An abnormal LPA/LPAR1-NHE1 axis leads to the autophagy deficiency of trophoblast cells in recurrent spontaneous abortion.

In brief: Autophagy is important for trophoblast cells at the maternal-fetal interface during early pregnancy. This study suggests that trophoblast cells can promote the autophagy under a regulation of the LPA/LPAR 1-NHE1 axis. Abstract: The autophagy of trophoblasts is necessary for developing and maintaining a healthy pregnancy. Autophagy dysfunction in trophoblast cells is linked to recurrent spontaneous abortion (RSA). However, the mechanism underlying trophoblast autophagy is unknown. In this study, we investigated the expression of autophagy-related genes in both normal and RSA villi. We also examined the production of LPA and LPAR1 in trophoblast cells during early pregnancy. We found that the activation of the LPA-LPAR1 axis triggered the autophagy of trophoblast cells and increased the expression of NHE1. Inhibition of NHE1 suppressed the autophagy in trophoblast cells and we confirmed that NHE1 regulates LPA production in trophoblast cells. Additionally, we found decreased expression of autophagy-related genes and LPAR1 in villi from RSA patients. These observations indicate that the LPA/LPAR1-NHE1 axis regulates the autophagy of trophoblast cells during pregnancy. Insufficient autophagy and poor expression of LPAR1 in trophoblast cells may result in the dysfunction of the trophoblasts and an increased risk of spontaneous abortion. Overall, our research elucidated that a positive LPA/LPAR1-NHE1 axis can promote the autophagy of trophoblast cells and the abnormal axis leads to the autophagy deficiency of trophoblast cells in recurrent spontaneous abortion.

Galectins: Important Regulators in Normal and Pathologic Pregnancies.

Galectins (Gal) are characterized by their affinity for galactoside structures on glycoconjugates. This relationship is mediated by carbohydrate recognition domains, which are multifunctional regulators of basic cellular biological processes with high structural similarity among family members. They participate in both innate and adaptive immune responses, as well as in reproductive immunology. Recently, the discovery that galectins are highly expressed at the maternal-fetal interface has garnerd the interest of experts in human reproduction. Galectins are involved in a variety of functions such as maternal-fetal immune tolerance, angiogenesis, trophoblast invasion and placental development and are considered to be important mediators of successful embryo implantation and during pregnancy. Dysregulation of these galectins is associated with abnormal and pathological pregnancies (e.g., preeclampsia, gestational diabetes mellitus, fetal growth restriction, preterm birth). Our work reviews the regulatory mechanisms of galectins in normal and pathological pregnancies and has implications for clinicians in the prevention, diagnosis and treatment of pregnancy-related diseases.

FBXO17 Inhibits the Wnt/ß-Catenin Pathway and Proliferation of Ishikawa Cells.

Uterine corpus endometrial carcinoma (UCEC) is one of the most common types of cancer in women, and the incidence is rapidly increasing. Studies have shown that various signaling pathways serve crucial roles in the tumorigenesis of UCEC, amongst which the Wnt/ß-catenin pathway is of great interest due to its crucial role in cell proliferation and the huge potential as a therapeutic target. In the present study, it was shown that FBXO17, which is a member of the F-box family, is abnormally downregulated in UCEC tissues compared with non-tumor endometrial tissues, and this was significantly associated with the clinical histological grade, as well as the abnormal proliferation of the UCEC cell line, Ishikawa, both in vitro and in vivo. Besides, the results suggested that FBXO17 may inhibit the Wnt/ß-catenin signaling pathway and influence the expression of adhesion molecules, such as E-cadherin and N-cadherin in Ishikawa cells. In conclusion, these findings indicate that FBXO17 is a novel inhibitor of endometrial tumor development and it likely exerts effects via regulation of the Wnt signaling pathway.

Macrophage polarization in placenta accreta and macrophage-trophoblast interactions.

