Application of NETs in diagnosis and treatment of endometriosis

Abstract

The invention relates to an application of a NeuMME subgroup in preparation of a diagnostic kit for evaluating NETosis occurrence degree or endometriosis severity. The invention also provides an application of the NETosis inhibitor or the DNase I in preparation of a medicine for treating endometriosis. It is proved that NETs in the abdominal cavity promote colonization, migration and proliferation capacity of endometrial cells through the capture effect, so that occurrence and development of endometriosis focuses are promoted, the NeuMME subgroup is a main source of the NETs, and therefore detection of the NeuMME subgroup and evaluation of the occurrence degree of NETosis have important significance. In addition, based on clinical sample analysis, it is found that NeuMME subgroups of peritoneal fluid are closely related to clinical rASRM scores of endometriosis; the analysis of a mouse endometriosis model also finds that the NeuMME subgroup is closely related to the occurrence and development of an endometriosis focus. Therefore, the NeuMME-based subgroup detection for evaluating the severity of endometriosis and the focus progress has remarkable advantages and application prospects. The development of endometriosis can be remarkably inhibited by inhibiting NETosis or clearing NETs in a mouse model through a drug.

Description

Technical Field

[0001] The present invention belongs to the fields of disease diagnosis and drug targets, and particularly relates to the application of NETs in the diagnosis and treatment of endometriosis. Background Art

[0002] Endometriosis (EM) is a chronic gynecological disease that affects approximately 10% of reproductive-aged women globally, characterized by the ectopic growth of endometrial-like tissue. The disease is often accompanied by severe pelvic pain, infertility, and a decline in quality of life. However, its pathogenesis has not been fully elucidated, which has hindered effective diagnosis and treatment. Since Sampson proposed the "retrograde menstruation" theory in 1927, that is, the implantation of shed endometrial cells in the abdominal cavity, several theories have emerged successively. These theories include hematogenous / lymphatic dissemination, hormone-driven metaplasia, genetic susceptibility, and immune dysregulation, indicating that microenvironmental differences and undefined regulatory factors play a key role in disease progression. Increasing evidence supports that the microbiota, especially the gut and peritoneal microbiota, is involved in the pathogenesis and progression of endometriosis through inflammatory pathways.

[0003] The gut microbiota is a complex microbial community present in the gastrointestinal tract that regulates systemic inflammation and immune responses. Dysbiosis, that is, the imbalance of the gut microbiota, is associated with various inflammatory and autoimmune diseases. In endometriosis, it has been reported that the gut microbiota composition changes, showing pro-inflammatory characteristics, such as an increase in Proteobacteria and a decrease in Firmicutes. These changes in the microbiota may be involved in the pathogenesis of endometriosis by producing pro-inflammatory metabolites and regulating host immune responses. The peritoneal cavity is the main site of endometriosis lesions. Traditionally considered sterile, but recent studies have shown that the peritoneal cavity of endometriosis patients has different microbiota characteristics compared to healthy controls. These characteristics include an increase in pathogenic bacteria (such as Ruminococcus and Pseudomonas aeruginosa), and an increase in the level of endotoxin (such as lipopolysaccharide, LPS). These microbiota changes may promote chronic inflammation through mechanisms (such as LPS-TLR4 signaling), thus potentially promoting the formation of ectopic lesions. Previous studies have reported that the microbiota in the gut and peritoneal fluid share common genus-level taxonomic units, which can be used as potential biomarkers for endometriosis, suggesting that there may be cross-talk between the gut-peritoneal microbiota. However, the regulatory role of the microbiota factors in the peritoneal immune microenvironment has not been clarified.

[0004] Neutrophils are the most numerous type of immune cells in the body and play a key role as the first line of defense in immune responses and against infections and injuries. In endometriosis, neutrophils are recruited to the peritoneal cavity and participate in the formation of ectopic lesions by releasing pro-inflammatory cytokines. Activated neutrophils release a variety of bioactive substances, such as cytokines, chemokines, proteolytic enzymes, reactive oxygen species, and neutrophil extracellular traps (NETs). NETs are composed of decondensed DNA and antimicrobial proteins. NETs play a key role in innate immunity, for example, preventing the ascending of urinary tract pathogenic bacteria because they can efficiently capture pathogenic microorganisms. However, the overproduction of NETs may exacerbate tissue damage and inflammation. Importantly, microbial products (such as lipopolysaccharide, LPS) and dysregulated gut or peritoneal flora may activate neutrophils through pattern recognition receptors (such as TLR). However, the link between microbial triggers and NETosis as well as the development of endometriosis lesions and their exact signaling pathways remain unclear.

[0005] The present invention aims to explore the role of gut and peritoneal flora in inducing NETosis and the pathogenesis of endometriosis. Through single-cell RNA sequencing, functional experiments, and mouse models, we aim to clarify the complex interactions between immune cells and microbial factors in endometriosis. Understanding these mechanisms may provide insights into potential therapeutic targets and improve the management strategies for endometriosis patients. Summary of the Invention

[0006] The object of the present invention is to provide the application of NETs in the diagnosis and treatment of endometriosis in view of the deficiencies in the prior art.

[0007] In a first aspect, the present invention provides the application of NETs as biomarkers in the preparation of a diagnostic kit for endometriosis.

[0008] As a preferred example, the NETs refer to those released from the Neu_MME subset.

[0009] As another preferred example, the sample to be detected is the polymer in peritoneal fluid, and the polymer is composed of neutrophils, NETs, and endometrial cells.

[0010] In a second aspect, the present invention provides the application of a reagent for detecting the content of NETs in the preparation of a diagnostic kit for endometriosis.

[0011] As a preferred example, the NETs refer to those released from the Neu_MME subset.

[0012] As another preferred example, the sample to be detected is the polymer in peritoneal fluid, and the polymer is composed of neutrophils, NETs, and endometrial cells.

[0013] In a third aspect, the present invention provides the use of the Neu_MME subset in the preparation of a diagnostic kit for evaluating the degree of NETosis or the severity of endometriosis.

[0014] As a preferred example, the detection marker of the Neu_MME subset in human samples is CD45 + CD11b + CD16b + MME + ; the detection markers in mouse samples are CD45 + CD11b + Ly6G + MME + .

[0015] As a preferred example, the Neu_MME subset is the source cell that releases and generates NETs in the form of NETosis.

[0016] As another preferred example, the degree of NETosis refers to the level of NETs generation.

[0017] As another preferred example, the severity of endometriosis refers to the clinical score of endometriosis (rASRM score).

[0018] In a fourth aspect, the present invention provides the use of a NETosis inhibitor or DNase I in the preparation of a drug for treating endometriosis.

[0019] As a preferred example, the NETosis inhibitor is GSK484.

[0020] As another preferred example, the endometriosis is NETs-induced endometriosis.

[0021] As another preferred example, the endometriosis is LPS-induced endometriosis derived from abdominal cavity bacteria.

[0022] The advantages of the present invention are as follows: The present invention demonstrates that intraperitoneal NETs promote the colonization, migration and proliferation of endometrial cells through capture, thus promoting the occurrence and development of endometriosis lesions, and discovers that the Neu_MME subset is the main source of NETs. Therefore, detecting the Neu_MME subset to evaluate the degree of NETosis is of great significance. However, the current indicators for evaluating NETosis are single (such as the commonly used PADI4). Our data show that MME is expressed earlier and more sensitively than PADI4 during the occurrence of NETosis. Therefore, detecting MME to evaluate the degree of NETosis has significant advantages.

[0023] In addition, analysis based on clinical samples found that the Neu_MME subset in peritoneal fluid was closely related to the clinical rASRM score of endometriosis; analysis of the mouse endometriosis model also found that the Neu_MME subset was closely related to the occurrence and development of endometriosis lesions. Therefore, detecting the Neu_MME subset has significant advantages and application prospects for evaluating the severity of endometriosis and the progression of lesions.

