Abnormal HCK/glutamine/autophagy axis promotes endometriosis development by impairing macrophage phagocytosis.

The presence of extensive infiltrated macrophages with impaired phagocytosis is widely recognised as a significant regulator for the development of endometriosis (EMs). Nevertheless, the metabolic characteristics and the fundamental mechanism of impaired macrophage phagocytosis are yet to be clarified. Here, we observe that there is the decreased expression of haematopoietic cellular kinase (HCK) in macrophage of peritoneal fluid from EMs patients, which might be attributed to high oestrogen and hypoxia condition. Of note, HCK deficiency resulted in impaired macrophage phagocytosis, and increased number and weight of ectopic lesions in vitro and in vivo. Mechanistically, this process was mediated via regulation of glutamine metabolism, and further upregulation of macrophage autophagy in a c-FOS/c-JUN dependent manner. Additionally, macrophages of EMs patients displayed insufficient HCK, excessive autophagy and phagocytosis dysfunction. In therapeutic studies, supplementation with glutamine-pre-treated macrophage or Bafilomycin A1 (an autophagy inhibitor)-pre-treated macrophage leads to the induction of macrophage phagocytosis and suppression of EMs development. This observation reveals that the aberrant HCK-glutamine-autophagy axis results in phagocytosis obstacle of macrophage and further increase the development risk of Ems. Additionally, it offers potential therapeutic approaches to prevent EMs, especially patients with insufficient HCK and macrophage phagocytosis dysfunction.

Hypoxia-hindered methylation of PTGIS in endometrial stromal cells accelerates endometriosis progression by inducing CD16- NK-cell differentiation.

Prostacyclin (PGI2) plays key roles in shaping the immune microenvironment and modulating vasodilation, whereas its contribution to endometriosis (EMs) remains largely unclear. Our study suggested that prostacyclin synthase (PTGIS)-dependent PGI2 signaling was significantly activated in EMs, which was involved in the hypoxic microenvironment of ectopic lesions and deficient methylation status of the PTGIS promoter. Notably, in vitro assays, hypoxia promoted PTGIS expression through DNA methyltransferase 1 (DNMT1)-mediated DNA methylation deficiency in endometrial stromal cells (ESCs); PTGIS overexpression enhanced the adhesive ability of ESCs and led to elevated PGI2 production, and PGI2 triggered CD16- (encoded by FCGR3, Fc fragment of IgG receptor IIIa) natural killer (NK)-cell differentiation through PGI2 receptor (IP, PTGIR) in an ESC/NK-cell coculture system. Our rodent model experiment suggested that treatment with the PGI2 analog iloprost and adoptive transfer of fcgr3 knockout (fcgr3-/-) NK cells aggravated EMs progression and that genetic ablation of ptgis (ptgis-/-) in ectopic lesions and treatment with the PTGIR antagonist RO1138452 partially rescued this outcome. Thus, our findings identified the contribution of PGI2 to EMs progression via enhancement of the adhesive ability of ESCs and inhibition of the activity of NK cells. We hypothesized that PGI2 is a target for EMs intervention and provide a rationale for studying pharmacological PTGIR inhibition and PTGIS genetic depletion therapies as therapeutic strategies for EMs.

Downregulating HK2 inhibits proliferation of endometrial stromal cells through a noncanonical pathway involving phosphorylation of signal transducer and activator of transcription 1 in endometriosis.

BACKGROUND: Endometriosis is a benign gynecologic disease that causes chronic pelvic pain, dysmenorrhea and infertility and shares several characteristics with malignant tumors, afflicting women of reproductive age. Hexokinase 2 plays an essential role as the first rate-limiting enzyme in the metabolic glycolysis pathway, and its abnormal elevation in tumors is associated with tumor genesis and metastasis. However, the expression and role of hexokinase 2 in endometriosis remain unclear. METHODS: We sequenced the primary endometrial stromal cells from patients with endometrioma and utilized immunohistochemistry, quantitative real-time PCR, and western blot to determine the expression of hexokinase 2. Then wound healing assays, cell invasion assays, and cell proliferation assays were performed to explore the functions of hexokinase 2 in endometrial stromal cells. Furthermore, mice models of endometriosis were used to observe the effects of hexokinase 2 inhibitors in vivo. Lastly, glycolysis metabolism detection and transcriptome sequencing were carried out in hexokinase 2-knockdown endometrial stromal cells to analyze the mechanism of hexokinase 2 affecting cell function. RESULTS: Endometrial stromal cells of endometrioma displayed active glycolysis metabolism and elevated expression of hexokinase 2. Downregulating hexokinase 2 reduced the migration, invasion, and proliferation capacity of endometrial stromal cells. Knockdown of hexokinase 2 induced upregulation of signal transducer and activator of transcription 1 and their phosphorylation to attenuate the proliferation of endometrial stromal cells. CONCLUSIONS: Hexokinase 2 is associated with the migration, invasion, and proliferation of endometrial stromal cells, which might provide new insights into the pathogenesis and treatment of endometriosis. SUMMARY SENTENCE: HK2 is upregulated in ovarian endometrioma and knockdown of HK2 induced upregulation of signal transducer and activator of transcription 1 (STAT1) and their phosphorylation to attenuate the proliferation of endometrial stromal cells.

