An Estrogen-NK Cells Regulatory Axis in Endometriosis, Related Infertility, and Miscarriage.

Endometriosis is a common estrogen-dependent condition that impacts 8-10% of women in their reproductive age, resulting in notable pain, morbidity, and infertility. Despite extensive research endeavors, the precise cause of endometriosis remains elusive, and the mechanisms contributing to its associated infertility are still not well comprehended. Natural killer (NK) cells, vital innate immune cells crucial for successful pregnancy, have been investigated for their potential involvement in the pathogenesis of endometriosis. Prior research has mainly concentrated on the diminished cytotoxicity of NK cells in endometrial fragments that evade the uterus. Interestingly, accumulating evidence suggests that NK cells play multifaceted roles in regulating the biology of endometrial stromal cells (ESCs), promoting local immune tolerance, influencing endometrial receptivity, oocyte development, and embryo implantation, thereby contributing to infertility and miscarriage in patients with endometriosis. In this comprehensive review, our goal is to summarize the current literature and provide an overview of the implications of NK cells in endometriosis, especially concerning infertility and pregnancy loss, under the influence of estrogen.

A Galectin-9-Driven CD11chigh Decidual Macrophage Subset Suppresses Uterine Vascular Remodeling in Preeclampsia.

BACKGROUND: Preeclampsia is a serious disease of pregnancy that lacks early diagnosis methods or effective treatment, except delivery. Dysregulated uterine immune cells and spiral arteries are implicated in preeclampsia, but the mechanistic link remains unclear. METHODS: Single-cell RNA sequencing and spatial transcriptomics were used to identify immune cell subsets associated with preeclampsia. Cell-based studies and animal models including conditional knockout mice and a new preeclampsia mouse model induced by recombinant mouse galectin-9 were applied to validate the pathogenic role of a CD11chigh subpopulation of decidual macrophages (dMφ) and to determine its underlying regulatory mechanisms in preeclampsia. A retrospective preeclampsia cohort study was performed to determine the value of circulating galectin-9 in predicting preeclampsia. RESULTS: We discovered a distinct CD11chigh dMφ subset that inhibits spiral artery remodeling in preeclampsia. The proinflammatory CD11chigh dMφ exhibits perivascular enrichment in the decidua from patients with preeclampsia. We also showed that trophoblast-derived galectin-9 activates CD11chigh dMφ by means of CD44 binding to suppress spiral artery remodeling. In 3 independent preeclampsia mouse models, placental and plasma galectin-9 levels were elevated. Galectin-9 administration in mice induces preeclampsia-like phenotypes with increased CD11chigh dMφ and defective spiral arteries, whereas galectin-9 blockade or macrophage-specific CD44 deletion prevents such phenotypes. In pregnant women, increased circulating galectin-9 levels in the first trimester and at 16 to 20 gestational weeks can predict subsequent preeclampsia onset. CONCLUSIONS: These findings highlight a key role of a distinct perivascular inflammatory CD11chigh dMφ subpopulation in the pathogenesis of preeclampsia. CD11chigh dMφ activated by increased galectin-9 from trophoblasts suppresses uterine spiral artery remodeling, contributing to preeclampsia. Increased circulating galectin-9 may be a biomarker for preeclampsia prediction and intervention.

Rab27b promotes endometriosis by enhancing invasiveness of ESCs and promoting angiogenesis.

PROBLEM: Endometriosis (EMS) is an estrogen-dependent disease which is characterized with estrogen-dependent growth of ectopic endometrium and increased local estrogen production. EMS performs tumor-like biological functions such as invasiveness and angiogenesis. Rab27b is a member of the Rab family of GTPases, which is strongly associated with the growth, invasion and metastasis of a variety of tumors. However, little is known about the function of Rab27b in EMS. In this study, we intended to investigate the impact of Rab27b and its downstream molecule in the development of EMS. METHOD OF STUDY: Normal endometrium and endometriotic lesions were collected to investigate the expression levels of Rab27b. Then, ESCs were transfected with Rab27b siRNA. We analyzed the influence of Rab27b on the proliferation and invasive activity of ESCs. Conditioned media harvested from Rab27b siRNA-treated ESCs were used to treat HUVECs. HUVEC Tube formation and ELISA were performed to explored the interactions between ESCs and HUVEC. In addition, ESCs were treated with different concentrations of estrogen. Based on biological database predictions, we explored possible mechanisms through which estrogen regulates the expression of Rab27b. RESULTS: The expressions of Rab27b were significantly higher in endometriotic lesions than that in normal endometrium. Rab27b can promote the cell proliferation, migration and invasion of ESCs. The elevated expression of Rab27b, on the one hand, promotes the secretion of MMP9 and increases the invasiveness of ESCs. On the other hand, Rab27b may play a key role in the communication between ESC and endothelial cells, by simulating VEGF secretion and neovascularization. Besides, estrogen upregulated phosphorylated FOXO1 levels in ectopic ESCs, resulting in the promotion of Rab27b expression levels. CONCLUSION: Rab27b plays a key role in the development of EMS, which may provide new insights into the pathogenesis of EMS. Our findings may also contribute to the development of therapeutic interventions for EMS.

