1 (Th1) and Th2 cells in the progress of endometriosis have remained to be fully elucidated. The present study reported that the interferon (IFN)-γ levels and the percentage of IFN-γ+CD4+ cells were significantly increased in the peritoneal fluid (PF) at the early stage and maintained at a higher level at the advanced stage of endometriosis; furthermore, interleukin (IL)-10 and IL-10+CD4+ cells were elevated in the advanced stage of endometriosis. In addition, IL-2 levels in the PF at the advanced stage of endometriosis were elevated and negatively associated with IFN-γ expression. In a co-culture system of ectopic endometrial stromal cells (ESCs) and macrophages, elevated IL-2 was observed, and treatment with cytokines IL-2 and transforming growth factor-β led to upregulation of the ratio of IL-2+ macrophages. IL-27-overexpressing ESCs and macrophages were able to induce a higher ratio of IL-10+CD4+ T cells. Blocking of IL-2 with anti-IL-2 neutralizing antibody led to upregulation of the ratio of IFN-γ+CD4+ T cells in the co-culture system in vitro. Recombinant human IL-10 and IFN-γ promoted the viability, invasiveness and transcription levels of matrix metalloproteinase (MMP)2, MMP9, and prostaglandin-endoperoxide synthase 2 of ESCs, particularly combined treatment with IL-10 and IFN-γ. These results suggest that IL-2 and IL-27 synergistically promote the growth and invasion of ESCs by modulating the balance of IFN-γ and IL-10 and contribute to the progress of endometriosis.
Jun Shao, Bing Zhang, Jia-Jun Yu, Chun-Yan Wei, Wen-Jie Zhou, Kai-Kai Chang, Hui-Li Yang, Li-Ping Jin, Xiao-Yong Zhu, and Ming-Qing Li
Macrophages play an important role in the origin and development of endometriosis. Estrogen promoted the growth of decidual stromal cells (DSCs) by downregulating the level of interleukin (IL)-24. The aim of this study was to clarify the role and mechanism of IL-24 and its receptors in the regulation of biological functions of endometrial stromal cells (ESCs) during endometriosis. The level of IL-24 and its receptors in endometrium was measured by immunohistochemistry. In vitro analysis was used to measure the level of IL-24 and receptors and the biological behaviors of ESCs. Here, we found that the expression of IL-24 and its receptors (IL-20R1 and IL-20R2) in control endometrium was significantly higher than that in eutopic and ectopic endometrium of women with endometriosis. Recombinant human IL-24 (rhIL-24) significantly inhibited the viability of ESCs in a dosage-dependent manner. Conversely, blocking IL-24 with anti-IL-24 neutralizing antibody promoted ESCs viability. In addition, rhIL-24 could downregulate the invasiveness of ESCs in vitro. After co-culture, macrophages markedly reduced the expression of IL-24 and IL-20R1 in ESCs, but not IL-22R1. Moreover, macrophages significantly restricted the inhibitory effect of IL-24 on the viability, invasion, the proliferation relative gene Ki-67, proliferating cell nuclear antigen (PCNA) and cyclooxygenase2 (COX-2), and the stimulatory effect on the tumor metastasis suppressor gene CD82 in ESCs. These results indicate that the abnormally low level of IL-24 in ESCs possibly induced by macrophages may lead to the enhancement of ESCs’ proliferation and invasiveness and contribute to the development of endometriosis.
Li Nie, Li-xue Zhang, Yi-cheng Wang, Yun Long, Yong-dan Ma, Lin-chuan Liao, Xin-hua Dai, Zhi-hui Cui, Huan Liu, Zhao-qi Wang, Zi-yang Ma, Dong-zhi Yuan, and Li-min Yue
Uterine receptivity to the embryo is crucial for successful implantation. The establishment of uterine receptivity requires a large amount of energy, and abnormal energy regulation causes implantation failure. Glucose metabolism in the endometrium is tissue specific. Glucose is largely stored in the form of glycogen, which is the main energy source for the endometrium. AMP-activated protein kinase (AMPK), an important energy-sensing molecule, is a key player in the regulation of glucose metabolism and its regulation is also tissue specific. However, the mechanism of energy regulation in the endometrium for the establishment of uterine receptivity remains to be elucidated. In this study, we aimed to investigate the energy regulation mechanism of mouse uterine receptivity and its significance in embryo implantation. The results showed that the AMPK, p-AMPK, glycogen synthase 1, and glycogen phosphorylase M levels and the glycogen content in mouse endometrial epithelium varied in a periodic manner under regulation by the ovarian hormone. Specifically, progesterone significantly activated AMPK, promoted glycogenolysis, and upregulated glycogen phosphorylase M expression. AMPK regulated glycogen phosphorylase M expression and promoted glycogenolysis. AMPK was also found to be activated by changes in the energy or glycogen of the endometrial epithelial cells. The inhibition of AMPK activity or glycogenolysis altered the uterine receptivity markers during the window of implantation and ultimately interfered with implantation. In summary, consistency and synchronization of AMPK and glycogen metabolism constitute the core regulatory mechanism in mouse endometrial epithelial cells involved in the establishment of uterine receptivity.