PROBLEM: Placenta accreta (PA) is defined by an abnormal invasion of placental trophoblasts into the myometrium, which can lead to serious postpartum complications. Macrophages play an important role in the regulation of trophoblast function. Both granulocyte colony-stimulating factor (G-CSF) and its receptor (granulocyte colony-stimulating factor receptor, G-CSFR) have effects on trophoblast invasion. However, the current understanding of G-CSF secretion, G-CSFR expression, abnormal polarization of decidual macrophages (dMϕ) in PA and the abnormal invasion of placental trophoblasts into the myometrium are limited. METHOD OF STUDY: The polarization of dMϕ in PA was analyzed by flow cytometry (FCM), and the expression of G-CSFR in placental trophoblasts in PA was evaluated by immunohistochemistry. In an in vitro co-culture model, we investigated the effects of HTR-8/SVneo trophoblasts cell line (HTR-8) on macrophage human monocyte cell line (THP-1) polarization and G-CSF secretion, and we also analyzed the effects of THP-1 cells, especially M2-like subtype, on primary trophoblasts and HTR-8 proliferation, invasion, and adhesion. FCM, transwell assays, adhesion assays, and proliferation assays were used in the above model. RESULTS: Compared with controls (n = 9), dMϕ showed significantly lower levels of M1 markers CD80 and CD86 and higher levels of the M2 markers CD163 and CD206, and G-CSFR expression of placental trophoblasts was increased in PA (n = 5). In vitro experiments showed that the trophoblast HTR-8 cell line induced polarization of THP-1 cells to an M2-like subtype and increased their secretion of G-CSF. Furthermore, IL-4/IL-13-induced M2-like THP-1 macrophages were able to increase the expression of G-CSFR, proliferation, invasion and adhesion of both primary trophoblasts and HTR-8 trophoblasts. CONCLUSIONS: There is an altered immune imbalance at the maternal-fetal interface in PA, which further may lead to abnormal trophoblast function. G-CSF and its receptors may play important roles in abnormal polarization of macrophages and abnormal invasion of trophoblasts.

IL-17: an important pathogenic factor in endometriosis.

Interleukin-17 (IL-17) is known as a Th17-cell-derived proinflammatory cytokine, which plays a pivotal role in several inflammatory and autoimmune diseases such as systemic lupus erythematosus (SLE), rheumatoid arthritis, and psoriasis. Emerging evidence has shown that IL-17 is linked to endometriosis, although the etiology of endometriosis is still unknown. The IL-17 expression is up-regulated in serum, peritoneal fluid (PF) and endometriotic lesions from patients with endometriosis but the related regulation mechanisms are complex and obscure. Meanwhile, the specific roles of IL-17 in endometriosis are also worthy of further exploration. Through the integration and summary of literature, we conclude that the secretion of IL-17 increases under the regulation of ectopic microenvironment and other factors, and then IL-17 is deeply involved in endometriosis in the regulation of immune microenvironment, the invasion and growth of ectopic lesions, and so on, which implies its therapeutic value in this disorder.

CCL2: An important cytokine in normal and pathological pregnancies: A review.

C-C motif ligand 2 (CCL2), also known as monocytic chemotactic protein 1 (MCP-1), is an integral chemotactic factor which recruits macrophages for the immune response. Together with its receptors (e.g., CCR2, ACKR1, and ACKR2), they exert noticeable influences on various diseases of different systems. At the maternal-fetal interface, CCL2 is detected to be expressed in trophoblasts, decidual tissue, the myometrium, and others. Meanwhile, existing reports have determined a series of physiological regulators of CCL2, which functions in maintaining normal recruitment of immunocytes, tissue remodeling, and angiogenesis. However, abnormal levels of CCL2 have also been reported to be associated with adverse pregnancy outcomes such as spontaneous abortion, preeclampsia and preterm labor. In this review, we concentrate on CCL2 expression at the maternal-fetal interface, as well as its precise regulatory mechanisms and classic signaling pathways, to reveal the multidimensional aspects of CCL2 in pregnancy.

HIF1A-induced heme oxygenase 1 promotes the survival of decidual stromal cells against excess heme-mediated oxidative stress.

