Extracellular Succinate Derived From Ectopic Milieu Drives Adhesion and Implantation Growth of Endometrial stromal cells via the SUCNR1 signal in endometriosis

Extracellular Succinate Derived From Ectopic Milieu Drives Adhesion and Implantation Growth of Endometrial stromal cells via the SUCNR1 signal in endometriosis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Extracellular Succinate Derived From Ectopic Milieu Drives Adhesion and Implantation Growth of Endometrial stromal cells via the SUCNR1 signal in endometriosis Qi Tian, JingYao Ruan, Yuning Wang, Yinping Xiao, Qi Cheng, Yun Chen, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3303001/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 30 Jan, 2024 Read the published version in Cell Communication and Signaling → Version 1 posted 7 You are reading this latest preprint version Abstract 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. Meanwhile, extracellular succinate released into the microenvironment is considered an inflammatory alarm that can be sensed by its membrane receptor SUCNR1, boosts proinflammatory responses and acts akin to classical hormones and cytokines. Succinate has been reported to play an important role in inflammatory disease. It is worth exploring whether succinate can facilitate the progress of endometriosis (EMs), which is characterized by chronic inflammation and peritoneal adhesion. Objective To evaluate the main source and potential role of succinate in endometriosis, we mimics the ectopic milieu in vitro and in vivo . The molecular and functional effects of succinate on macrophages and peritoneal mesothelial cells in peritoneal cavity were assessed. The succinate/SUCNR1 signal acting 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 assay to assess whether there was an obvious accumulation of succinate in the peritoneal fluid of EMs patients and its correlated with disease severity, Visual Analogue Scale (VAS), and the Endometriosis Fertility Index (EFI). Flow cytometry, Enzyme linked immunosorbent assay (ELISA), western-blot assay, and quantitative real-time PCR, and other molecular biology techniques were used for exploring the potential mechanisms. Results By mimicking the ectopic milieu, we constructed an in vitro co-culture system and found that M1 polarized macrophages and the peritoneal mesothelial cell line (HMrSV5) mainly released succinate into their microenvironment and activated the succinate receptor (SUCNR1) signal, which further polarizes macrophages and significantly enhances the invasive survival of ESCs, and the adhesion with peritoneum. We further investigated the pathological effect of extracellular succinate in vivo using xenograft mouse models of endometriosis. Conclusions Collectively, the succinate-SUCNR1 signal facilitates in creating the inflammatory nice and plays a vital role in EMs progression and peritoneal adhesion. Our work on the molecular mechanism of succinate accumulation and function will be helpful to elucidate the phenotypic mystery of pain and infertility in EMs. Endometriosis Succinate Peritoneal Mesothelial cell Endometrial stromal cells (ESCs) Macrophage SUCNR1 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction Endometriosis (EMs) is a heterogeneous clinical syndrome characterized by a chronic inflammatory process that is strongly linked to peritoneal adhesion, infertility, dysmenorrhea, and chronic pelvic pain ( 3 ). Endometrial stromal cells (ESCs) and epithelial cells within the retrograde menstrual endometrium commonly attached to the pelvic peritoneum. Once an ectopic lesion is formed, ectopic tissue with periodic bleeding is exposed to immune surveillance, leading to chronic inflammation and repeated tissue repair. The presence of cytokines and shifts in circulating immune cell populations creates a widespread inflammatory environment and peritoneal adhesions (PA) extending outside the pelvis ( 4 ). Monolayers of peritoneal-mesothelial cells and macrophages form major cell populations in the peritoneal fluid, which may play a central role in lesion establishment and maintenance by driving chronic inflammation and tissue remodeling. Although the pathogenesis of EMs remains unclear, genetics and the microenvironment are its key drivers ( 5 ). Succinate occupies an extremely vital position in the metabolism because of its direct connection to the Krebs cycle and the mitochondrial respiratory chain ( 1 ) ( 6 ). The final stage of the Krebs cycle involves the regeneration of oxaloacetate, involves a process in which succinate is oxidated to fumarate via succinate dehydrogenase (SDH) ( 7 ). Dynamic changes in SDH under physiological or pathological metabolic