[0024] The present invention found that the recruitment and activation of MME+ neutrophils (Neu_MME) in peritoneal fluid are related to the development of endometriosis. Neutrophil extracellular traps (NETs) released by Neu_MME are induced by bacterial lipopolysaccharide (LPS). NETs promote the development of endometriosis by capturing endometrial cells and enhancing their proliferation and migration. Inhibiting NETosis or clearing NETs by drugs in a mouse model can significantly inhibit the development of endometriosis. These findings highlight the key role of NETs in the pathogenesis of endometriosis, suggesting that targeting NETosis and NETs may provide a new treatment strategy to block the key links of adhesion, colonization, proliferation, and migration of free endometrial fragments or cells in the peritoneal cavity during the formation of endometriosis lesions, thereby reducing the occurrence and progression of lesions. Brief Description of the Drawings

[0025] Figure 1 : The recruitment and activation of MME+ neutrophils (Neu_MME) in the peritoneal cavity are related to the pathogenesis of endometriosis. a) Flow chart of the experiment of single-cell RNA sequencing (scRNA-seq), showing the method of sorting immune cells using CD45 magnetic beads from peritoneal fluid (EM group: peritoneal fluid of endometriosis patients, n = 9; Con group: peritoneal fluid of control patients, n = 3). b) Immunocytes (CD45 + magnetic beads) in peritoneal fluid. +) UMAP plots for dimensionality reduction analysis of the clusters. Stacked bar charts show the proportions of immune cells in peritoneal fluid. c) Facet visualization is used to show the cell UMAP plots of different sample groups. d) Comparison of neutrophils, dendritic cells (DCs), T cells and natural killer cells (T-NKs), macrophages and monocytes (Ma-Monos) between the endometriosis group and the control group. e) Comparison of the proportions of macrophages and monocytes (Ma-Monos) and neutrophils in rASRM stages I–II and III–IV. f) UMAP plot of neutrophil subsets. Stacked bar charts show the proportions of neutrophil subsets. g) Facet visualization is used to show the neutrophil UMAP plots of different sample groups. h) Comparison of the Neu_MME ratio between rASRM stages I-II and III-IV. i) Immunocytes in peritoneal fluid were analyzed by flow cytometry. The proportions of neutrophils and MME+ neutrophils (Neu_MME, CD45 + CD11b + CD16b + MME + ) were compared, as well as the differences between rASRM stages I–II and III–IV (PF_Con group: peritoneal fluid of control patients, n = 20; PF_EM group: peritoneal fluid of endometriosis patients, n = 20). j) UMAP plot and box plot of CytoTRACE analysis of neutrophils. k) Pseudotime analysis of neutrophils, and the faceted plot shows the distribution of different states and subsets of neutrophils. l) Gene Ontology (GO) biological process analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed on the Neu_MME subset. Results are expressed as mean ± standard deviation (SD). Student’s t-test was used for the analysis in parts d, e and h. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns: not significant. Abbreviations: EM, endometriosis; Con, control; PF, peritoneal fluid; CD16b (also known as FCGR3B, Fc Gamma receptor IIIb); rASRM, revised American Society for Reproductive Medicine; DC, dendritic cell; T-NK, T cells and natural killer cells; Ma-Mono, macrophages and monocytes.

[0026] Figure 2 : compared with Figure 1Related supplementary results. a-c) The samples were normalized. The count distribution and the percentage of mitochondrial genes were shown. d-e) The correlation plot between the count and the percentage of mitochondrial genes was drawn. f) The heatmap of the marker genes of immune cell populations was drawn. g) The heatmap of the marker genes of neutrophil subsets was drawn. h) The differences in eosinophils and mast cells (Eo-mast cells), B cells and plasma cells (B-plasma cells), basophils, mast cells, and proliferating cells between endometriosis patients and the control group were compared. i) The immune cells in the ascites were analyzed by flow cytometry. The proportion of myeloid cells between endometriosis patients and the control group, and between rASRM stages I-II and III-IV was compared (PF_Con group: ascites of control patients, n = 20; PF_EM group: ascites of endometriosis patients, n = 20). The results were expressed as mean ± standard deviation (SD). Student’s t-test was used in the analysis of parts m and n. *P<0.05; **P<0.01; ns: not significant. Abbreviations: EM, endometriosis; Con, control; PF, ascites; UMAP, Uniform Manifold Approximation and Projection; rASRM, Revised American Society for Reproductive Medicine.

[0027] Figure 3: Neutrophil extracellular traps (NETs) from Neu_MME are induced and released by bacterial lipopolysaccharide (LPS). a) The expression levels of myeloperoxidase (MPO) and citrullinated histone H3 (citH3) in peritoneal fluid cells of the control group and patients with endometriosis (EM) were detected by immunofluorescence (PF_Con group: n = 21; PF_EM group: n = 52). b) The content of citrullinated histone H3 (citH3) in peritoneal fluid was detected by enzyme-linked immunosorbent assay (ELISA) (PF_Con group: n = 20; PF_EM group: n = 67). c) The co-expression of Fc Gamma receptor IIIb (CD16b), membrane metalloendopeptidase (MME) and citrullinated histone H3 (citH3) in peritoneal fluid cells was detected by immunofluorescence. d) Neutrophils were treated with peritoneal fluid (PF_SN), and the expression levels of myeloperoxidase (MPO) and citrullinated histone H3 (citH3) in the control group and patients with endometriosis (EM) were detected (PF_SN_Con group: n = 16; PF_SN_EM group: n = 23). e) The content of lipopolysaccharide (LPS) in peritoneal fluid of the control group and patients with endometriosis (EM) was detected (PF_SN_Con group: n = 20; PF_SN_EM group: n = 60). In addition, the content of lipopolysaccharide-binding protein (LBP) in the serum of the control group and patients with endometriosis (EM) was detected (PF_SN_Con group: n = 24; PF_SN_EM group: n = 51). f) The proportion of Neu_MME in neutrophils after lipopolysaccharide (LPS) treatment was calculated. Three independent experiments were performed. g) The expression levels of Toll-like receptor 4 (TLR4), tumor necrosis factor α (TNF-α), peptidylarginine deiminase 4 (PADI4), membrane metalloendopeptidase (MME) and citrullinated histone H3 (citH3) in neutrophils after lipopolysaccharide (LPS) treatment were detected by Western blotting. h) Clinical serial sections were prepared for hematoxylin and eosin (H&E) staining and immunofluorescence analysis of myeloperoxidase (MPO), citrullinated histone H3 (citH3) and estrogen receptor 1 (ESR1). The expression levels of MPO and citH3 in normal endometrium, eutopic endometrium and ectopic lesions were analyzed (normal endometrium: n = 17; eutopic endometrium: n = 21; ectopic lesions: n = 82). i) ROC curves of MPO and citH3 were generated to distinguish normal / eutopic endometrium from endometrial lesions, and the area under the curve (AUC), Youden index, cut-off value, sensitivity and specificity were shown. j) Pairwise comparisons of MPO and citH3 were performed between estrogen receptor 1 (ESR1)-positive regions and ESR1-negative regions.k) Using fluorescence in situ hybridization (FISH) technology, simultaneously detect the bacterial content in ectopic lesions (using the Eubacteria probe 338 (EUB338, composed of a mixture of EUB338 I, II, and III) labeled with GFP), and the contents of lipopolysaccharide (LPS) and lipoteichoic acid (LTA) (n = 82). l) Analyze the expression levels of myeloperoxidase (MPO), citrullinated histone H3 (citH3), Eubacteria probe 338 (EUB338), and lipopolysaccharide (LPS) in stage I–II and stage III–IV lesions of rASRM. m) Draw a heat map of the correlation analysis of Eubacteria probe 338 (EUB338), lipopolysaccharide (LPS), lipoteichoic acid (LTA), myeloperoxidase (MPO), citrullinated histone H3 (citH3), and rASRM score. n) Use a Sankey diagram to show the combined analysis of Eubacteria probe 338 (EUB338), lipopolysaccharide (LPS), lipoteichoic acid (LTA), myeloperoxidase (MPO), citrullinated histone H3 (citH3), and rASRM stage. The results are expressed as mean ± standard deviation (SD). Parts a, b, d–f, h, j, and l were analyzed using Student’s t-test. Part m was analyzed using Pearson correlation analysis. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns: not significant. Abbreviations: MPO, myeloperoxidase; citH3, citrullinated histone H3; DAPI, 4’,6-diamidino-2-phenylindole; PF, peritoneal fluid; EM, endometriosis; Con, control; PF_SN, peritoneal fluid supernatant; MME, membrane metalloendopeptidase; CD16b (also known as FCGR3B, Fc Gamma receptor IIIb); LPS, lipopolysaccharide; PADI4, peptidylarginine deiminase 4; FOSB, FosB proto-oncogene, AP-1 transcription factor subunit; TNF-α, tumor necrosis factor α; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; EUB338, Eubacteria probe 338; LTA, lipoteichoic acid; AUC, area under the curve.