Synergy between Th1 and Th2 responses during endometriosis: A review of current understanding.

Endometriosis is widely perceived as an estrogen-dependent chronic disorder with infertility and pelvic pain. Although the etiology of endometriosis has remained elusive, many studies have proclaimed the relevance of immune system disorders with endometriosis. With the discovery that the dysregulation of multiple biological functions in endometriosis is caused by the aberrant differentiation of T helper cells, a shift towards Th2 immune response may account for the disease progression. This review attempts to present mechanisms of cytokines, chemokines, signal pathways, transcription factors and some other factors related with the derivation of Th1/Th2 immune response involved in the development of endometriosis. The current understanding of treatment approaches and potential therapeutic targets will also be outlined with brief discussion.

Paradoxical expression of NRP1 in decidual stromal and immune cells reveals a novel inflammation balancing mechanism during early pregnancy.

OBJECTIVE AND DESIGN: To investigate the balancing mechanisms between decidualization-associated inflammation and pregnancy-related immunotolerance. MATERIAL OR SUBJECTS: Decidual samples from women with normal pregnancy (n = 58) or unexplained spontaneous miscarriage (n = 13), peripheral blood from normal pregnancy and endometria from non-pregnancy (n = 10) were collected. Primary endometrial stromal cells (ESCs), decidual stromal cells (DSCs), decidual immune cells (DICs) and peripheral blood mononuclear cells (PBMCs) were isolated. TREATMENT: The plasmid carrying neuropilin-1 (NRP1) gene was transfected into ESC for overexpression. To induce decidualization in vitro, ESCs were treated with a combination of 10 nM estradiol, 100 nM progesterone and 0.5 mM cAMP. Anti-Sema3a and anti-NRP1 neutralizing antibodies were applied to block the ligand-receptor interactions. METHODS: RNA-seq analysis was performed to identify differentially expressed genes in DSCs and DICs, and NRP1 expression was verified by Western blotting and flow cytometry. The secretion of inflammatory mediators was measured using a multifactor cytometric bead array. The effects of Sema3a-NRP1 pathway on DICs were determined by flow cytometry. Statistical differences between groups were compared using the T test and one way or two-way ANOVA. RESULTS: Combined with five RNA-seq datasets, NRP1 was the only immune checkpoint changing oppositely between DSCs and DICs. The decreased expression of NRP1 in DSCs allowed intrinsic inflammatory responses required for decidualization, while its increased expression in DICs enhanced tolerant phenotypes beneficial to pregnancy maintenance. DSC-secreted Sema3a promoted immunosuppression in DICs via NRP1 binding. In women with miscarriage, NRP1 was abnormally elevated in DSCs but diminished in decidual macrophages and NK cells. CONCLUSION: NRP1 is a multifunctional controller that balances the inflammatory states of DSCs and DICs in gravid uterus. Abnormal expression of NRP1 is implicated in miscarriage.

Decidual Stromal Cell Ferroptosis Associated with Abnormal Iron Metabolism Is Implicated in the Pathogenesis of Recurrent Pregnancy Loss.