Heme oxygenase 1 (HO-1, encoded by the HMOX1 gene) is the rate-limiting enzyme that catalyzes heme degradation, and it has been reported to exert antioxidative effects. Recently, decidualization has been reported to confer resistance to environmental stress signals, protecting against oxidative stress. However, the effects and regulatory mechanism of HO-1 in decidual stromal cells (DSCs) during early pregnancy remain unknown. Here, we verified that the levels of HO-1 and heme in DSCs are increased compared with those in endometrial stromal cells. Additionally, the upregulation of HIF1A expression led to increased HMOX1 expression in DSCs possibly via nuclear factor erythroid 2-related factor (encoded by the NFE2L2 gene). However, addition of the competitive HO-1 inhibitor zinc protoporphyrin IX resulted in an increase in HIF1A expression. Hydrogen peroxide (H2O2) induced the production of reactive oxygen species (ROS), decreased the cell viability of DSCs in vitro, and upregulated the level of heme. As an HO-1 inducer, cobalt protoporphyrin IX decreased ROS production and significantly reversed the inhibitory effect of H2O2 on cell viability. More importantly, patients with unexplained spontaneous abortion had low levels of HO-1 that were insufficient to protect against oxidative stress. This study suggests that the upregulation of HO-1 expression via HIF1A protects DSCs against excessive heme-mediated oxidative stress. Furthermore, the excessive oxidative stress injury and impaired viability of DSCs associated with decreased HO-1 expression should be associated with the occurrence and/or development of spontaneous abortion.

Changes in subsets of immunocytes in endometrial hyperplasia.

PROBLEM: Endometrial hyperplasia (EH) is characterized by an endometrial gland-to-stroma ratio >1 and is one of the most common gynecological diseases in the world. The role of immunocyte subsets in the development of EH remains unknown. METHODS: Patients who underwent dilatation and curettage due to abnormal uterine bleeding were recruited in the present study. Alterations in the numbers of different types of immune cell subsets in the endometrium of patients were analyzed by flow cytometry. RESULTS: The present study included 48 patients who were divided into three groups, based on the pathological results: (a) proliferative period (PP, n = 12); (b) simple EH (SEH, n = 30); and (c) complex EH (CEH, n = 6). The results showed that immune cell subpopulations were significantly different between these three groups. Compared with the PP group, the proportion of CD45+ cells and neutrophils and the subtypes of T cells and macrophages were significantly increased in the SEH patients. Compared with the PP and SEH groups, subsets of immunocytes in the CEH group were significantly decreased, including the population of CD45+ cells and the subtypes of T cells and natural killer cells; in contrast, the proportion of macrophages was significantly increased. There were no significant differences between the other cell subsets in each group. CONCLUSION: The changes in immune cell subsets may be closely associated with the progression of EH. Although the specific role of different immune cell subsets in the development of the diseases requires further study, the changes in the proportions of immune cell subsets should not be ignored.

Myeloid-derived suppressor cells in obstetrical and gynecological diseases.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous group of myeloid-origin cells which have immunosuppressive activities in several conditions, such as cancer and inflammation. Recent research has also associated MDSCs with numerous obstetrical and gynecological diseases. During pregnancy, MDSCs accumulate to ensure maternal-fetal immune tolerance, whereas they are decreased in patients who suffer from early miscarriage or pre-eclampsia. While the etiology of endometriosis is still unknown, abnormal accumulation of MDSCs in the peripheral blood and peritoneal fluid, alongside an increased level of reactive oxygen species (ROS), has been observed in these patients, which is central to the cellular immune regulations by MDSCs. Additionally, the regulation of MDSCs observed in tumours is also applicable to gynecologic neoplasms, including ovarian cancer and cervical cancer. More recently, emerging evidence has shown that there are high levels of MDSCs in premature ovarian failure (POF) and in vitro fertilization (IVF), but the underlying mechanisms are unknown. In this review, the generation and mechanisms of MDSCs are summarized. In particular, the modulation of these cells in immune-related obstetrical and gynecological diseases is discussed, including potential treatment options targeting MDSCs.