conditions are associated with succinate accumulation ( 8 , 9 ). Over the past 10 years, the new roles of extracellular succinate have expanded beyond metabolism and into signaling. Beyond its metabolic role in conditions of stress and damage, an increasing body of evidence points to additional immunological functions, especially in subacute inflammatory conditions, such as Inflammatory Bowel Disease (IBD), Crohn’s disease(CD) ( 10 , 11 ), gestational diabetes ( 12 ), and non-alcoholic fatty liver disease (NAFLD) ( 13 ). Succinate accumulation could be followed by succinate release from cells, which would then act on other cell types via the SUCNR1, driving inflammation or type 2 immunity ( 6 ).These multiple functions indicate that succinate plays an extensive role in cellular activation and inflammation. Succinate triggers macrophage polarization and subsequent inflammation. However, the role of succinate-SUCNR1 signal in EMs, which is characterized by chronic inflammation, remains unclear. On this basis, our study hypothesized that stimulation with cytokines, such as IL-6, or contact with endometrial stromal cells during retrograde menstruation triggers succinate release from peritoneal mesothelial cells and macrophages. Extracellular succinate polarizes macrophages into the M1-like type and continuously recruits macrophages via CCL2 secretion from peritoneal mesothelial cells. Thus, the crosstalk between these cells leads to massive succinate accumulation and an inflammatory microenvironment. Eventually, accumulated succinate enhances the survival, adhesion and deep infiltration of ESCs via SUCNR1 signaling, leading to the acceleration of EMs progress. 2. Materials and Methods 2.1 Patients and Tissues collection Premenopausal women diagnosed with EMs or benign gynecological diseases (such as fibroids and benign teratomas) underwent laparoscopic surgery at the Obstetrics and Gynecology Hospital of Fudan University between January 2020 and December 2022. Normal endometrial samples were obtained from 6 patients with EMs who underwent combined laparoscopy and hysteroscopy for tubal infertility. EMs was diagnosed based on clinical symptoms and imaging findings. Symptoms related to EMs include pelvic masses, chronic pelvic pain, dysmenorrhea, dyspareunia, infertility, and cyclical alterations in bowel and urinary habits that occur only during menstruation. A total of 2–10 ml of undiluted peritoneal fluid was drawn at the beginning of the laparoscopy, and biopsies from ectopic lesions were obtained from each patient. Finally, according to the rAFS (ASRM, 1997), patients with pathologically confirmed EMs were grouped according to disease stage(stages I-II, n = 12; stages III-IV, n = 24). Thirty, patients without EMs were included in the control group, and peritoneal fluid was collected during laparoscopic surgery. The enrolled patients were free of hormonal medication for at least 6 months. Patients with acute and subacute inflammatory diseases, autoimmune disorders, pregnancy, or malignant tumors were excluded from this study. The clinical and demographic characteristics of all the participants are shown in Supplementary Table 1. 2.2 Peritoneal Fluid Isolation Peritoneal fluid was placed on ice at acquisition and then transferred to the laboratory within 30 minutes for further experiments. Fresh peritoneal fluid was centrifuged at 1500 rpm(4℃) for 5 minutes. The pellet was resuspended and the erythrocyte lysis solution (1×) added according to the instructions. After centrifugation (1000 rpm) for three times, the fresh cell pellets were immediately prepared for flow cytometry. The supernatant of the PF cells and debris was packaged in 1.5mL centrifugal tubes and store in a refrigerator at − 80°C until the metabolomics detection and ELISA analysis. 2.3 Targeting Organic Acid Metabolomics Analysis Shanghai Lu-Ming Biotech Company Limited (Shanghai, China) provided an experimental platform and assistance for the target organic acid metabolomics analysis. Briefly, a mixture of acetonitrile and methanol (2:1, V/V, containing seven isotopes internal standards) was used to collect 0.1ml per sample. After shaking and centrifugation, 100µl of supernatant per sample was freeze-dried. Finally, a mixture of BSTFA and n-hexane (4:1 by volume) was added to the sample, vortexed vigorously for 2 min, and derivatized at 70° C for 60 min. The samples were analyzed using a gas chromatography system Trace1310 coupled to a TSQ9000 Mass spectrometer equipped with an Electron ionization (EI) source (Thermo Fisher Scientific, USA). The raw data exported by UPLC-MS/MS were processed using the QuanMET software (v1.0, Metabo-Profile, Shanghai, China). The concentrations and peak areas of the standards were used to construct a standard curve and calculate the sample concentration. The calculated concentrations of bile acids in all samples were imported into the SIMCA-P + software (v. 14.1, Umetrics, Sweden) for multivariate analysis, including principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA). An independent sample non-parametric test judgment was used to test for significant differences between the groups (P < 0.05) and the variables of importance (VIP) values in the OPLS-DA model were used to identify potential biomarkers. 