Iron is necessary for various critical biological processes, but iron overload is also dangerous since labile iron is redox-active and toxic. We found that low serum iron and decidual local iron deposition existed simultaneously in recurrent pregnancy loss (RPL) patients. Mice fed with a low-iron diet (LID) also showed iron deposition in the decidua and adverse pregnancy outcomes. Decreased ferroportin (cellular iron exporter) expression that inhibited the iron export from decidual stromal cells (DSCs) might be the reason for local iron deposition in DSCs from low-serum-iron RPL patients and LID-fed mice. Iron supplementation reduced iron deposition in the decidua of spontaneous abortion models and improved pregnancy outcomes. Local iron overload caused ferroptosis of DSCs by downregulating glutathione (GSH) and glutathione peroxidase 4 levels. Both GSH and cystine (for the synthesis of GSH) supplementation reduced iron-induced lipid reactive oxygen species (ROS) and cell death in DSCs. Ferroptosis inhibitor, cysteine, and GSH supplementation all effectively attenuated DSC ferroptosis and reversed embryo loss in the spontaneous abortion model and LPS-induced abortion model, making ferroptosis mitigation a potential therapeutic target for RPL patients. Further study that improves our understanding of low-serum-iron-induced DSC ferroptosis is needed to inform further clinical evaluations of the safety and efficacy of iron supplementation in women during pregnancy.

Towards a Better Understanding of Endometriosis-Related Infertility: A Review on How Endometriosis Affects Endometrial Receptivity.

Endometriosis is the most common cause of infertility. Endometrial receptivity has been suggested to contribute to infertility and poor reproductive outcomes in affected women. Even though experimental and clinical data suggest that the endometrium differs in women with endometriosis, the pathogenesis of impaired endometrial receptivity remains incomplete. Therefore, this review summarizes the potential mechanisms that affect endometrial function and contribute to implantation failure. Contemporary data regarding hormone imbalance, inflammation, and immunoregulatory dysfunction will be reviewed here. In addition, genetic, epigenetic, glycosylation, metabolism and microRNA in endometriosis-related infertility/subfertility will be summarized. We provide a brief discussion and perspectives on their future clinical implications in the diagnosis and therapy to improve endometrial function in affected women.

ANP promotes HTR-8/SVneo cell invasion by upregulating protein kinase N 3 via autophagy inhibition.

Preeclampsia is a gestational disease characterized by two major pathological changes-shallow trophoblast invasion and impaired spiral artery remodeling. Atrial natriuretic peptide (ANP) is a kind of peptide hormone that regulates blood pressure, while the lack of active ANP participates in preeclampsia pathogenesis. However, the underlying mechanism of how ANP modulates trophoblasts function remains unclarified. Here, we performed isobaric tags for relative and absolute quantification (iTRAQ) in ANP-treated HTR-8/SVneo cells and identified Protein Kinase 3 (PKN3) as the downstream factor of ANP, which was downregulated in preeclamptic placenta. Chromatin immunoprecipitation analysis and luciferase assays showed that NFYA was one of the transcription factors for the PKN3 promoter, which was also regulated by ANP treatment in HTR-8/SVneo cells. Transmission electron microscopy and Western Blotting in HTR-8/SVneo cells indicated that ANP inhibited autophagy via AMPK-mTORC1 signaling, while excess autophagy was observed in preeclamptic placenta. The increased expression of PKN3 and enhanced cell invasion ability in HTR-8/SVneo cells induced by ANP could be abolished by autophagy activation or transfection with PKN3 shRNA or NFYA shRNA or NPR-A shRNA via regulating the invasion-related genes and the epithelial mesenchymal transition molecules. Our results demonstrated that ANP could enhance trophoblast invasion by upregulating PKN3 via NFYA promotion through autophagy inhibition in an AMPK/mTORC1 signaling-dependent manner.

Tim-3 Coordinates Macrophage-Trophoblast Crosstalk via Angiogenic Growth Factors to Promote Pregnancy Maintenance.

T-cell immunoglobulin mucin-3 (Tim-3) is an important checkpoint that induces maternal-fetal tolerance in pregnancy. Macrophages (Mφs) play essential roles in maintaining maternal-fetal tolerance, remodeling spiral arteries, and regulating trophoblast biological behaviors. In the present study, the formation of the labyrinth zone showed striking defects in pregnant mice treated with Tim-3 neutralizing antibodies. The adoptive transfer of Tim-3+Mφs, rather than Tim-3-Mφs, reversed the murine placental dysplasia resulting from Mφ depletion. With the higher production of angiogenic growth factors (AGFs, including PDGF-AA, TGF-α, and VEGF), Tim-3+dMφs were more beneficial in promoting the invasion and tube formation ability of trophoblasts. The blockade of AGFs in Tim-3+Mφs led to the narrowing of the labyrinthine layer of the placenta, compromising maternal-fetal tolerance, and increasing the risk of fetal loss. Meanwhile, the AGFs-treated Tim-3-Mφs could resolve the placental dysplasia and fetal loss resulting from Mφ depletion. These findings emphasized the vital roles of Tim-3 in coordinating Mφs-extravillous trophoblasts interaction via AGFs to promote pregnancy maintenance and in extending the role of checkpoint signaling in placental development. The results obtained in our study also firmly demonstrated that careful consideration of reproductive safety should be taken when selecting immune checkpoint and AGF blockade therapies in real-world clinical care.