[0028] Figure 4 : compared with Figure 3Related supplementary results. a) Co-expression of CD16b, MME, and citH3 was detected in LPS-treated white blood cells and isolated neutrophils. b) Volcano plots were generated showing differentially expressed genes between endometriosis patients and controls in the Neu_MME cluster. c) Plots were generated for analyzing the correlations of EUB338, LPS, LTA, MPO, citH3, and rASRM scores. Pearson correlation analysis was performed on the data in c. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Abbreviations: MPO, myeloperoxidase; citH3, citrullinated histone H3; MME, membrane metalloprotease; CD16b (also known as FCGR3B, Fc Gamma Receptor IIIb); DAPI, 4’,6-diamidino-2-phenylindole; WBC, white blood cell; EUB338, Eubacteria probe 338; LPS, lipopolysaccharide; LTA, lipoteichoic acid.

[0029] Figure 5NETs promote the development of endometriosis by capturing endometrial cells and enhancing their proliferation and migration. a) Aggregates of "neutrophils-NETs-endometrial cells" were observed in peritoneal fluid. Neutrophils were labeled with MPO, NETs were labeled with citH3, and endometrial cells were labeled with ESR1. b) A "NETs-endometrial cell" capture experiment was conducted. Fluorescence co-localization analysis curves were shown. F-actin in cells was stained with GFP-Phalloidin. c) Transwell migration experiments were performed on endometrial epithelial cells (EECs) and endometrial stromal cells (ESCs). Ten fields of view were randomly selected for counting per well. Scale bar = 62.2 μm. d-e) The proliferation ability of endometrial cells (ECs) and stromal cells (SCs) was evaluated using colony formation assays (d) and CCK-8 assays (e). Three independent experiments were conducted. f) The experimental flow chart shows an endometriosis model induced in C57BL / 6 mice by intraperitoneal injection of LPS, GSK484, and DNase I. g) The number of endometriosis lesions in mice of each group was shown. h) Immunohistochemical images of pelvic lesion appearance and Ki67 expression were provided. i-j) The number of lesions in each mouse and the thickness of the stromal layer in the lesions were compared. k) The expression levels of Ki67 were compared. l) The number of CD31-positive capillaries per high power field in the lesions was calculated. m-n) The expression levels of Ly6G and citH3 in the lesions were analyzed. o) The level of citH3 in mouse peritoneal fluid was detected by ELISA. p) Immunofluorescence images showed the expression of Ly6G and citH3 in the lesions. q) Flow cytometry was used to analyze neutrophils and MME+ neutrophils (Neu_MME, CD45 + CD11b + Ly6G + MME +)Ratio. Results are expressed as mean ± standard deviation. Student's t-test was used to analyze parts c-e, i-o, and q. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns: not significant. Abbreviations: MPO, myeloperoxidase; citH3, citrullinated histone H3; ESR1, estrogen receptor 1; DAPI, 4’,6-diamidino-2-phenylindole; EECs, endometrial epithelial cells; ESCs, endometrial stromal cells; LPS, lipopolysaccharide; PF, peritoneal fluid; EM, endometriosis; Con, control; NETs, neutrophil extracellular traps; PBS, phosphate buffer solution; DNase I, deoxyribonuclease I; OVX, ovariectomy; E2, estradiol; LPS, lipopolysaccharide; GSK484, N-[(1R,2R)-2-(2,5-difluorophenyl)cyclopropyl]-N-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine; DNase I, deoxyribonuclease I; ELISA, enzyme-linked immunosorbent assay; Ly6G, lymphocyte antigen 6 complex; citH3, citrullinated histone H3; MME, membrane metalloproteinase.

[0030] Figure 6 : Supplementary results related to Figure 5 Isolation and identification of neutrophils and endometrial cells. a) Schematic experimental flowchart for isolating neutrophils from human peripheral blood. b) Detection of myeloperoxidase (MPO) expression in neutrophils using immunofluorescence (IF). c) Observation of neutrophils isolated from human peripheral blood under bright-field microscopy. d) Evaluation of the expression of Fc gamma receptor IIIb (CD16b) in neutrophils using immunofluorescence combined with bright-field microscopy. e) Observation of the primary culture of endometrial cell clusters under bright-field microscopy. f) Observation of the morphology of primary endometrial epithelial cells (EECs) and stromal cells (ESCs) under bright-field microscopy. g) Immunofluorescence staining identification of endometrial epithelial cells (EECs) and stromal cells (ESCs) using markers vimentin, keratin, and estrogen receptor 1 (ESR1). Abbreviations: RBC, red blood cell; MPO, myeloperoxidase; CD16b (also known as FCGR3B, Fc gamma receptor IIIb); DAPI, 4’,6-diamidino-2-phenylindole; ESR1, estrogen receptor 1; EECs, endometrial epithelial cells; ESCs, endometrial stromal cells.

[0031] Figure 7 : Supplementary results related to Figure 5Related supplementary results. a) Schematic experimental flowchart of the endometriosis animal model in NCG mice treated by intraperitoneal injection of neutrophil extracellular traps (NETs) (n = 5 per group). b) Examination of the appearance of pelvic endometriosis lesions in NCG mice and representative hematoxylin and eosin (H&E) staining images. The volumes of the lesions in each group were compared. c) Representative hematoxylin and eosin (H&E) staining and immunohistochemistry images showing the staining of estrogen receptor 1 (ESR1) and platelet and endothelial cell adhesion molecule 1 (CD31) in endometriosis lesions of C57BL / 6 mice. d) Flow cytometry was used to analyze the proportions of immune cells in the peritoneal fluid of C57BL / 6 mice. The proportions of T cells, myeloid cells, and macrophages in the peritoneal fluid of each group were compared. The results are expressed as mean ± standard deviation (SD). Student's t-test was used for the statistical analysis in b and d. *P < 0.05; **P < 0.01; ***P < 0.001; ns: not significant. Abbreviations: H&E, hematoxylin and eosin staining; ESR1, estrogen receptor 1; NCG mice, NOD / ShiLtJGpt-Prkdcem26Il2rgem26 / Gpt mice; CD31 (also known as PECAM1, platelet and endothelial cell adhesion molecule 1); PBS, phosphate-buffered saline; LPS, lipopolysaccharide; DNase I, deoxyribonuclease I; GSK484, N-[(1R,2R)-2-(2,5-difluorophenyl)cyclopropyl]-N-methyl-7H-pyrrolo[2,3-d]pyrimidin-4-amine. Detailed Description of the Invention

[0032] The present invention will be further described below in conjunction with the specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. In addition, it should be understood that after reading the content recorded in the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of this application.

[0033] Example 1

[0034] 1 Method

[0035] 1.1 Clinical Samples

[0036] Clinical samples used in this study included freshly collected samples (such as peripheral blood, peritoneal fluid, and endometrial tissue), as well as paraffin-embedded tissue sections obtained from the pathology department's storage. All cases in the endometriosis group were diagnosed based on intraoperative findings and confirmed by postoperative pathological examination. The control group consisted of individuals without endometriosis lesions or disease-related symptoms. All paraffin-embedded tissue sections and peripheral blood samples were from the First Affiliated Hospital of Guangzhou Medical University. Fecal and peritoneal fluid samples were from the First Affiliated Hospital of Guangzhou Medical University, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Qingyuan Hospital Affiliated to Guangzhou Medical University, Zhuhai People's Hospital, and the First Affiliated Hospital of Sun Yat-sen University. All participants signed a written informed consent form, and this study was approved by the Ethics Committee of the First Affiliated Hospital of Guangzhou Medical University (approval number: 2020074).