2.4 Cell Culture and Treatments Primary Human Endometrial stromal cells(hESCs) from the endometrium of patients with or without EMs were isolated using collagenase digestion, as described previously( 21 ), and cultured in DMEM F12 supplemented with 10% fetal bovine serum (FBS) for FCM analysis. The cell line used in this study, ESCs, was a gifted from Professor Ming-qing Li of the Reproductive Immunology Laboratory of Obstetrics and Gynecology Hospital, Fudan University. ESCs were cultured in DMEM F12(10% FBS) for the co-culture system and other assays. HMrSV5 and THP-1 cells were obtained from the National Collection of Authenticated Cell Cultures of China. HMrSV5 cells cultured in DMEM F12(10% FBS) and THP-1 cells cultured in RPMI-1640(10%FBS) were incubated with or without various concentrations of drugs for the predefined times before each experiment, according to the cell experiment protocol. For macrophage polarization, THP-1 cells were differentiated and polarized by using 100 ng/ml phorbol 12-myristate13-acetate (PMA; Sigma-Aldrich) for 48 h to obtain M0, and M0 were transformed into M1 through LPS (Peprotech) (100ng/ml) and IFN-γ (PeproTech)(20ng/ml) stimulation or M2 through IL-4 (PeproTech) (20ng/ml) and IL-13 (PeproTech) (20ng/ml) stimulation for 48h. FCM was performed to verify successful induction via CD80, CD86, CD163, and CD206. Non-adherent macrophages were cleaned using phosphate-buffered saline (PBS), and adherent cells were cultured in fresh RPMI-1640 medium. The details of cytokines used in this study are as follows: IFN-γ Peprotech Cat#300-02 lot#091927; LPS Peprotech Cat#M9524; IL-4 Peprotech Cat#200-04 lot#051914; IL-10 Peprotech Cat#200 − 10 lot#11021; IL-6 Peprotech Cat#200-06; IL-13 Peprotech Cat#200 − 13 lot#102123. 2.5 Cell Viability Assays The cell viability was measured using the CCK-8 assay. ESCs or HMrSV5 cells (four replicates per group) were seeded in 96-well plates (Corning) with 100ml medium (10% FBS) and then incubated at 37℃ overnight. The cell supernatants were removed after stimulation with succinate for48h. Into each well the CCK-8 solution (10 µl) and culture medium(100 µl) were added. After incubation for another 1h at 37℃, the plates were measured via a microplate reader at an absorbance of 450 nm (Bio-Rad 680, Bio-Rad, USA). 2.6 Apoptosis Assays For apoptosis assay, ESCs or HMrSV5 cells were respectively seeded in 24-well plates and cultured with LPS or succinate for 48 h. Cells were then co-stained with Annexin V-PE (BD Pharmingen, Heidelberg, Germany) and 7AAD. Flow cytometer (Beckman) was performed to obtain data on apoptosis, and analyzed using the FlowJo software. 2.7 Scratch Wound Assay Human ESCs (1×10 5 cells/well, four replicates per group) were seeded into a 12-well plate. After reaching confluence, the cells were scratched using a sterile tip to mimic the shape of a wound. An FBS-free medium was used to wash and remove loose cells. ESCs were then treated with succinate (0, 1, 2.5, or 5 mM) and photographed at 0, 24, and 48 h by using a light microscope. The closure area of wound was calculated as follows: Wound Closure (%) = ((Primary wound size-Final wound size)*100%/ Primary wound size. 2.8 Matrigel Invasion, Chemotaxis and Adhesion Assays, For the transwell assay, ESCs or HMrSV5 (1×10 4 cells/well, three replicates per group) were seeded into the upper chamber of 24-well transwell plates (8 µm pore filters) (Corning, USA). The lower chamber was supplemented with medium (10% FBS) with or without succinate (0, 1, 2.5, 5mM) or CCL-2 (Abclone Cat#RP01411) (100ng/ml). After 48 h, the migrated cells on the lower surface were stained and observed under a microscope. For chemotaxis assay, ESCs (1×10 4 cells/well, three replicates per group) were seeded in the upper chamber of 24-well transwell plates(8µm pore filters) (Corning, USA), and HMrSV5 cells (1×10 4 cells/well) stimulated with succinate (0, 2.5mM) or CCL2 (100 ng/ml) were put in lower chamber. CCL2 treatment was used as the positive control. After 48 hours, ESCs were replaced with THP-1 cells which were