The risk of intrauterine exposure to SARS-CoV-2 in female COVID-19 patients: A comprehensive review.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a new type of coronavirus that has caused fatal infectious diseases and global spread. This novel coronavirus attacks target cells through the interaction of spike protein and angiotensin-converting enzyme II (ACE2), leading to different clinical symptoms. However, for a successful pregnancy, a well-established in-uterine environment includes a specific immune environment, and multi-interactions between specific cell types are prerequisites. The immune-related changes in patients infected with novel coronavirus could interfere with the immune microenvironment in the uterus, leading to fetal loss. We first reviewed the intrauterine environment in the normal development process and the possible pregnancy outcome in the infection state. Then, we summarized the immune response induced by SARS-CoV-2 in patients and analyzed the changes in ACE2 expression in the female reproductive system. Finally, the present observational evidence of infection in pregnant women was also reviewed.

Dysfunction of CCR1+ decidual macrophages is a potential risk factor in the occurrence of unexplained recurrent pregnancy loss.

Recurrent pregnancy loss (RPL) puzzles 1-3% of women of childbearing age worldwide. Immunological factors account for more than 60% of cases of unexplained RPL (URPL); however, the underlying mechanism remains unclear. Here, using single-cell sequencing data and functional experiments with clinical samples, we identified a distinct population of CCR1+ decidual macrophages (dMφ) that were preferentially enriched in the decidua from normal early pregnancies but were substantially decreased in patients with URPL. Specific gene signatures endowed CCR1+ dMφ with immunosuppressive and migration-regulatory properties, which were attenuated in URPL. Additionally, CCR1+ dMφ promoted epithelial-to-mesenchymal transition (EMT) to promote trophoblast migration and invasion by activating the ERK1/2 signaling pathway. Decidual stromal cell (DSC)-derived CCL8 was the key regulator of CCR1+ dMφ as CCL8 recruited peripheral CCR1+ monocytes, induced a CCR1+ dMφ-like phenotype, and reinforced the CCR1+ dMφ-exerted modulation of trophoblasts. In patients with URPL, CCL8 expression in DSCs was decreased and trophoblast EMT was defective. Our findings revealed that CCR1+ dMφ play an important role in immune tolerance and trophoblast functions at the maternal-fetal interface. Additionally, decreased quantity and dysregulated function of CCR1+ dMφ result in URPL. In conclusion, we provide insights into the crosstalk between CCR1+ dMφ, trophoblasts, and DSCs at the maternal-fetal interface and macrophage-targeted interventions of URPL.

An active glutamine/α-ketoglutarate/HIF-1α axis prevents pregnancy loss by triggering decidual IGF1+GDF15+NK cell differentiation.

Deficiency of decidual NK (dNK) cell number and function has been widely regarded as an important cause of spontaneous abortion. However, the metabolic mechanism underlying the crosstalk between dNK cells and embryonic trophoblasts during early pregnancy remains largely unknown. Here, we observed that enriched glutamine and activated glutaminolysis in dNK cells contribute to trophoblast invasion and embryo growth by insulin-like growth factor-1 (IGF-1) and growth differentiation factor-15 (GDF-15) secretion. Mechanistically, these processes are dependent on the downregulation of EGLN1-HIF-1α mediated by α-ketoglutarate (α-KG). Blocking glutaminolysis with the GLS inhibitor BPTES or the glutamate dehydrogenase inhibitor EGCG leads to early embryo implantation failure, spontaneous abortion and/or fetal growth restriction in pregnant mice with impaired trophoblast invasion. Additionally, α-KG supplementation significantly alleviated pregnancy loss mediated by defective glutaminolysis in vivo, suggesting that inactivated glutamine/α-ketoglutarate metabolism in dNK cells impaired trophoblast invasion and induced pregnancy loss.

Low-density lipoprotein receptor deficiency reduced bone mass in mice via the c-fos/NFATc1 pathway.

