The dysfunction of CD8+ T cells triggered by endometriotic stromal cells promotes the immune survival of endometriosis.

Endometriosis is defined as an oestrogen-dependent and inflammatory gynaecological disease of which the pathogenesis remains unclear. This study aimed to investigate the cellular heterogeneity and reveal the effect of CD8+ T cells on the progress of endometriosis. Three ovarian endometriosis patients were collected, and single-cell RNA sequencing (scRNA-seq) progressed and delineated the cellular landscape of endometriosis containing five cell clusters. The endometrial cells (EMCs) were the major component, of which the mesenchymal cells were preponderant and characterized with increased inflammation and oestrogen synthesis in endometriosis. The proportion of T cells, mainly CD8+ T cells rather than CD4+, was reduced in endometriotic lesions, and the cytokines and cytotoxicity of ectopic T cells were depressed. CD8+ T cells depressed the proliferation of ESCs through inhibiting CDK1/CCNB1 pathway to arrest the cell cycle and triggered inflammation through activating STAT1 pathway. Correspondingly, the coculture with ESCs resulted in the dysfunction of CD8+ T cells through upregulating STAT1/PDCD1 pathway and glycolysis-promoted metabolism reprogramming. The endometriotic lesions were larger in nude mouse models with T-cell deficiency than the normal mouse models. The inhibition of T cells via CD90.2 or CD8A antibody increased the endometriotic lesions in mouse models, and the supplement of T cells to nude mouse models diminished the lesion sizes. In conclusion, this study revealed the global cellular variation of endometriosis among which the cellular count and physiology of EMCs and T cells were significantly changed. The depressed cytotoxicity and aberrant metabolism of CD8+ T cells were induced by ESCs with the activation of STAT1/PDCD1 pathway resulting in immune survival to promote endometriosis.

Effect of interleukin-1ß and lipoxin A4 in human endometriotic stromal cells: Proteomic analysis.

AIM: Lipoxin A4 (LXA4 ) can function as an endogenous 'breaking signal' in inflammation and plays an important role in the progression of endometriosis. The proteome responses to interleukin-1ß (IL-1ß) or LXA4 in human endometriotic stromal cells (ESC) are not well understood. METHODS: In this study, primary ESC were cultured from ovarian endometriosis tissue. Three groups were established: the control group; the IL-1ß stimulation group; and the IL-1ß and LXA4 incubation group. Proteins were assessed on 2-D polyacrylamide gel electrophoresis (2D-PAGE), and differentially expressed protein spots were further identified on matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MALDI-TOF-MS). Wound healing and transwell assays were performed to assess the migration and invasion of ESC after treatment. RESULTS: In total, 40 differentially expressed protein spots were identified successfully on MALDI-TOF-MS. The proteins identified were related to cell structure, metabolism, signal transduction, protein synthesis and membrane structure, processes that may be involved in the development of endometriosis. Vinculin and IL-4 were further analyzed on western blot and quantitative real-time polymerase chain reaction. Moreover, LXA4 could suppress the migration and invasion of ESC induced by IL-1ß. CONCLUSION: LXA4 may inhibit the progression of endometriosis partly by lowering or raising the effect of IL-1ß, mediated via some inflammation-related proteins (e.g. vinculin) and immune response-related protein (e.g. IL-4) in vitro.

Lipoxin A4 Suppresses Estrogen-Induced Epithelial-Mesenchymal Transition via ALXR-Dependent Manner in Endometriosis.

OBJECTIVE: Epithelial-mesenchymal transition (EMT) is essential for embryogenesis, fibrosis, and tumor metastasis. Aberrant EMT phenomenon has been reported in endometriotic tissues of patients with endometriosis (EM). In this study, we further investigated the molecular mechanism of which lipoxin A4 (LXA4) suppresses estrogen (E2)-induced EMT in EM. STUDY DESIGN: The EMT markers were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot in eutopic endometrial epithelial cells (EECs) or investigated by immunohistochemistry and qRT-PCR in endometriotic lesion of EM mice. The invasion and migration under different treatments were assessed by transwell assays with or without Matrigel. The messenger RNA (mRNA) and activities of matrix metalloproteinase 2 (MMP-2) and MMP-9 were determined by qRT-PCR and gelatin zymography, respectively. Luciferase reporter assay was used to measure the activity of zinc finger E-box binding homeobox 1(ZEB1) promoter. The level of E2 in endometriotic tissues was assessed by enzyme-linked immunosorbent assay. RESULTS: In eutopic EECs, stimulatory effects of E2 on EMT progress, migration, and invasion were all diminished by LXA4. Lipoxin A4 reduced E2-induced ZEB1 promoter activity. Lipoxin A4 also attenuated the phosphorylation of extracellular signal-regulated kinase and p38 mitogen-activated protein kinase induced by E2. Co-incubation with Boc-2 rather than DMF antagonized the influence of LXA4. Animal experiments showed that LXA4 inhibited the EMT progress, MMP expression, and proteinase activities of endometriotic lesion in an LXA4 receptor (ALXR) manner, which suppressed the progression of EM. ZEB1 mRNA expression was upregulated and well correlated with E2 level in human endometrium. CONCLUSION: Lipoxin A4 suppresses E2-induced EMT via ALXR-dependent manner in eutopic EECs, which reveals a novel biological effect of LXA4 in EM.