pre-labeled with CellTracker red (DiO, Beyotime, C1995S, China), and then continuously incubated for another 12 hours with 3 µm pore filters. Finally, the traced THP-1 cells were collected and calculated as cell number per field (scale bar-100 µm) (red tracer staining THP-1 cell). For the adhesion assay, HMrSV5 cells (2×10 5 cells/well, three replicates per group) were treated with succinate(0, 1, 2.5, 5mM) and seeded in 6-well plates. ESCs(1×10 4 cells/well, three replicates per group) pre-labeled with CellTracker green (DiO, Beyotime, C1993S, China) were further seeded into each HMrSV5-well. Finally, the traced ESCs were calculated as cell number per field by using a fluorescence microscope (scale bar-200µm) (green tracer staining ESCs). 2.9 Flow cytometry Peritoneal cell pellets collected from the PE were suspended in PBS and stained with the following antibodies: anti-human CD14 -PerCy5.5 (BioLegend,325621), anti-human CD45 APC-Cy7 (BioLegend, 368515) and anti-human GPR91/SUCNR1 FITC (Alomone, ASR-090-F). After staining for half an hour, the cells were washed and prepared for FCM (Beckman Coulter). Data were analyzed using the FlowJo(v10) software. Flow cytometry was also performed to analyze the expression of CD80, CD86, CD163, and CD206 in macrophages in vivo or in THP-1 cells in vitro , as well as SUCNR1 levels in ESCs. The FCM antibodies used were as follows: anti-human CD86 Percp/cy5.5 (BioLegend, 305419); anti-human/Mouse GPR91/SUCNR1 FITC (Alomone, ASR-090-F); anti-mouse CD45 percp (BioLegend, 103129); anti-human/mouse GPR91/SUCNR1 FITC (Alomone, ASR-090-F); anti-ouse CD80 PE (BioLegend, 104707), anti-mouse CD86 ALexa fluor 700 (BioLegend, 105024); anti-mouse CD163 BV421 (BioLegend, 155309); anti-mouse CD206 BV605 (BioLegend, 141721), anti-mouse CD11b PCy 7(BioLegend, 101215); anti-mouse F4/80 APC (BioLegend, 123116). 2.10 ELISA Assay The level of succinate in the PF or cell supernatants were measured using ELISA assay. Briefly, HMrSV5 cells, ESCs, and macrophages polarized from THP-1 cells were seeded into 6-well plates. After culturing for 48 h, the cells were treated with BMDM alone, IL-6 (100 ng/ml) or CCL2 (50 ng/ml) for another 48 h. The cell supernatant was centrifuged at 1000 rpm (4° C) for 10 minutes. Peritoneal fluid was collected as described above. PF was put on ice at acquisition and then was centrifuged twice at 1500 rpm for 5 minutes at 4° C to remove the cells. All the samples were diluted twice and analyzed according to the specifications of the ELISA kit (Abcam, ab204718). 2.11 Quantitative Real-time PCR (RT-qPCR) Analysis Total RNA was extracted via RNA Purification Kit (EZBioscience, USA) and reverse-transcribed into cDNA by using Hifair®II 1st Strand cDNA Synthesis SuperMix for qPCR (Yeasen, Shanghai, China). The quantitative PCR (RT-qPCR) was performed according to the protocol (Hieff UNICON® Universal Blue qPCR SYBR Green Master Mix, Yeasen). Data analysis was repeated three times and analyzed using 2 −ΔΔ Ct method. The primer sequences used in this study are listed in Supplementar y Table 2. 2.12 Immunohistochemistry (IHC) Immunohistochemistry for SDHB was performed according to the manufacturer’s instructions. In brief, after dewaxing and antigen repair, the primary antibody for SDHB (Abcam, ab178423) was incubated overnight(4° C) at a 1:150 dilution. Subsequently, the membranes were incubated with the secondary antibody at room temperature for half an hour. Sections were stained with hematoxylin and photographed under a microscope. 2.13 Western Blotting Assay HMrSV5 cells (2×10 5 cells/well) were seeded in 6-well plates and treated with different concentrations of succinate (0, 0.5, 1, 2.5 and 5 mM) for 48 h. Proteins were extracted by cell lysis. The protein sample (15 µg/lane) was evaluated with electrophoresis and transfected into 0.45 µm polyvinylidene fluoride (PVDF) which was stained with ECL after incubating with the primary antibody anti-ICAM1 (Abcam, ab53013) (1:1000) at 4° C overnight and with a secondary antibody(1:5000) at 24°C for 2 h. The total gray scale of each strip was quantified using ImageJ software with the values normalized based on housekeeping proteins (i.e., β -actin). 2.14 Mouse Model of EMs Thirty adult C57BL/6 female mice (6–8 weeks, weight 20 ± 2g) were purchased from the Laboratory Animal Facility of Fudan University and used for this study. The animal protocols were approved by the Ethics Committee of the Obstetrics and Gynecology Hospital, Fudan University. All the mice were randomly assigned to one of the three groups. Intraperitoneal EMs-like lesions were surgically induced by injecting fragments of uterine tissue into the peritoneal cavity. 