AIM: The low-density lipoprotein receptor (LDLR) plays a crucial role in regulating lipid metabolism. However, whether LDLR deficiency affects bone mass and morphology remains controversial. This study aimed to analyze the bone phenotypes of LDLR knockout (LDLR-/-) mice. MAIN METHODS: Eight-week-old LDLR-/- and wild-type (WT) mice were subjected to microcomputed tomography to detect bone phenotypes. Enzyme-linked immunosorbent assay kits were used to detect the serum estrogen levels and matrix metalloproteinase 9 (MMP-9) levels in tissue homogenates. Von Kossa, toluidine blue, tartrate-resistant acid phosphatase (TRAP) staining, and calcein labeling were performed to explore bone turnover parameters. In vitro, osteoclastogenesis was induced in bone marrow cells from LDLR-/- mice and WT mice in the presence or absence of 17ß-estradiol. The microphotographs and number of osteoclasts were validated using TRAP staining. Relative gene expression during osteoclast differentiation and maturation was determined by quantitative real-time polymerase chain reaction. KEY FINDINGS: LDLR deficiency results in reduced bone mineral density of the tibial cancellous bone, indicating bone loss to some extent in LDLR-/- mice. LDLR deficiency significantly increased the number of osteoclasts, but not osteoblasts. In vitro, bone marrow cells from LDLR-/- mice displayed enhanced osteoclastic potential along with increased expression of TRAP, cathepsin K, nuclear factor of activated T-cells 1 (NFATc1), c-fos, and MMP-9 and inhibited dendritic cell-specific transmembrane protein expression. Moreover, 17ß-estradiol treatment can inhibit osteoclastogenesis in vitro. SIGNIFICANCE: Our data demonstrated that LDLR deficiency promoted osteoclastogenesis by upregulating c-fos and NFATc1 expression, reducing cancellous bone mass in LDLR-/- mice.

RhoB Promotes Endometrial Stromal Cells Decidualization Via Semaphorin3A/PlexinA4 Signaling in Early Pregnancy.

Endometrial decidualization refers to a series of morphological changes and functional remodeling of the uterine endometrium to accept the embryo under the effect of estrogen and progesterone secreted by ovaries after ovulation. During decidualization, endometrial stromal cells (ESCs) proliferate and differentiate into decidual stromal cells, undergoing cytoskeletal rearrangement-mediated morphological changes and expressing decidualization markers, such as insulin-like growth factor-binding protein-1 and prolactin. Ras homology (Rho) proteins, a family of small G proteins, are well known as regulators of cellular morphology and involved in multiple other cellular processes. In this study, we found ras homolog family member B (RHOB) was the most significantly upregulated gene in the Rho protein family after the in vitro decidualization of human primary ESCs. RhoB expression was induced mainly by 3',5'-cyclic adenosine 5'-monophosphate (cAMP) / protein kinase A (PKA) / cyclic adenosine monophosphate-response element binding protein signaling and partly by progesterone signaling. Knockdown of RhoB in ESCs greatly inhibited actin cytoskeletal rearrangement, cell morphological transformation, and upregulation of insulin-like growth factor-binding protein-1, suggesting an indispensable role of RhoB in decidualization. Mechanistically, the downstream target of RhoB was semaphorin3A (Sema3A), which mediated its signaling via interacting with the receptor, plexinA4. More importantly, decreased expression of RhoB, Sema3A, and plexinA4 were detected in deciduas from patients with unexplained spontaneous miscarriage. Collectively, our results indicate that RhoB/Sema3A/plexinA4 signaling plays a positive role in endometrial decidualization and relates to unexplained spontaneous miscarriage, which is worthy of further exploration so as to provide new insights into therapeutic strategies for pregnancy diseases associated with poor decidualization.

SCM-198 Prevents Endometriosis by Reversing Low Autophagy of Endometrial Stromal Cell via Balancing ERα and PR Signals.