[0037] 1.2 Mice

[0038] Six-week-old female NCG (NOD / ShiLtJGpt-Prkdcem26Il2rgem26 / Gpt) mice were purchased from GemPharmatech and housed in a specific pathogen-free (SPF) environment. Before the experiment, peripheral blood was collected via the tail vein, and the immune cell profile was analyzed by flow cytometry (CD45 represents total immune cells, CD3 represents T cells, NK1.1 represents NK cells, and CD11b represents myeloid cells) to verify the defective state of immune cells. Six-week-old C57BL / 6 mice were purchased from the Guangdong Provincial Center for Laboratory Animals. After the experiment, the mice were sacrificed by cervical dislocation under pentobarbital sodium anesthesia. All animal experiments were conducted in accordance with the "Guidelines for the Management and Use of Laboratory Animals" of Guangzhou Medical University and were approved by the Ethics Committee of Guangzhou Medical University (approval numbers: 2021036, 2021143, 2021228, and 2022062).

[0039] 1.3 Reagents and Antibodies

[0040] The antibodies used in this study are as follows: anti-CD10 (MME) antibody (Catalog No.: ET1611-82, Huabiao Biotech, Hangzhou, China); anti-CD31 antibody (Catalog No.: ZA-0568, Zhongshan Golden Bridge, Beijing, China); anti-ESR1 antibody (Catalog No.: ab259427, Abcam, Cambridge, UK; Catalog No.: DF6094, Affinity, Cambridge, UK); anti-FOSB antibody (Catalog No.: AF2392, Beyotime, Shanghai, China); anti-GAPDH antibody (Catalog No.: ab9485, Abcam, Cambridge, UK); anti-Histone3 (citrulline R2+R8+R17) antibody (Catalog No.: ab5103, Abcam, Cambridge, UK); anti-Ki67 antibody (Catalog No.: GB111499, Sevier Biotech, Wuhan, China); anti-LPS antibody (Catalog No.: HM6011-1217, Hycult Biotech, Uithoorn, Netherlands); anti-LTA antibody (Catalog No.: HM2048, Hycult Biotech, Uithoorn, Netherlands); anti-Ly6G antibody (Catalog No.: MAB1037, R&D Systems, Minneapolis, MN, USA); InVivoMAb anti-Ly6G antibody (Catalog No.: BE0075-1, Bio XCell, West Lebanon, NH, USA); anti-MPO antibody (Catalog No.: AF3667, R&D Systems, Minneapolis, MN, USA); anti-PADI4 antibody (Catalog No.: A16188, Abclonal, Wuhan, China); anti-TLR4 antibody (Catalog No.: AF8187, Beyotime, Shanghai, China); anti-TNF-α antibody (Catalog No.: AF8208, Beyotime, Shanghai, China); anti-ZO-1 antibody (Catalog No.: GB111402, Sevier Biotech, Wuhan, China). The fluorescently labeled antibodies used for immunofluorescence staining and flow cytometry are as follows: donkey anti-rabbit IgG H&L (Alexa 647) antibody (Catalog No.: ab150075, Abcam, Cambridge, UK), donkey anti-goat (Alexa 488) antibody (Catalog No.: ab150129, Abcam, Cambridge, UK), donkey anti-mouse IgG H&L (Alexa 568) antibody (Catalog No.: ab175472, Abcam, Cambridge, UK), goat anti-rat IgG (H+L) Cy3 antibody (Catalog No.: 33308ES60, YEASEN, Shanghai, China), donkey anti-mouse IgG H&L Cy3 antibody (Catalog No.: GB21401, Sevier Biotech, Wuhan, China), eBioscience TMFixable Viability Dye eFluor TM 780 (Catalog No.: 65-0865-14, ThermoFisher, Waltham, MA, USA), PerCP / Cyanine5.5 anti-mouse / human CD11b (Catalog No.: 101228, Biolegend, San Diego, CA, USA), Brilliant Violet 510 TM anti-mouse CD45 (Catalog No.: 103138, Biolegend, San Diego, CA, USA), APC anti-human CD45 (Catalog No.: 304012, Biolegend, San Diego, CA, USA), PE anti-mouse F4 / 80 (Catalog No.: 123110, Biolegend, San Diego, CA, USA), Brilliant Violet510 TM anti-mouse CD3 (Catalog No.: 100234, Biolegend, San Diego, CA, USA), PE / Cyanine7 anti-human CD3 (Catalog No.: 300316, Biolegend, San Diego, CA, USA), FITC anti-human CD16b (Catalog No.: 302005, Biolegend, San Diego, CA, USA; Catalog No.: #130-109-191, Miltenyi Biotec, Bergisch Gladbach, Germany), FITC anti-human CD10 (Catalog No.: 312207, Biolegend, San Diego, CA, USA), anti-CD16b-FITC antibody.

[0041] 1.4 Single-cell dissociation

[0042] Ascites fluid samples were collected from 6 patients with endometriosis and 3 control individuals without the disease for single-cell RNA sequencing. The single-cell RNA sequencing experiments were performed by laboratory technicians at NovelBio Co., Ltd. After passing the samples through a 70-μm cell strainer, the samples were centrifuged at 300 g for 5 minutes. After removing the supernatant, the pelleted cells were resuspended in red blood cell lysis buffer (Catalog No.: 130094183, Miltenyi Biotec, USA) to lyse red blood cells. Subsequently, the cell pellet was washed with PBS containing 0.04% BSA and resuspended in PBS containing 0.04% BSA, and then filtered through a 35-μm cell strainer. Immunocytes were isolated from ascites cells using a CD45+ magnetic bead cell isolation kit (Catalog No.: 130045801, Miltenyi Biotec, USA) according to the manufacturer's instructions. Subsequently, the isolated CD45+ single cells were stained with Calcein-AM (C3099, ThermoFisher Scientific, USA) and Draq7 (Catalog No.: 564904, BD Biosciences, USA) to evaluate cell viability.

[0043] 1.5 Single-cell RNA sequencing

[0044] In this study, the BD Rhapsody system was used to capture the transcriptomic features of peritoneal fluid single cells. The single-cell suspension was randomly assigned to more than 200,000 microwells by limited dilution to complete single-cell capture. Magnetic beads containing oligonucleotide barcodes were added until saturation to ensure that each bead was paired with a single cell in the microwell. Subsequently, cell lysis buffer was added to bind polyadenylated RNA molecules to the magnetic beads. The magnetic beads were collected into a single tube for reverse transcription. During cDNA synthesis, a unique molecular identifier (UMI) and a cell barcode were assigned to the 5′ end of each cDNA molecule (i.e., the 3′ end of mRNA transcription) to label its source cell. The whole-transcriptome library was constructed using the BD Rhapsody single-cell whole-transcriptome amplification workflow. Briefly, the second-strand cDNA was first synthesized, and then WTA adapters were ligated to enable universal amplification. The adapter-ligated cDNA products were amplified by 18 cycles of PCR. The whole-transcriptome amplification products were amplified by random-prime PCR to enrich the 3′ ends of transcripts associated with cell barcodes and UMIs, generating a sequencing library. The sequencing library was quantitatively analyzed on a Bioanalyzer 2200 using Agilent's high-sensitivity DNA chip and Thermo Fisher Scientific's Qubit high-sensitivity DNA kit. The library of each sample was sequenced in a 150-bp paired-end manner on an Illumina NovaSeq 6000 (Illumina, San Diego, CA).