17-β-Estradiol-3-benzoate (30 \(\mu\) g/kg, Sigma) was administered to each postoperative mouse every 3 days for 14 days. Three days after surgery, each mouse in the experimental group received succinate (100 mg/kg, Sigma) intraperitoneally every 3 days for 14 days. PBS was used instead of succinate for the sham-operated group. In the control group, no surgery was performed and PBS was used instead of succinate. Fourteen days after the operation, endometrial-like lesions were established, the mice were sacrificed, and peritoneal lavage fluids and ectopic lesions were harvested. SUCNR1 and M1/M2 macrophage markers were measured and analyzed via FCM. MMP9 and ICAM-1 of lesions were detected via IHC. 3. Results 3.1 Succinate Accumulation in Peritoneal Fluid and Clinical Relevance in EMs In order to assess the level of metabolites in the peritoneal fluid(PF), we applied targeted organic acid metabolomic analysis to study the differences in organic acid profiles in healthy and EMs subjects. The results showed significantly difference in the peritoneal fluid between EMs and non-EMs (Fig. 1 A). As found by assessing metabolomics results of PF, four organic acid metabolites, including 5-hydroxymethy-2-furancarbosylic acid, 2-hydroxyhippuric acid, succinic acid, and 2-hydroxy-3-methylpentanoic acid, were clearly elevated in EMs patients (Fig. 1 B-D). Specifically, succinate was significantly increased in EMs patients (328.65 ± 105.4ng/ml) in comparison with those in the non-EMs group (244.27 ± 43.76 ng/ml), which corresponded with the ELISA results (Supplementary Fig. 1A) . Succinate dehydrogenase (SDH), with unique characteristic of oxidation of succinate to fumarate, is a classic mitochondrial enzyme. Immunohistochemical staining revealed a decreased expression of SDHB in ectopic lesions when compared to that in the normal endometrium (Supplementary Fig. 1B) . Metabolic pathway enrichment analysis showed that pathways were differentially regulated between EMs and non-EMs, oxidative phosphorylation and citrate cycle (TCA cycle) pathways were significantly upregulated in EMs group. ( Fig. 1 E). Succinate is an important intracellular metabolic intermediate. Although normally regarded as an intermediate, succinate accumulates under certain pathophysiological conditions, especially at the sites of inflammation and metabolic stress ( 1 , 6 , 10 , 14 – 16 ). Numerous studies have shown that succinate is not simply an inert byproduct of metabolism, but that it also plays an active role in downstream cellular responses and can have tissue-specific and systemic effects as a proinflammatory mediator ( 1 , 6 , 17 ). Similarly, we found that higher levels of succinate accumulate in the peritoneal fluid of patients with severe EMs (stages Ⅲ-Ⅳ) than in those with mild EMs (stages Ⅰ-Ⅱ)(Fig. 2 A). Therefore, we speculated that succinate levels may reflect disease severity. To some extent, succinate has potential clinical value in reflecting EM severity (AUC = 0.951) (Fig. 2 B). Furthermore, Pearson's correlation analysis was conducted on the EMs clinical data, and the results revealed a linear correlation between succinate and clinical symptoms/indicators, such as pain (VAS, R2 = 0.46, P < 0.001, 95% confidence interval: 0.15–0.68), EMs stage according to the revised American Fertility Society (rAFS,1985) score (rAFS, R2 = 0.38, P = 0.02, 95% confidence interval: 0.06–0.63), and fertility prediction after EMs surgical staging (EFI, R2 = -0.44, P < 0.01, 95% confidence interval: -0.67– -0.13)(Fig. 2 C-E). Supplementary Table 1 presents the clinical parameters of patients with EMs and non-EMs subjects. 3.2 Succinate is Prone to Polarize M1-Like Macrophages in the Endometriotic Milieu As succinate triggers inflammatory changes in macrophages, changes in gene expression triggered by succinate in M0 macrophages derived from THP-1 cells were compared with the marker gene expression observed in human M1 and M2 macrophages. By using a real-time quantitative polymerase chain reaction (PCR) analysis of macrophages exposed to succinate (0, 0.5, 1, 2.5, and 5 mM) for 48h, we compared the gene expression changes of M1 markers (CD80, CD86) with M2 markers (CD206, CD163) triggered in M0 macrophages by succinate. As illustrated in Supplementary Fig. 2A-E , in resting M0 macrophages without lipopolysaccharide (LPS) stimulation exposed to succinate, genes that were preferentially expressed by M1 macrophages were upregulated by succinate, and genes that were preferentially expressed in M2 macrophages were downregulated by succinate. Thus, succinate exposure causes polarization of naïve macrophages toward M1. As previously reported( 17 , 18 ), the effects of