The IL-33-ST2 axis plays a vital role in endometriosis via promoting epithelial-mesenchymal transition by phosphorylating ß-catenin.

OBJECTIVES: Interleukin 33 (IL-33) is a crucial inflammatory factor that functions as an alarm signal in endometriosis (EMs). Epithelial-mesenchymal transition (EMT), a process related to inflammatory signals, intracellular reactive oxygen species (ROS) production, and lipid peroxidation, have been proposed as potential mechanisms that contribute to the development and progression of EMs. IL-33 is highly upregulated in the ectopic milieu. Moreover, ectopic endometrial cells constitutively express interleukin-33 receptor ST2 (IL-33R). However, the role of IL-33/ST2 in the EMT of EMs remains largely unknown. In this study, we aimed to mechanistically determine the role of IL-33/ST2 in EMs-associated fibrosis. MATERIALS AND METHODS: We established a non-lethal oxidative stress model to explore the conditions that trigger IL-33 induction. We performed α-smooth muscle actin (α-SMA) protein detection, cell counting kit-8 (CCK-8) assays, and scratch assays to analyze the impact of IL-33 on primary endometrial stromal cells (ESCs) proliferation and invasion. Clinical samples from patients with or without EMs were subjected to immunohistochemical (IHC) and and immunofluorescence(IF) staining to assess the clinical relevance of IL-33 receptor ST2 and EMT-related proteins. Furthermore, we used the ectopic human endometrial epithelial cell line 12Z and normal human epithelial cell line EEC to evaluate the effects of IL-33 on Wnt/ß-catenin signaling. The effect of IL-33 on EMT-associated fibrosis was validated in vivo by intraperitoneal injections of IL-33 and antiST2. RESULTS: We observed that ectopic milieu, characterized by ROS, TGF-ß1, and high level of estrogen, triggers the secretion of IL-33 from ectopic ESCs. Ectopic endometrial lesions exhibited higher level of fibrotic characteristics and ST2 expression than that in the normal endometrium. Exogenous recombinant human (rhIL-33) enhanced ESC migration and survival. Similarly, 12Z cells displayed a higher degree of EMT characteristics with elevated expression of CCN4 and Fra-1, downstream target genes of the WNT/ß-catenin pathway, than that observed in EECs. Conversely, blocking IL-33 with neutralizing antibodies, knocking down ST2 or ß-catenin with siRNA, and ß-catenin dephosphorylation abolished its effects on EMT promotion. In vivo validation demonstrated that IL-33 significantly promotes EMs-related fibrosis through the activation of Wnt/ß-catenin signaling. CONCLUSION: Our data strongly support the vital role of the IL-33/ST2 pathway in EMs-associated fibrosis and emphasize the importance of the EMT in the pathophysiology of fibrosis. Targeting the IL-33/ST2/Wnt/ß-catenin axis may hold promise as a feasible therapeutic approach for controlling fibrosis in EMs.

Extracellular succinate derived from ectopic milieu drives adhesion and implantation growth of ectopic endometrial stromal cells via the SUCNR1 signal in endometriosis.

BACKGROUND: As a dual-function metabolite, succinate has emerged in cell function and plays a key signaling role in linking mitochondrial function to other cellular functions. Succinate accumulation in the cytoplasm is commonly associated with hypoxia in the microenvironment and immune cell activation. Extracellular succinate released into the microenvironment is considered an inflammatory alarm that can be sensed by its membrane receptor SUCNR1, which boosts proinflammatory responses and acts akin to classical hormones and cytokines. Succinate plays an important role in the development of inflammatory diseases. Whether succinate facilitates the progression of endometriosis (EMs), characterized by chronic inflammation and peritoneal adhesion, is worth exploring. OBJECTIVE: We mimicked the ectopic milieu in vitro and in vivo to evaluate the main source and potential role of succinate in endometriosis. We assessed the molecular and functional effects of succinate on macrophages and peritoneal mesothelial cells in peritoneal cavity. The effect of succinate/SUCNR1 signaling on ectopic endometrial stromal cells (ESCs) was further explored in this study. METHODS: In this study, we used targeted organic acid metabolomics analysis and in vitro assays to assess the potential accumulation of succinate in the peritoneal fluid of EMs patients. We examined its correlation with disease severity, Visual Analogue Scale, and the Endometriosis Fertility Index. Flow cytometry, enzyme linked immunosorbent assay, western blot assay, quantitative real-time PCR, and other molecular biology techniques were used to explore the potential mechanisms. RESULTS: By mimicking the ectopic milieu, we constructed an in vitro co-culture system and found that M1 polarized macrophages and that the peritoneal mesothelial cell line (HMrSV5) mainly released succinate into their microenvironment and activated the succinate receptor (SUCNR1) signal, which further polarized the macrophages and significantly enhanced the invasive survival of ESCs, and the adhesion to the peritoneum. We further investigated the pathological effects of extracellular succinate in vivo using a xenograft mouse models of endometriosis. CONCLUSIONS: Succinate-SUCNR1 signaling facilitates the creation of inflammatory cells and plays a vital role in EMs progression and peritoneal adhesion. Our work on the molecular mechanisms underlying succinate accumulation and function will help elucidate the phenotypic mysteries of pain and infertility in EMs. Video Abstract.