[0045] 1.6 Single-cell RNA sequencing data analysis

[0046] Single-cell RNA sequencing (scRNA-seq) data analysis was completed by NovelBio Co., Ltd. using the NovelBrainCloud analysis platform. We used fastp for processing, filtering adapter sequences and removing low-quality reads with default parameters to generate clean data. Single-cell transcriptome analysis was performed using UMI-tools to determine the whitelist of cell barcodes. The UMI-based clean data was aligned to the human genome (Ensembl version 91) using STAR mapping (combined with custom parameters in the UMI-tools standard pipeline) to obtain UMI counts for each sample. Cells with more than 200 expressed genes and a mitochondrial UMI rate lower than 20% passed the cell quality filter, and mitochondrial genes were removed from the expression table. Cell normalization and regression analysis were performed using the Seurat package (version 2.3.4, https: / / satijalab.org / seurat / ) based on the expression table, considering the UMI counts and mitochondrial read percentages of each sample to obtain normalized data. Based on the normalized data, principal component analysis (PCA) was performed using the top 2000 highly variable genes, and t-distributed stochastic neighbor embedding (tSNE) construction and uniform manifold approximation and projection (UMAP) construction were performed using the top 10 principal components.

[0047] 1.7 Pseudotime analysis

[0048] CytoTRACE and Monocle 2 were used to predict the differentiation status of cells along the pseudotime sequence. We performed the analysis using Monocle2 (https: / / github.com / MaxMeieran / monocle2), combined with DDR-Tree and default parameters. Before performing Monocle analysis, we selected marker genes from the Seurat clustering results and the original expression counts of cells passing through the filtering process. Based on the pseudotime analysis, branch expression analysis modeling (BEAM analysis) was performed to analyze the genes determining branch fate.

[0049] 1.8 Differential gene expression and gene enrichment analysis

[0050] To identify the differentially expressed genes (DEGs) between endometriosis (EM) samples and control samples, we used the FindMarkers function combined with the Wilcoxon rank-sum test algorithm and analyzed them according to the following criteria: 1. lnFC > 0.25; 2. p-value < 0.05; 3. min.pct > 0.1. Based on the Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases, all pathway terms involved by genes were annotated. The significance level (p-value) of each pathway was calculated using Fisher's exact test to screen out the significant pathway terms enriched with genes.

[0051] 1.9 Flow cytometry analysis

[0052] Flow cytometry analysis was performed on BD LSRFortessa X-20 and BD FACSVerse flow cytometers according to the standard procedures. The data were analyzed using FlowJo (v10.9.0). Doublet cells were excluded by comparing FSC-H with FSC-A and SSC-H with SSC-A. Dead cells were excluded using the fixable viability dye 780 (Catalog No.: 565388, BD Biosciences, USA). The list of antibodies used in the experiment is shown in Section 1.3. For neutrophils isolated from the peritoneal fluid and blood of patients, MME-positive neutrophils (Neu_MME) were detected using CD45, CD11b, CD16b, and MME antibodies. For the peritoneal fluid of mice, Neu_MME was detected using CD45, CD11b, Ly6G, and MME antibodies, and T cells and macrophages were detected using CD3 and F4 / 80 antibodies.

[0053] 1.10 Immunofluorescence

[0054] Immunofluorescence staining was performed according to the previously reported method. The samples included fresh cell smears, cells on slides, and paraffin-embedded samples. For paraffin-embedded samples, antigen retrieval was performed using an antigen retrieval solution (Tris-EDTA buffer, pH 9.0 or citrate buffer, pH 6.0) in a pressure cooker for 5–10 minutes. For antigens expressed in the cytoplasm or nucleus, tissue sections were pre-treated with 0.1% Triton X-100. After blocking non-specific antigens, the tissue sections were placed in a humidified sealed container and incubated overnight at 4°C with primary antibodies against ESR1 (1:100), MPO (1:40), MME (1:200), Ly6G (1:50), citH3 (1:200), vimentin (1:200), and cytokeratin (1:200). After incubation, fluorescently labeled secondary antibodies and 4’,6-diamidino-2-phenylindole (DAPI) were added, and images were acquired using a fluorescence microscope (Leica DM6; Zeiss Axio Imager 2). The fluorescence intensity and fluorescence co-localization of the images were analyzed using ImageJ software.

[0055] 1.11 Enzyme-linked immunosorbent assay

[0056] Enzyme-linked immunosorbent assay (ELISA) was used to determine the concentrations of citrullinated histone H3 (JM-0611H1, Jinmei, Jiangsu, China; MM-1005M1, Enzyme Immunoassay, Jiangsu, China) and lipopolysaccharide (LPS, MM-0634M1, Enzyme Immunoassay, Jiangsu, China) in peritoneal fluid, and the concentration of LPS-binding protein (LBP, MM44515M1, Enzyme Immunoassay, Jiangsu, China) in serum. The specific operation was carried out according to the kit instructions. The brief steps were as follows: 100 μl of the standard and ten-fold diluted samples were added to a 96-well plate and incubated at 37°C for 30 minutes. After incubation, the plate was emptied and washed 5 times with washing buffer. Subsequently, 100 μl of the HRP-labeled working solution was added to each well and incubated at 37°C for another 30 minutes. After the second incubation, the plate was emptied again and washed 5 times with washing buffer. Then, 100 μl of the TMB-B substrate solution was added to each well and incubated at 37°C for 10 minutes for color development. To terminate the reaction, 100 μl of the HRP stop solution was added to each well, turning the blue mixture yellow. Finally, the absorbance was measured using an enzyme-linked immunosorbent assay reader at a wavelength of 450 nm. The concentration of the measured sample was estimated by comparison with normal control samples.

[0057] 1.12 Western blot

[0058] The Western blot experiment was performed according to the previously reported method. Proteins in neutrophils were extracted using RIPA protein lysis buffer (P0013C, Beyotime, Shanghai, China) and quantified using a BCA protein quantification reagent (P0011, Beyotime, Shanghai, China). After electrophoresis and protein transfer, the polyvinylidene fluoride (PVDF) membrane was blocked and incubated overnight at 4 °C with primary antibodies against MME (1:2000), PADI4 (1:1000), FOSB (1:1000), TNF-α (1:1000), citH3 (1:1000), and GAPDH (1:20000). After washing and incubation with an enzyme-labeled secondary antibody, chemiluminescence signals were detected using an enhanced chemiluminescence (ECL) kit (FD8000, Fdbio Science, Hangzhou, China) and a chemiluminescence imaging analysis system (Tanon5200, Tanon, Shanghai, China). To detect proteins with similar molecular weights, the membrane was treated with an antibody stripping solution (BL1382B, Baitiao, Anhui, China) and then incubated again with another primary antibody.

[0059] 1.13 Fluorescence in situ hybridization (FISH) localization of bacteria

[0060] A bacterial direct fluorescence in situ hybridization (FISH) detection kit (D0016, Exonbio, Guangzhou, China) was used to detect bacteria in the lesions, and the operation steps were referred to the kit instructions. The EUB338 probe sequence was 5’-GCTGCCTCCCGTAGGAGT-3’ (see SEQ ID NO:1). The brief steps were as follows: Paraffin-embedded tissue sections were first dewaxed and rehydrated. Subsequently, the sections were placed in Solution A (0.2 N hydrochloric acid) and incubated at room temperature for 15 minutes. After aspirating the excess Solution A, Solution B (containing 50 μg / ml proteinase K) was added and incubated at 37 °C for 5 minutes. After the reaction was terminated, the sections were washed with phosphate-buffered saline (PBS) and air-dried. 30 μl of 25% hybridization buffer containing the FISH probe was added dropwise onto the dried sections, and the slides were placed in a dark and humid environment and incubated overnight at 37 °C. Subsequently, the slides were placed in pre-warmed wash buffer and incubated at 37 °C for 15 minutes, and then air-dried for 20 minutes. Under light-proof conditions, 20 μl of DAPI anti-fading solution was added dropwise, a coverslip was covered, and incubated for 10 minutes. Finally, a fluorescence microscope (Leica DM6) was used for observation.