Background: Endometriosis (EMS), an endocrine-related inflammatory disease, is characterized by estrogen and progesterone imbalance in ectopic lesions. However, its pathogenic mechanism has not been fully elucidated. While SCM-198 is the synthetic form of leonurine and has multiple pharmacological activities such as antioxidation and anti-inflammation, it remains unknown whether it could inhibit the progress of EMS by regulating estrogen signaling and inflammation. Methods: The therapeutic effects of SCM-198 on EMS and its potential mechanism were analyzed by establishing EMS mouse models and performing an RNA sequencing (RNA-seq) assay. ELISA was performed to detect estrogen and tumor necrosis factor (TNF) -α concentrations in normal endometrial stromal cells (nESCs) and ectopic endometrial stromal cells (eESCs) with or without SCM-198 treatment. Western blotting, RNA silencing, and plasmid overexpression were used to analyze the relationship between inflammation, endocrine factors, and autophagy and the regulatory activity of SCM-198 on the inflammation-endocrine-autophagy axis. Results: Increased estrogen-estrogen receptor (ER) α signaling and decreased progesterone receptor isoform B (PRB) expression synergistically led to a hypo-autophagy state in eESCs, which further inhibited the apoptosis of eESCs. The high expression of TNF-α in eESCs enhanced the antiapoptotic effect mediated by low autophagy through the activation of the aromatase-estrogen-ERα signaling pathway. SCM-198 inhibited the growth of ectopic lesions in EMS mice and promoted the apoptosis of eESCs both in vivo and in vitro. The apoptotic effect of SCM-198 on eESCs was attained by upregulating the autophagy level via the inhibition of the TNF-α-activated aromatase-estrogen-ERα signal and the increase in PRB expression. Conclusion: Inflammation facilitated the progress of EMS by disrupting the estrogen regulatory axis. SCM-198 inhibited EMS progression by regulating the inflammation-endocrine-autophagy axis.

Tim-3+ decidual Mφs induced Th2 and Treg bias in decidual CD4+T cells and promoted pregnancy maintenance via CD132.

T-cell immunoglobulin mucin-3 (Tim-3) plays roles in the functional regulation of both adaptive and innate immune cells and is greatly involved in many diseases. However, the precise roles of Tim-3 on macrophages (Mφs) in pregnancy remain unstated. In the current study, we found the higher frequency of Tim-3+ decidual Mφs (dMφs) in response to trophoblasts. The reduced abundance of Tim-3 on Mφs was accompanied by disordered anti- and pro-inflammatory cytokine profiles in miscarriage. Adoptive transfer of Tim-3+Mφs, but not Tim-3-Mφs, relieved murine embryo absorption induced by Mφ depletion. Our flow cytometry results and the extensive microarray analysis confirmed that Tim-3+ and Tim-3-dMφs were neither precisely pro-inflammatory (M1) nor anti-inflammatory (M2) Mφs. However, with higher CD132 expression, Tim-3+dMφs subset induced Th2 and Treg bias in decidual CD4+T cells and promoted pregnancy maintenance. Blockade of Tim-3 or CD132 pathways leaded to the dysfunction of maternal-fetal tolerance and increased fetal loss. These findings underscored the important roles of Tim-3 in regulating dMφ function and maintaining normal pregnancy, and suggested that Tim-3 on Mφs is a potential biomarker for diagnosis of miscarriage. Our study also emphasized the importance of careful consideration of reproductive safety when choosing immune checkpoint blockade therapies in real world clinical care. Though IL-4 treated Tim-3-Mφs could rescue the fetal resorption induced by Mφ depletion, whether IL-4 represent novel therapeutic strategy to prevent pregnancy loss induced by checkpoint inhibition still needs further research.

Identification of Cancer-Associated Fibroblast Subtype of Triple-Negative Breast Cancer.

Background: There is limited knowledge about the role of cancer-associated fibroblasts (CAF) in the tumor microenvironment of triple-negative breast cancer (TNBC). Methods: Three hundred and thirty-five TNBC samples from four datasets were retrieved and analyzed. In order to determine the CAF subtype by combining gene expression profiles, an unsupervised clustering analysis was adopted. The prognosis, enriched pathways, immune cells, immune scores, and tumor purity were compared between CAF subtypes. The genes with the highest importance were selected by bioinformatics analysis. The machine learning model was built to predict the TNBC CAF subtype by these selected genes. Results: TNBC samples were classified into two CAF subtypes (CAF+ and CAF-). The CAF- subtype of TNBC was linked to the longer overall survival and more immune cells than the CAF+ subtype. CAF- and CAF+ were enriched in immune-related pathways and extracellular matrix pathways, respectively. Bioinformatics analysis identified 9 CAF subtype-related markers (ADAMTS12, AEBP1, COL10A1, COL11A1, CXCL11, CXCR6, EDNRA, EPPK1, and WNT7B). We constructed a robust random forest model using these 9 genes, and the area under the curve (AUC) value of the model was 0.921. Conclusion: The current study identified CAF subtypes based on gene expression profiles and found that CAF subtypes have significantly different overall survival, immune cells, and immunotherapy response rates.