PrPC Promotes Endometriosis Progression by Reprogramming Cholesterol Metabolism and Estrogen Biosynthesis of Endometrial Stromal Cells through PPARα Pathway.

Endometriosis (EMs) is characterized as an estrogen-dependent disease. Whereas, the underlying mechanism for activated estrogen biosynthesis in EMs lesions is largely unknown. We analyzed cholesterol metabolism and estrogen biosynthesis condition of EMs lesions by biological information analysis of GEO datasets, and further verified both in vitro and in vivo by constructing EMs models with uterus fragments from donors of PRNP knockout mouse (Prnp-/-, KO119), Octapeptide repeat region of PRNP knockout mouse (KO120) and PRNP transgenic mouse (Tg20). We found that transcriptome of cholesterol metabolism and estrogen-converting enzymes were disturbed in EMs patients, and cellular cholesterol concentration and local estradiol level were substantially increased in EMs lesions, as well as the high level of prion (PrPC, encoded by PRNP). Notably, 17-ß estradiol stimulation significantly up-regulated PrPC expression in endometrial stromal cells (ESC) and PrPC promoted the proliferative, migratory and invasive abilities of ESC, and was further verified to accelerate EMs progression in mouse models. More importantly, PrPC promoted cholesterol accumulation and activated estrogen biosynthesis of ESC in a PPARα pathway-dependent manner. Taken together, this study suggests that PrPC-cholesterol metabolism/estrogen biosynthesis contributes to the progression of EMs by negatively regulating PPARα pathway, and could be potential therapeutic targets for EMs intervention.

Follistatin-like I promotes endometriosis by increasing proinflammatory factors and promoting angiogenesis.

Endometriosis (EMS) is a chronic benign inflammatory disease characterized by the growth of endometrial-like tissue in aberrant locations outside of the uterine cavity. Angiogenesis and abnormal immune responses are the fundamental requirements of endometriotic lesion survival in the peritoneal cavity. Follistatin-like I (FSTL1) is a secreted glycoprotein that exhibits varied expression levels in cardiovascular disease, cancer and arthritis. However, the role of FSTL1 in the development of EMS remains to be fully elucidated. Results of the present study demonstrated that the expression of FSTL1 was significantly increased in ectopic endometrial stromal cells (ESCs) and peritoneal fluid from patients with EMS, compared to the control group. Both conditions of hypoxia and estrogen treatment induced human ESCs to produce increased levels of FSTL1 and disco-interacting protein 2 homolog A (DIP2A). Furthermore, the expression levels of DIP2A, IL8 and IL1ß were increased in FSTL1 overexpressed HESCs. Additionally, FSTL1 treatment increased the proliferation of HUVECs in a dose-dependent manner in vitro and markedly increased the tube formation of HUVECs. Moreover, treatment with FSTL1 facilitated M1 polarization of macrophages, increased the secretion of proinflammatory factors and inhibited the expression of scavenger receptor CD36. Results of the present study suggested that the elevated expression of FSTL1 may play a key role in accelerating the development of EMS via enhancing the secretion of proinflammatory factors and promoting angiogenesis.

Systematic oxidative stress is not associated with live birth rate in young non-obese patients with polycystic ovarian syndrome undergoing assisted reproduction cycles: A prospective cohort study.