[0061] 1.14 Immunohistochemistry

[0062] Immunohistochemistry (IHC) staining was performed according to the previously reported method. Briefly, antigen retrieval was carried out in a pressure cooker using a target antigen retrieval solution (Tris-EDTA buffer, pH 9.0 or citrate buffer, pH 6.0) for 5 - 10 minutes. Subsequently, tissue sections were blocked with 3% hydrogen peroxide (H2O2) and 5% bovine serum albumin (BSA), and then incubated overnight at 4°C with antibodies against ZO-1 (1:200), Claudin4 (1:4000), LPS (1:1000), LTA (1:400), ESR1 (1:100), Ki67 (1:150), CD31 (undiluted), CD4 (1:300), CD8 (1:1000), CD68 (1:200), and Ly6G (1:100) in a humidified sealed environment. Subsequently, detection was performed using a DAB detection kit (PV-6001, PV-6002, and PV-9004, Zsgb-Bio, Beijing, China) according to the kit instructions. After hematoxylin staining and mounting with neutral resin, images were acquired using a microscope (Leica DM2500). H score analysis was performed using the IHC Profiler plugin in ImageJ software. CD31 + Microvessels were quantified by the particle analysis function of ImageJ software, and the size threshold was set from 100 pixels to infinity.

[0063] 1.15 Isolation of endometrial cells

[0064] Fresh endometrial tissues were obtained from women who underwent hysterectomy for the treatment of uterine fibroids (without endometriosis), and primary endometrial epithelial cells (EECs) and stromal cells (ESCs) were isolated. The brief steps were as follows: After washing the tissue fragments, they were cut into small pieces with scissors and then digested in DMEM / F12 medium (Gibco, CA, USA) containing 1 mg / mL type IV collagenase (Sigma, MO, USA) at 37°C for 35 minutes. The dispersed cells were filtered through a 400-mesh sieve. The cell filtrate was collected, centrifuged, and resuspended in DMEM / F-12 medium containing 10% fetal bovine serum (FBS). Enriched stromal cells (ESCs) were obtained by plastic adherent culture. To obtain endometrial epithelial cells (EECs), the cells rinsed back from the 400-mesh sieve were collected and cultured. The purity of EECs and ESCs was determined by immunofluorescence staining for vimentin, cytokeratin, and ESR1 markers.

[0065] 1.16 Isolation of neutrophils and NETs formation assay

[0066] Peripheral blood neutrophils from healthy donors (without endometriosis) were isolated using a neutrophil isolation kit (P9040, Solarbio, Beijing, China) according to the manufacturer's instructions. After obtaining written informed consent from the donors, blood was collected into EDTA anticoagulant tubes and processed at room temperature. The brief steps were as follows: Neutrophil isolation medium (4 mL of reagent A, followed by 2 mL of reagent C) was added to a 15 mL conical tube, and then the peripheral blood layer was carefully layered on top of the lower layer medium to form a clear interface. Centrifuge at 1200×g for 30 minutes at room temperature. The neutrophil layer was transferred to a new 15 mL conical tube and washed with phosphate-buffered saline (PBS). The supernatant was discarded after centrifugation, and the pellet contained neutrophils and a small amount of red blood cells (RBC). Lysis buffer was added to lyse the remaining red blood cells. After 2 - 3 wash - centrifuge cycles, the pellet mainly consisted of neutrophils. Freshly isolated neutrophils were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) for subsequent experiments. The purity of neutrophils was determined by immunofluorescence staining for myeloperoxidase (MPO) and CD16b markers.

[0067] Preparation of purified neutrophil extracellular traps (NETs). The brief steps were as follows: Freshly isolated neutrophils were stimulated with phorbol 12-myristate 13-acetate (PMA, 100 nmol / L; 10008014; Cayman Chemical; Michigan, USA) or lipopolysaccharide (LPS, 10 μg / mL; L2880; Sigma; Shanghai, China) for 4 hours to induce NETs formation. The bottom adherent layer containing NETs and neutrophils was collected and centrifuged at 450×g for 10 minutes at 4°C. Then, the cell-free, NETs-rich supernatant was obtained and centrifuged at 18000×g for 10 minutes at 4°C. The supernatant was discarded, and the precipitated NETs were resuspended in ice-cold phosphate-buffered saline (PBS). Subsequently, the NET-DNA concentration was measured and adjusted to 100 μg / mL, and the prepared NETs were stored at -80°C for future experiments.

[0068] 1.17 'NETs-Endometrial cell' capture experiment

[0069] A "NETs-Endometrial cell" capture experiment was designed to explore the interaction between neutrophil extracellular traps (NETs) and endometrial cells. First, a coverslip was placed at the bottom of a 24-well plate. Then, 1×10 5 freshly isolated neutrophils and lipopolysaccharide (LPS) were added to induce NETosis. After 2 hours, NETs would adhere to the coverslip. The culture medium was gently aspirated, and then 1×104 Primary endometrial cells. After incubation in a cell culture incubator at 37 °C for 1 hour, floating cells were removed from the supernatant. The coverslips were taken out for fixation and immunofluorescence staining. F-actin was labeled with Phalloidin-GFP. NETs and endometrial cells were labeled with anti-citH3 and anti-ESR1 antibodies, respectively. Fluorescence co-localization analysis was performed using ImageJ software.

[0070] 1.18 Transwell migration assay

[0071] Transwell migration assay. Briefly, 5×10 4 Endometrial epithelial cells (EECs) or endometrial stromal cells (ESCs) were seeded into the upper chamber, and 800 μL of medium containing 10% fetal bovine serum (FBS) was added to the lower chamber. After 8 hours of incubation, the cells attached to the upper surface of the membrane were gently wiped off with a cotton swab. The cells that migrated to the lower surface of the membrane were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Images were taken under white light using a microscope (EVOS M5000, Thermo Fisher Scientific, USA), and the number of cells in 10 fields of view was counted.

[0072] 1.19 Cell proliferation assay

[0073] Cell proliferation was determined by colony formation assay and cell counting kit-8 (CCK-8) assay, and the methods were referred to the previously reported ones. For the colony formation assay, endometrial epithelial cells (EECs) or stromal cells (SCs) were seeded into 6-well plates at a density of 500 cells per well and cultured for 1 week. Subsequently, the colonies were fixed with 4% paraformaldehyde solution and stained with 0.2% crystal violet at room temperature for 15 minutes. The cell colonies were counted and images were taken. For the CCK-8 assay, endometrial epithelial cells (EECs) or stromal cells (SCs) were seeded into 96-well plates at a density of 1000 cells per well. According to the manufacturer's instructions (CK04-5000, DOJINDO, Japan), the optical density at 450 nm was measured daily for 7 days (days 0-6). 10 μL of CCK-8 solution was added to each well, and the absorbance was measured after incubation in a humidified incubator for 2 hours.

[0074] 1.20 Establishment of an endometriosis model with human endometrium in NCG mice

[0075] In this study, a xenograft model of endometriosis in NCG mice was used. Before intraperitoneal injection of endometrial tissue, the mice were subjected to bilateral ovariectomy (OVX), and this day was designated as day 0. The mice were anesthetized with sodium pentobarbital (2%, 45 mg / kg, intraperitoneal injection). One week later (day 7), the mice were subcutaneously implanted with a pellet of 21-day sustained-release β-estradiol-17-acetate (E2) (0.1 mg per pellet, E271, Innovative Research of America, USA). Two weeks later (day 14), a xenograft model of endometriosis was established in NCG mice by intraperitoneal injection of human endometrial tissue (40 mg per mouse). Fresh endometrial tissue was obtained from women who underwent total hysterectomy due to uterine fibroids, and endometriosis was not detected in these tissues. From day 14 to day 28, 10 μg of NETs was intraperitoneally injected into each mouse daily. On day 35, the mice were euthanized, and the lesion sites were observed and fixed in 4% paraformaldehyde, followed by paraffin embedding to prepare paraffin blocks for subsequent analysis.