extracellular succinate and LPS can be superimposed, and can especially augment the LPS-driven M1 phenotype ( Supplementary Fig. 2C). Additionally, we found that succinate also markedly induced the proinflammatory cytokine IL-8, IL-1β, and IL-6 transcription in macrophages, however, a higher concentration of succinate (5 mM) created an opposite effect in IL-8 and IL-6 (Supplementary Fig. 2F-I). To evaluate the expression of SUCNR1 in M0 macrophages exposed to succinate, we chose to to detect the transcription of SUCNR1 via real-time PCR. The results showed a dose-dependent expression of SUCNR1 mRNA in M0 macrophages exposed to succinate as compared to that in the vehicle ( Supplementary Fig. 2J ). Succinate appears to activate inflammatory pathways and the switch to the M1-like phenotype, at least in part, via SUCNR1. These data demonstrate that exposing monocyte-derived macrophages to relevant concentrations of extracellular succinate unequivocally regulates the expression of immune function genes, resulting in the polarization of the M1-like phenotype or synergism with LPS. 3.3 Extracellular Succinate is Mainly Released From M1-Polarized Macrophages and Peritoneal Mesothelial Cells Macrophage were the most common population (~ 60%) of PF leukocytes in patients with EMs, and the peritoneal mesothelial cell (PMC) monolayer that lines the abdominal cavity is the first barrier encountered by menstrual fragments. To understand the capacity of extracellular succinate secretion among these cell types, THP-1 cells were respectively polarized into the M1 phenotype with LPS and IFNγ, or into the M2 phenotype with IL-4 and IL-13 as previously described ( 19 , 20 ). Macrophages in different polarizing states were cultured for 48 h, and the levels of extracellular succinate in the cell supernatant were measured by ELISA. Consistently with previous reports that M1 polarized macrophages are major producers of succinate, our study showed that the production of succinate in M1 polarized macrophages and peritoneal mesothelial cells was higher than that in naïve and M2-polarized macrophages (Fig. 3 A). To identify the main source of succinate in the endometriotic milieu, a co-culture model with ESC, peritoneal mesothelial cell line HMrSV5, or THP-1 cells was constructed to imitate the ectopic immune microenvironment of EMs. High levels of succinate were observed in the culture supernatants of M1-polarized macrophages after being co-cultured with HMrSV5 cells or ESCs (Fig. 3 B). As reported in our previous studies, some inflammatory cytokines (such as IL-6 and CCL-2) are significantly elevated in the peritoneal fluid of patients with EMs ( 21 ). Interestingly, co-culture of resting M0 cells with HMrSV5 cells or ESCs also triggered succinate release in macrophages, but not when stimulated with IL-6 or CCL2 (Fig. 3 C), showing that contact with ESCs or HMrSV5 cells, rather than the M0 phenotype, determines succinate secretion. In parallel, MCP-1 from the endometriotic milieu failed to induce succinate secretion in HMrSV5 cells; however, IL-6 stimulation or interaction with ESCs significantly increased succinate production (Fig. 3 D). Based on these findings, such changes in the ectopic milieu establish a vicious circle, with each condition promoting the other and accelerating succinate accumulation. 3.4 SUCNR1 is Elevated in Ectopic ESCs and Macrophages From EMs Patients To analyze the expression and distribution of SUCNR1 in human tissues, we found, via the Human Protein Atlas/Dataset (data available from proteinatlas.org), that SUCNR1 expression in normal female tissues was low under physiological homeostasis but high in their immune system and gastrointestinal tract (Fig. 4 A). To analyze whether the accumulation of succinate participates in EMs progression, we further measured SUCNR1 expression in CD45 + CD14 + macrophages, PMCs, and ESCs from patients with or without EMs via FCM. Serosal healing involves free-floating mesothelial cells ( 24 , 25 ), and our results showed the macroscopic shed mesothelial cells float in clumps in peritoneal fluid (Fig. 4 E), and found a 16-fold increase of SUCNR1 expression in mesothelial cell mass of EMs patients (Fig. 4 B-C). Macrophages from the PF of EMs patients underwent significant phenotypic changes, with a 1.8 to 3.9-fold increase of SUCNR1 expression in macrophages (Fig. 4 B, D), and a parallel 2-fold increase in SUCNR1 expression in ectopic ESCs (hESC.D) compared with ESC cell line (ESC.L) and primary normal ESCs (hESC.N) (Fig. 4 F-G). Accordingly, we evaluated the effect of succinate on SUCNR1 expression in HMrSV5 cells. Increasing the succinate concentrations induced a dose-dependent increase in SUCNR1 expression in HMrSV5 cells (Fig. 4 H-I), indicating an important role of succinate in mesothelial cell function during EMs lesion formation. Using this assay, the optimal concentration of 2.5mM was determined. 