OBJECTIVE: Oxidative stress may be associated with polycystic ovary syndrome (PCOS) and poor outcomes after assisted reproduction treatment. It is unclear if systematic oxidative stress is correlated with live birth rate in patients with PCOS after controlled ovarian stimulation. This study aimed to investigate the serum oxidative stress markers on the day of trigger hCG and its relationship with live birth rate in young non-obese women with or without PCOS undergoing in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) treatment. STUDY DESIGN: In this prospective cohort study, 50 patients with PCOS and 50 patients without PCOS aged less than 35 years and undergoing IVF/ICSI between February 2017 and January 2018 were studied. The body mass index (BMI) of all participants was less than 28 kg/m2. Serum malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GPx) levels on the day of hCG were measured. Demographic and cycle characteristics, embryo laboratory data and pregnancy outcomes after fresh embryo transfer were analyzed. Univariate analysis and logistic regression analysis were used to analyze independent variables predicting the live birth rate. RESULTS: After conventional ovarian stimulation, the patients with PCOS had higher serum MDA and GPx concentrations (P < 0.03) and lower serum SOD levels (P < 0.03) on the hCG trigger day than the controls. Further, the serum MDA and GPx levels on the trigger day were also positively correlated with the number of good quality embryos (R = 0.26, P = 0.01; R = 0.21, P = 0.04; respectively). However, cycle characteristics, IVF/ICSI outcomes and pregnancy outcomes were comparable between the two groups. Multivariate logistic regression analysis showed that the rate of good quality embryos (OR 1.04, 95 %CI 1.01-1.06, P = 0.005) and the duration of infertility (OR 0.74, 95 %CI 0.56-0.98, P = 0.04) were predictive factors of live birth rate. The ROC curve analysis showed the AUC for the model was 0.76 (95 %CI: 0.67-0.86, P < 0.001). CONCLUSION: The data suggest that moderate systemic oxidative stress on the hCG trigger day had no detrimental effects on live birth rate in young and non-obese patients with PCOS after IVF/ICSI treatment.

Estrogen-regulated CD200 inhibits macrophage phagocytosis in endometriosis.

OBJECTIVES: Endometriosis (EMS) is a benign disease that is related to estrogen, immune disorders and inflammation. The purpose of this research was to determine the expression of CD200 in EMS and to clarify its role in the pathogenesis of the disease. METHODS: The levels of serum CD200 in patients with and without EMS were determined by ELISA. Furthermore, the expression of CD200 in normal eutopic endometrium and ectopic endometrium was detected by immunohistochemistry and western blotting. The CD200 receptor (CD200R) in macrophages in peritoneal fluid (pMØ) obtained from controls and patients with EMS was examined by western blotting. CD200 expression in human endometrial stromal cells (HESCs) stimulated with 17ß-estradiol (E2) was measured by western blotting. Furthermore, macrophages were stimulated with different concentrations of CD200 and the effect on phagocytosis was analyzed. RESULTS: The plasma CD200 levels of patients with EMS was significantly increased compared with controls (P = 0.0173, 95%CI [18.75, 159.6]). Compared with normal eutopic endometrium, the expression of CD200 was significantly increased in ectopic endometrial tissues. The CD200R expression in pMØ obtained from patients with EMS was increased compared with the controls (P = 0.0244). CD200 expression in HESCs stimulated with E2 was up-regulated. As the levels of CD200 increased, macrophage phagocytosis in vitro gradually decreased. CONCLUSIONS: CD200 is an estrogen-induced molecule that impairs macrophage phagocytosis and may contribute to the immune escape of ectopic lesions in EMS.

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.

H2S promotes proliferation of endometrial stromal cells via activating the NF-κB pathway in endometriosis.

Hydrogen sulfide (H2S) is substantially converted from cysteine by the enzymes cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CSE). H2S can profoundly affect most organ systems in animals and humans by inducing a wide range of physiological functions. However, the roles of H2S in the progression of endometriosis remain unknown. The aim of the current study was to test the hypothesis that H2S might play a role in the pathogenesis of endometriosis via modulating the biological behavior of endometrial stromal cells (ESCs). First, we explored the expression level of CBS and CSE in ESCs via immunohistochemistry and immunocytochemistry. Second, cell Count Kit-8 (CCK-8) assays were utilized to investigate the cell viability of human ESCs (HESCs) in vitro. Third, we studied the potential effects of H2S in a rodent model of endometriosis. Both CBS and CSE were overexpressed in endometriotic lesions. Exogenous and endogenous H2S could promote HESC proliferation in vitro. Furthermore, this pro-proliferation effect could be reversed by treating with inhibitors of CBS, CSE, or the NF-κB pathway. In vivo, we uncovered that inhibitors of CBS and CSE could remarkably reduce the number and weight of mouse endometriotic lesions. These data suggested that H2S promotes ESC proliferation via activation of the NF-κB pathway, which provides a scientific basis for the clinical application of blocking H2S to treat endometriosis.