[0076] 1.21 Establishment of an endometriosis model with allogeneic endometrium of C57BL / 6 mice

[0077] To establish an autologous endometriosis model, endometrial tissue from C57BL / 6 donor mice was injected into the peritoneal cavity of recipient mice. All mice were subjected to bilateral ovariectomy (OVX), and this day was designated as day 0. In donor mice, 100 ng of E2 was intraperitoneally injected daily from day 7 to day 9, and then 5 ng of E2 was injected daily from day 13 to day 15. On day 13, all mice were subcutaneously implanted with a progesterone (P4, P0130, Sigma, USA) sustained-release capsule made of a silicone tube. Progesterone was dissolved in corn oil at a concentration of 1 g / mL. On day 19, 4 hours before endometrial transplantation, the P4 implant was removed. The endometrial tissue from one donor mouse was sufficient to be injected into two recipient mice. In recipient mice, a pellet of 60-day sustained-release β-estradiol-17-acetate (E2) (0.25 mg per pellet, SE-271, Innovative Research of America, USA) was subcutaneously implanted on day 7. In the NETosis inhibition model, GSK484 (at a concentration of 0.5 mg / mL, 200 μL) or DNase I (at a concentration of 1 mg / mL, 100 μL) was intraperitoneally injected daily from day 15 to day 29. LPS (at a concentration of 200 μg / mL, 50 μL) was intraperitoneally injected every other day from day 17 to day 25.

[0078] 1.22 Statistical analysis

[0079] All data were analyzed using SPSS Statistics 24.0 (IBM Corporation, Armonk, NY, USA) and presented as mean ± standard deviation (SD). The differences between the two groups were evaluated using Student’s t-test. For comparisons among multiple groups, the chi-square test was used. Pearson or Spearman correlation analysis was used to evaluate the relationship between two variables. Wilcoxon rank-sum test was used to identify differentially abundant microbial features. Statistical significance was defined as two-sided P < 0.05.

[0080] 2 Results

[0081] 2.1 Recruitment and activation of MME+ neutrophils (Neu_MME) in peritoneal fluid are associated with the development of endometriosis.

[0082] Immune cells were isolated from peritoneal fluid of 9 endometriosis patients and 3 disease-free control subjects, separated using CD45+ magnetic beads and subjected to single-cell RNA sequencing ( Figure 1 a). Based on the marker genes of immune cell clusters ( Figure 2 f), 8 distinct clusters were identified: B cells and plasma cells (B-Plasma), dendritic cells (DC), eosinophils and basophils (Eo-Basophil), macrophages and monocytes (Ma-Mono), mast cells, neutrophils, T cells and natural killer cells (T-NK), and proliferating cells ( Figure 1 b). Among these clusters, Ma-Mono was the major cluster, accounting for 53.7% in the control group and 67.5% in the endometriosis (EM) group ( Figure 1 b). Notably, the proportions of Ma-Mono and neutrophils were significantly higher in the EM group, while the proportions of T-NK and mast cells were significantly lower ( Figure 1 c-d, Figure 2 a-h). In addition, comparative analysis of EM patients with different revised American Society for Reproductive Medicine (rASRM) scores found that the proportion of neutrophils was significantly higher in rASRM III-IV stage patients ( Figure 1 e).

[0083] Subsequently, neutrophils were further divided into subsets ( Figure 2 f). 5 distinct subsets were identified: Neu_CD83, Neu_IFIT1, Neu_PTGS2, Neu_LCN2, and Neu_MME. Notably, the proportion of the Neu_MME subset was significantly increased in the endometriosis (EM) group ( Figure 1g-h). To verify these results, flow cytometry analysis was performed on 40 peritoneal fluid samples, 20 from EM patients and 20 from the control group. The results showed that the proportions of neutrophils and Neu_MME (CD45+CD11b+CD16b+MME+) in the EM group were significantly higher than those in the control group. In addition, the proportions of neutrophils and Neu_MME were also higher in patients with rASRM stage III-IV ( Figure 1 i, Figure 2 i), indicating that the increase in the Neu_MME subset in peritoneal fluid may be positively correlated with the progression of endometriosis.

[0084] According to the pseudotime analysis results of neutrophils, the cluster with the lowest CytoTRACE score (Neu_CD83) was designated as the starting point, while Neu_LCN2 and Neu_MME were identified as the endpoints of the trajectory ( Figure 1 j-k). Gene Ontology (GO) biological process and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that Neu_MME was significantly enriched in pathways including neutrophil degranulation, chemotaxis, response to lipopolysaccharide (LPS), neutrophil extracellular trap formation (NETosis), cytoskeleton regulation, and leukocyte transendothelial migration ( Figure 1 l), suggesting that Neu_MME represents a population of mature neutrophils and may undergo NETosis in response to LPS stimulation in endometriosis (EM). In summary, the recruitment and activation of Neu_MME in peritoneal fluid are related to the occurrence and development of endometriosis and may be affected by bacterial components in the peritoneal microenvironment.

[0085] 2.2 Neutrophil extracellular traps (NETs) are mainly generated by the LPS of bacteria inducing the Neu_MME subset

[0086] In EM patients, the levels of myeloperoxidase (MPO) and histone H3 citrullination (citH3) in peritoneal fluid cells were significantly higher than those in the control group, indicating the presence of activated neutrophils and NETs ( Figure 3 a). The level of citH3 in peritoneal fluid of EM patients was also significantly higher than that in the control group ( Figure 3 b). The co-expression of MME, CD16b, and citH3 in peritoneal fluid cells ( Figure 3 c) was verified in leukocytes treated with LPS and isolated neutrophils in vitro ( Figure 4 a). These findings suggest that the Neu_MME subset may serve as the main source of NETs in this context.

[0087] Neutrophils exposed to peritoneal fluid (PF_SN) from patients with endometriosis (EM) showed significantly higher citH3 expression levels than those in the control group, suggesting the presence of pro-inflammatory stimulating components in EM peritoneal fluid ( Figure 3 d). In addition, the LPS levels in the peritoneal fluid and the LBP levels in the serum of EM patients were also significantly higher than those in the control group ( Figure 3 e). The proportion of Neu_MME in neutrophils treated with LPS was significantly increased compared with that in the control group, highlighting LPS as a strong inducer of neutrophil activation and NETosis ( Figure 3 f). In addition, by comparing patients with endometriosis (EM) and the control group and analyzing the differentially expressed genes (DEGs) in Neu_MME, it was found that FOSB, FN1, and CSTL were significantly upregulated in the EM group ( Figure 4 b). In in vitro experiments, neutrophils were treated with LPS, and it was found that TLR4, TNF-α, PADI4, MME, FOSB, and citH3 were significantly upregulated, further supporting their involvement in NETosis ( Figure 3 g). Taken together, these findings suggest that NETs derived from Neu_MME are significantly increased in EM peritoneal fluid, with LPS as a key inducer.

[0088] In ectopic endometrial lesions, the expressions of MPO and citH3 were significantly higher than those in normal or eutopic endometrium ( Figure 3 h). ROC curve analysis showed that MPO and citH3 had high diagnostic accuracy, with AUC values of 0.924 and 0.935, respectively, suggesting the potential of NETs as diagnostic biomarkers for endometriosis lesions ( Figure 3 i). In addition, higher levels of myeloperoxidase (MPO) and citrullinated histone H3 (citH3) were observed in estrogen receptor 1 (ESR1)-positive regions. ESR1 is a marker of endometrial glandular cells in endometriosis. These findings suggest that the recruitment and activation of neutrophils mainly occur in the glandular cell layer close to endometriosis lesions, implying that NETosis and NETs may play a role in the pathogenesis and progression of endometriosis (EM) ( Figure 3 j).

[0089] Multiple indicators were detected by using consecutive sections. The 16S rRNA of bacteria was detected by using the EUB338 probe, and LPS and LTA were detected by immunohistochemistry. The results showed that positive signals of EUB338 and LPS were observed in EM lesions, while the LTA levels were low or undetectable ( Figure 3 k). The levels of MPO, citH3, EUB338, and LPS in patients with rASRM stage III-IV were significantly higher than those in patients with stage I-II.Figure 3 l). Correlation analysis showed that there was a significant positive correlation between MPO, citH3, EUB338, LPS and the rASRM score ( Figure 3 m,n, Figure 4 c). These findings together suggest that NETs released by Neu_MME in EM ascites and lesions are induced by bacterial LPS.