Distinct subtypes of endometriosis identified based on stromal-immune microenvironment and gene expression: implications for hormone therapy.

Background: Endometriosis (EMs) is a chronic inflammatory condition that is highly heterogeneous. Current clinical staging fails to accurately predict drug responses and prognosis. In this study, we aimed to reveal the heterogeneity of ectopic lesions and investigate the possible underlying mechanisms using transcriptomic data and clinical information. Methods: The EMs microarray dataset GSE141549 was obtained from the Gene Expression Omnibus database. Unsupervised hierarchical clustering was performed to identify EMs subtypes, which was followed by the functional enrichment analysis and estimation of immune infiltrates. Subtype-associated gene signatures were identified and further validated in other independent datasets, including GSE25628, E-MTAB-694, and GSE23339. Additionally, tissue microarrays (TMAs) were generated from premenopausal patients with EMs to investigate the potential clinical implications of the two identified subtypes. Results: The unsupervised clustering analysis revealed that ectopic EMs lesions can be classified into two distinct subtypes: stroma-enriched (S1) and immune-enriched (S2). The functional analysis revealed that S1 correlated with fibroblast activation and extracellular matrix remodeling in the ectopic milieu, whereas S2 was characterized by the upregulation of immune pathways and a higher positive correlation with the immunotherapy response. Moreover, we identified a subtype signature composed of FHL1 and SORBS1, and constructed a subtype diagnostic model. Based on the cohort data from the TMAs, we found that S2 was strongly associated with the failure of/intolerance to hormone therapy. Conclusions: This study identified two distinct subtypes that are varyingly associated with hormone resistance, stroma-immunity, and molecular features, thereby highlighting the importance of this stromal-immune heterogeneity in identifying EMs subtypes and providing novel insights into future personalized hormone-free therapy in EMs.

Bowel wall thickness measured by MRI is useful for early diagnosis of bowel endometriosis.

OBJECTIVE: To evaluate MRI features of bowel endometriosis (BE) and verify its clinical significance compared with pathological diagnosis. MATERIALS AND METHODS: Since 2018, patients clinically diagnosed with deep endometriosis (DE) and planned to undergo surgery were enrolled prospectively. MRI parameters including traction, thickening sign of the rectum, obliteration of the Douglas Pouch, sign of adenomyosis, and pelvic adhesion were extracted. Uni- and multi-variate analyses were performed to explore their association with pathological diagnosis of BE. ROC curve was utilized to ascertain the appropriate cutoff value for predicting the presence and assessing the severity of BE. RESULTS: A total of 226 patients with DE were recruited, and 154 BE cases were pathologically confirmed. Logistic regression analysis revealed that thickness of the rectal wall, traction sign of the rectum, and obliteration of the Douglas Pouch were independent factors to predict the presence of BE with the OR 1.59 (95% CI: 1.29-1.96), 0.24 (95% CI: 0.09-0.67), and 0.17 (95% CI: 0.07-0.40), respectively (p all < 0.01). A cutoff value of 6.0 mm for the thickness of rectal wall resulted in the highest predictive value of BE (specificity: 90.3%; sensitivity: 78.6%). For patients with measured thickness of the rectal wall over 6.0 mm, 72.1% (93/129) was confirmed BE with lesions infiltrated more than muscular layer. CONCLUSION: This prospective study indicates that based on precise definition of visualized features on MRI images, BE could be recognized pre-operatively. DE patients with thickness of rectal wall exceeding 6.0 mm have a greater probability of BE. CLINICAL RELEVANCE STATEMENT: Based on precise definition of visualized features and accurate measurement on MRI images, bowel infiltrating among deep endometriosis patients could be recognized pre-operatively. KEY POINTS: • Precise definition of measurable MRI parameters made it possible for early detection of bowel endometriosis. • Thickening sign, traction sign of the rectum, and obliteration of the Douglas Pouch were typical radiological indicators for bowel endometriosis. • Bowel involvement is more sensitive to be detected among pelvic deep endometriosis patients with the thickness of the rectal wall over 6.0 mm.