3.5 Succinate Enhances ESCs Survival and Implantation Capacity via the SUCNR1 Given the importance of extracellular succinate as an immunometabolic signal in the ectopic milieu, we investigated whether succinate released by type 1 proinflammatory macrophages could regulate the activity of surrounding PMCs in the peritoneal cavity, including that of refluxed and colonized ESCs. Considering that endometriotic lesion formation is a multi-step process that includes not only endometrial tissue proliferation but also antiapoptosis and invasion, we investigated whether succinate might acts as a chemoattractant for ESCs by using a transwell migration assay. The average number of ESCs per field that migrated toward succinate after 48 h was significantly higher than that in the vehicle. This invasive effect was dose-dependent and equivalent to that of CCL2 (Fig. 5 A). Next, we analyzed the migration of ESCs into the wounded cell-free areas using a scratch assay. Time-lapse imaging revealed the ability of succinate to promote wound healing in ESCs in a concentration dependent manner, reaching statistical significance upon treatment with 1 mM of succinate relative to the vehicle, up to the maximal effect at the optimal concentration (Fig. 5 B). Furthermore, we performed cell viability assay on ESCs and HMrSV5 cells treated with succinate for 24 and 48 h. The OD450 analysis showed a significantly higher survival capacity of ESCs treated with succinate when compared to the control (Fig. 5 C), similarly to the effect found in HMrSV5 cells (Fig. 5 D). Finally, we performed the AnnexinV/propidium iodide staining of ESCs and HMrSV5 cells treated with a vehicle or succinate for 48 h. We evaluated the percentages of apoptotic and live cells and observed no differences with succinate compared to the vehicle (Fig. 5 E-F). 3.6 Succinate-Stimulated Peritoneal Mesothelial Cells Recruit Macrophages and Boost Ectopic Growth and Implantation of ESCs Based on the above assay, we showed that succinate promotes various endometriotic processes in ESCs, including survival, migration, adhesion and invasion. As macrophages are the principal mediators of pathological EMs, we considered whether succinate could recruit macrophages and promote the adhesion of ESCs to HMrSV5 cells. As we previously reported that endometrial stromal cells from the ectopic milieu continuously recruit monocytes and is beneficial to expansion of monocyte-derived CCR2 + macrophages ( 26 – 28 ), we then tested the ability of HMrSV5 cells to attract macrophages after succinate stimulation. Our results revealed that treatment of HMrSV5 cells with succinate (0, 1, and 2.5 mM) for 48 h remarkably induced CCL2 gene expression and protein secretion (Fig. 6 A-B). Next, in a co-culture system of ESCs in the upper chamber and HMrSV5 cells exposed to 2.5mM succinate in the lower chamber for 48 h, we then replaced ESCs with THP-1 cells that were was pre-labeled with the CellTracker red and cultured them for another 12h. The chemotaxis assay showed that HMrSV5 cells exposed to succinate readily attracted the THP-1 (CCL2 treatment was the positive control) ( Fig. 6 C ) , suggesting that this chemotactic effect on monocytes/macrophages might be mediated by the CCL2 which was secreted by mesothelial cells after stress. Although the theory of retrograde menstruation and immune disorders is helpful for understanding EMs, the mechanisms underlying the pathological factors and their roles in ectopic implantation and aggressive growth of ESCs are still poorly understood. In this study, we analyzed the effect of succinate on the adhesion of ESCs to peritoneal mesothelial cells. The adhesion assay showed that succinate triggered the adhesion of ESCs to HMrSV5 cells more strongly than the vehicle, and that enhanced adhesion occurred when HMrSV5 cells were exposed to 2.5mM succinate ( Fig. 6 D ) . Further investigations showed that succinate-driven adhesion of ESCs to HMrSV5 cells might depend on the expression of ICAM-1 because succinate induced a concentration-dependent increase of ICAM-1 expression and of the adhesion capacity of HMrSV5 cells ( Fig. 6 E-F ) . Thus, succinate may play a pivotal role in the implantation growth of ESCs. Next, we investigated whether succinate derived from the ectopic milieu could induce the invasion and migration of ESCs in the presence of HMrSV5 cells. This effect was evaluated using a Matrigel invasion assay. ESCs in the presence of HMrSV5 