[0090] 2.3 NETs promote the development of endometriosis by capturing endometrial cells and enhancing their proliferation and migration.

[0091] In the ascites of endometriosis patients, a polymerized cell mass composed of neutrophils, NETs and endometrial cells was found, characterized by the co-expression of markers such as MPO, citH3 and ESR1 in these cell masses ( Figure 5 a), suggesting that there may be an interaction between NETs and free peritoneal endometrial cells. To study the interaction between NETs and endometrial cells, we designed a "NETs-endometrial cell" capture experiment. First, we successfully isolated neutrophils and primary endometrial cells (including endometrial glandular cells and endometrial stromal cells), and confirmed the characteristics and types of cells through markers ( Figure 6 ). Fluorescence co-localization analysis showed that there was a significant overlap of F-actin, ESR1 and citH3 signals, which supported the hypothesis that NETs contribute to the capture of endometrial cells ( Figure 5 b).

[0092] In vitro experiments showed that NETs enhanced the migration and proliferation abilities of endometrial epithelial cells (EECs) and endometrial stromal cells (ESCs). This effect was eliminated by treatment with DNase I, which specifically degrades NETs ( Figure 5 c-e). In an endometriosis model using immunodeficient NCG mice, intraperitoneal injection of NETs significantly increased the lesion volume ( Figure 7 a,b). These findings highlight the key role of NETs in capturing endometrial cells, promoting their migration and proliferation, and promoting lesion formation.

[0093] To further explore the role of NETosis in endometriosis, we intraperitoneally injected C57BL / 6 mice with LPS, GSK484 (a NETosis inhibitor), DNase I or PBS ( Figure 5 f). LPS increased the size of the lesions and the thickness of the stroma. In contrast, inhibition of NETosis by GSK484 or clearance of NETs by DNase I significantly inhibited the size of the lesions and the thickness of the stroma ( Figure 5g, i, j). LPS also promoted cell proliferation (Ki67 expression) and angiogenesis (formation of CD31 - positive capillaries). These effects were attenuated by inhibiting NETosis and eliminating NETs ( Figure 5 h, k, l, Figure 7 c). Markers of neutrophil infiltration and NETosis (Ly6G and citH3) in the lesions increased in the LPS - treated group. However, by inhibiting NETosis and enzymatically eliminating NETs, the neutrophil infiltration and NET levels in the lesions were significantly reduced ( Figure 5 m - p). In peritoneal fluid, LPS increased neutrophil recruitment and the proportion of the Neu_MME subset, while GSK484 and DNaseI significantly reduced neutrophil recruitment and the proportion of the Neu_MME subset ( Figure 5 q, Figure 7 d). These findings suggest that intraperitoneal LPS - induced neutrophil infiltration and NETosis promote cell proliferation, angiogenesis, and lesion development. Targeted blockade of NETosis or degradation of NETs may be an effective approach for treating endometriosis.

[0094] 3 Discussion

[0095] Recently, the association between gut, genital tract, and peritoneal fluid microbiota and the pathogenesis of endometriosis has become a research focus. However, the regulatory mechanism of the interaction between peritoneal microbiota and neutrophils in endometriosis remains unclear. Based on clinical sample detection and analysis, cell function experiments, and validation in animal models, we found that Neu_MME in peritoneal fluid was induced by bacterial lipopolysaccharide (LPS) to undergo NETosis, thus promoting cell proliferation, angiogenesis, and lesion development. This study provides new insights into understanding the role of peritoneal microbiota in the pathogenesis of endometriosis and offers a new perspective for formulating prevention and treatment strategies.

[0096] The peritoneal immune microenvironment of patients with endometriosis (EM) exhibits unique inflammatory characteristics, acting as a "pro - endometriosis niche". Early studies preliminarily revealed the composition and activation status of immune cells in peritoneal fluid of endometriosis patients by mass cytometry. Recently, advances in single - cell sequencing technology have further elucidated the heterogeneity of immune cell populations in peritoneal fluid and endometriosis lesions, revealing the dynamics of immune cells and their potential roles in disease progression. However, the role of neutrophils in this process remains unclear.

[0097] In this study, macrophages remained the major cell population. However, we observed a significant increase in the number of neutrophils recruited in the peritoneal fluid of endometriosis patients, especially those with rASRM III-IV stage. Neutrophils, as key participants in innate immunity, are associated with the pathogenesis of endometriosis. Previous studies have shown that neutrophil infiltration is an early event in the formation of endometriosis lesions. Similarly, our data confirmed that neutrophil depletion effectively inhibited the development of lesions. We further characterized neutrophil subsets and found a new subset, Neu_MME. Functional analysis of this subset showed its involvement in the response to bacterial infection, LPS signaling, and NETosis. In addition, our additional data indicated that Neu_MME induced by bacterial LPS was the main source of NETs in the peritoneal fluid and lesions of endometriosis (EM) patients.

[0098] NETs have received attention due to their roles in various pathological conditions, such as cancer and inflammatory diseases. NETs may potentially regulate the tumor microenvironment by capturing and influencing metastatic cancer cells, which may have similar implications in the pathology of endometriosis. Although some previous studies have mentioned the potential role of NETs in endometriosis (EM), its regulatory mechanism has not been verified. We found that NETs were enriched in the regions with high ESR1 expression in endometriosis (EM) lesions and formed "neutrophil-NETs-endometrial cell" aggregates in the peritoneal cavity. We developed an experimental method to detect the capture of endometrial cells by NETs and confirmed that NETs could capture endometrial cells, promoting their proliferation and migration. In addition, the key roles of neutrophils and NETs were verified in NCG and C57BL / 6 mouse models. Our research results showed that inhibiting NETosis or clearing NETs could effectively inhibit the progression of endometriosis, suggesting new therapeutic targets. In addition, NETs are composed of DNA wrapped with histones and proteases. Our data support the view that NET-DNA is the main active component of NETs. Therefore, blocking or inhibiting the downstream signaling pathways related to NETs may be a potential therapeutic strategy. CCDC25 has been identified as the first receptor proven to bind to NET-DNA.

[0099] In summary, our research results showed that NETs released by MME-positive neutrophils (Neu_MME) induced by intraperitoneal bacteria-derived LPS played a key role in the progression of endometriosis, mainly by capturing endometrial cells to promote cell adhesion, colonization, and enhancing their proliferation and migration abilities, thus promoting the occurrence and development of lesions. These results indicated that targeted blocking of NETosis or clearing of NETs may provide potential therapeutic strategies to prevent the progression of endometriosis.

[0100] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the method of the present invention, several improvements and supplements can be made, and these improvements and supplements should also be regarded as the protection scope of the present invention.

Claims

1. Use of NETs as biomarkers in the preparation of a diagnostic kit for endometriosis.

2. Use of a reagent for detecting the content of NETs in the preparation of a diagnostic kit for endometriosis.

3. The application according to claim 1 or 2, characterized in that, The NETs referred to are those released from the Neu_MME subset.

4. The application according to claim 1 or 2, characterized in that, The sample to be detected is the polymer in peritoneal effusion, and the polymer is composed of neutrophils, NETs and endometrial cells.

5. Use of the Neu_MME subset in the preparation of a diagnostic kit for evaluating the degree of NETosis or the severity of endometriosis.

6. Use of a NETosis inhibitor or DNase I in the preparation of a drug for treating endometriosis.

7. The application according to claim 6, wherein The NETosis inhibitor is GSK484.

8. The application according to claim 6, wherein The endometriosis is endometriosis induced by NETs released by NETosis.

9. The application according to claim 6, wherein Endometriosis is endometriosis induced by LPS derived from abdominal cavity bacteria-induced NETosis.

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