Decidual stromal cells maintain decidual macrophage homeostasis by secreting IL-24 in early pregnancy.

PROBLEM: The state of self-renewal and self-maintain of decidual macrophages would be important for immune homeostasis at the maternal-fetal interface. The roles of interleukin (IL)-24 derived from decidual stromal cells (DSCs) on decidual macrophages have not been explored. METHOD OF STUDY: IL-24 expression in DSCs was interfered by lentivirus, and the transcription levels of IL-24 in DSCs were verified by real time (RT)-PCR. The levels of IL-24 receptors were determined by flow cytometry assays. The effect of recombination human IL-24 (rhIL-24) on the differentiation and apoptosis of macrophages was analyzed by flow cytometry in vitro. The viability of macrophages was detected by Cell Counting Kit-8 assays. RESULTS: The growth of DSCs was not affected obviously only by IL-24 knockdown while the growth of knockdown DSCs was inhibited significantly after co-cultured with decidual macrophages. The levels of IL-24 receptors (IL-20R1 and IL-22R1) were moderately to highly expressed on decidual macrophages and human macrophage cell line U937. The differentiation of decidual macrophages treated by rhIL-24 or co-cultured with IL-24 knockdown DSCs was not affected. Both apoptosis and viability of U937 cells were promoted by rhIL-24. The ratio of Bcl-2/Bax was down-regulated and Ki-67 was up-regulated by IL-24 treatment. The expression of Bcl-2/Bax was up-regulated while Ki-67 was down-regulated in U937 cells after co-cultured by IL-24 knockdown DSCs. CONCLUSION: IL-24 secreted by DSCs promotes the renewal and homeostasis of decidual macrophages possibly via down-regulating the ratio of Bcl-2/Bax and up-regulating of the expression of Ki-67 in early pregnancy.

Decidual stromal cells promote the differentiation of CD56bright CD16- NK cells by secreting IL-24 in early pregnancy.

PROBLEM: Decidual stromal cells (DSCs) are important origins of cytokines to modulate maternal-fetal immunotolerance and provide a feasible environment for embryo implantation and development. Interleukin (IL)-24 is a multifunctional cancer killing cytokine and a pleiotropic immunoregulator with complex potency according to tissue or cell types. Its role in establishment and maintenance of normal pregnancy is largely unknown. The aim of our study was to investigate the function and significance of IL-24 and its receptor in the coordination between DSCs and natural killer cells (NK) in early pregnancy. METHOD OF STUDY: The levels of IL-24 in DSC, endometrial stromal cell (ESC), peripheral blood NK cells (pNK), or decidual NK cells (dNK) culture supernatants were detected by enzyme-linked immunosorbent assay (ELISA), and the levels of IL-24 receptors were determined by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and flow cytometry assays. The effect of IL-24 on the functions of decidual NK cells was analyzed by flow cytometry assays in vitro. RESULTS: The concentration of IL-24 in culture supernatant of DSCs was significantly higher than that of ESCs. Both eNK (endometrial NK cells) and dNK highly expressed IL-24 receptors (IL-20R1 and IL-22R1), especially on CD56dim eNK. However, there were extremely low levels of IL-20R1 and IL-22R1 on pNK. Recombinant human IL-24 or DSCs-secreted IL-24 downregulated the levels of CD16, Granzyme B, perforin, and interferon (IFN)-γ and upregulated the levels of inhibitory receptors killer-cell immunoglobulin-like receptor (KIR)2DL1 and KIR3DL1, or immunotolerant or angiogenic cytokines (eg, transforming growth factor (TGF)-ß, IL-10, and IL-8), and elevated the percentage of CD56bright CD16- dNK in vitro. CONCLUSION: These data suggest that DSCs promote the differentiation of CD56bright CD16- NK with high levels of inhibitory receptors, immunotolerant, and angiogenic cytokines by secreting IL-24 during decidualization in early pregnancy.