SCM-198 Alleviates Endometriosis by Suppressing Estrogen-ERα mediated Differentiation and Function of CD4+CD25+ Regulatory T Cells.

Background: Endometriosis (EMS), a typical endocrine immune disorder, associates with dramatically increased estrogen production and disorganized immune response in ectopic focus. Peritoneal regulatory T cells (Tregs) expansion in women with EMS and their pathogenic role attributable to endometriotic immunotolerance has been reported. Whether local high estrogen promotes EMS by discipling Tregs needs to be further explored. Up to date, there is no effective medicine for the treatment of EMS. SCM-198 is a synthetic leonurine with multiple physiological activities. Whether SCM-198 could regulate Tregs via estrogen and facilitate the radical cure of EMS has not yet been reported. Methods: Proportion of Tregs in peritoneal fluid of patients with EMS was firstly analyzed via flow cytometry. Peritoneal estrogen concentration and the mRNA levels of estrogen receptor α (ERα) and estrogen receptor ß (ERß) of Tregs were detected by ELISA and RT-PCR, respectively. Grouped in vitro induction assays were performed to explore the effects of SCM-198 and estrogen signaling on Tregs. Cell invasion and viability assays were utilized to detect the crosstalk between Tregs and ectopic endometrial stromal cells (eESCs), with or without SCM-198 treatment. Furthermore, EMS mice models were established to verify the therapeutic effects of SCM-198. Results: Increased Tregs were found in peritoneal fluid of EMS patients, accompanied with estrogen-ERα overactivation. Estrogen-ERα triggered the expansion of Tregs and their cytokine production (IL-10 and TGF-ß1), which could be reversed by SCM-198 treatment. Moreover, SCM-198 abated the invasion and viability of eESCs enhanced by Tregs. In vivo experiments confirmed that SCM-198 obviously retarded the growth of ectopic lesions and downregulated the functions of Tregs via estrogen-ERα inactivation. Conclusions: These data suggest that SCM-198 attenuates Tregs expansion via the inhibition of estrogen-ERα signaling in EMS and offer a promising therapy for such a refractory disease.

Decidualization-derived cAMP promotes decidual NK cells to be angiogenic phenotype.

BACKGROUND: During early pregnancy, a large number of CD56bright natural killer cells (NKs) are accumulated in the decidua; unlike peripheral and cord blood NK cells (pNK and cNK), these decidual NK cells (dNK) display a great capacity to secrete a series of angiogenic/vascular factors, which are essential for placental development. However, the mechanism underlying the formation of dNK cells with an angiogenic phenotype remains unclear. METHODS: First, we compared the difference between dNK and cNK/pNK cells in terms of the expression of CD56 and VEGF, and the regulation of the tube formation. The effect of cAMP on the differentiation of NK cells was evaluated by its analog and inhibitor stimulation. We further analyzed the differences in the phenotype of dNK cells and the expression of VEGF in dNK cells from normal pregnancies and miscarriages. RESULTS: Different from cNK and pNK, dNK cells displayed high expression of CD56 and VEGF. And dNK cells showed a higher capacity of inducing tube formation of HUVEC by trophoblast. Meanwhile, we observed that cAMP-analogue increased the percentage of CD56bright NK population in cNK cells with up-regulated VEGF secretion and tube formation of HUVEC by trophoblast, which could be inhibited by pretreatment with VEGFR neutralizing antibody. Similar changes occurred when co-culturing cNK cells with DSCs but not ESCs. Interestingly, the inhibitor of cAMP signaling (Metadoxine, META) could significantly inhibit the upregulation of VEGF in cNK cells by DSCs. Furthermore, DSCs could secret much more cAMP than ESCs. Notably, decreased CD56bright NK population and VEGF secretion by dNK were related to pregnancy loss. CONCLUSIONS: These findings suggest that dNK cells display an angiogenic phenotype that can be induced by decidualized cAMP signaling. Our study indicates the significance of decidualization-derived cAMP in regulating angiogenesis of decidual NKs and reveals complex crosstalk between different cell types in a critical period during early pregnancy.