cells were stimulated with a vehicle or with 2.5 mM succinate for 48 h, and CCL2 treatment was used as a positive control. Indeed, we observed that 2.5 mM succinate strongly induced invasion of ESCs in the presence of HMrSV5 cells ( Fig. 6 G ) . Matrix metalloproteinases (MMPs) are vital regulators of invasion and extracellular matrix remodeling. We also demonstrated that succinate (0.5–2.5 mM) stimulation induced the expression of MMPs in HMrSV5 cells, such as MMP2, MMP9 and MMP11 ( Fig. 6 H ) . Further in vivo analysis of the role of succinate in EMs progression was performed using an EMs allograft model. The body weight of the mice and the number and weight of lesions were recorded after treatment with either succinate (100 mg/kg) or PBS ( Fig. 7 A-B ) . Our results showed that the intraperitoneal injection of succinate did not affect the body weight of BALB/c mice ( Fig. 7 A ) or the ectopic lesion weight ( Fig. 7 C ) . Interestingly, succinate-treated mice showed more ectopic lesions ( Fig. 7 D ) . To analyze the expression of SUCNR1 in M1 or M2 macrophages, SUCNR1 and CD80/CD163 on CD11b(+)F4/80(+) macrophages were assessed for mean fluorescence intensity (MFI) using Flow cytometry (FCM). The results revealed that SUCNR1 expression was higher in both on M1-like or M2-like peritoneal macrophages in the succinate exposed group than in the control and model groups ( Fig. 7 E-I ) . Similarly, succinate-exposure increaded the expression of ICAM-1) and MMP9 in ectopic lesions in compare with that of model group (Fig. 7 J). In summary, these data indicate that succinate accumulation induced by polarized macrophages and peritoneal mesothelial cells obviously enhances the aggressive implantation of ectopic ESCs and the adhesion between ESCs and peritoneal mesothelial cells, promoting the progression of EMs via SUCNR1 signaling. 4. Discussion Metabolites have been considered as the vital players in metabolism over the past few decades, and have been recently proven to have key immune regulatory functions in recent years. Among these, succinate stands out for its multifaceted roles in immune and metabolic function regulation ( 13 , 17 , 18 , 29 ), dynamic changes, and selective cell release( 30 ). Notably, several pathogenic states, such as obesity ( 14 ), diabetes ( 31 ), hypertension ( 32 ), and various inflammatory conditions ( 33 ), have been associated with elevated levels of extracellular succinate in the body. Our study identified an important axis that comprises succinate and its cognate receptor, SUCNR1, as the potential drivers of ectopic endometrial survival and adhesion in the EMs milieu. As EMs progresses, succinate accumulates in the PF of EMs patients. Hypoactivity/deficiency or overproduction of SDH may cause succinate accumulation in the ectopic milieu and is associated with a range of clinical symptoms in EMs, such as dysmenorrhea, and with the revised American Fertility Society (rAFS) classification and the Endometriosis Fertility Index (EFI). In the present study, we investigated the main sources of succinate in the extracellular fluid of patients with EMs. It has been previously revealed that under the stimulation or polarization with LPS, macrophages changed from oxidative phosphorylation to glycolysis, which was accompanied by the elevated level of intracellular and extracellular succinate ( 34 – 36 ). Our data revealed that succinate was mainly from the type 1 polarized macrophages. Surprisingly, peritoneal mesothelial cells produced high levels of succinate comparable to those produced by M1 macrophages. Conditions that trigger succinate secretion include stimulation by the inflammatory cytokine IL-6 and exposure of stromal cells. The elevations of succinate in the cell supernatant partly depended on the direct physical contact between the mesothelial cells/macrophages and the ESCs. However, mechanistic insights into the effects of dysregulated succinate levels on ectopic cellular functions in this pathology remain unavailable. SUCNR1 is widely and heterogeneously expressed in various cell types throughout the body. The most well-studied SUCNR1-expressing cells are monocytes and macrophages ( 18 , 34 ). In addition, many non-immune tissues, including the intestine ( 11 ), placenta ( 12 ), skeletal muscle satellite ( 37 ), and endothelial ( 38 ), express SUCNR1 and respond to paracrine signaling in the form of succinate secretion in response to local pathologies. It has been speculated that the inflammation and fibrosis associated with these pathologies are, at least in part, attributable to chronic SUCNR1 agonism in tissue-resident cell populations. Whereas succinate-SUCNR1 interplay has been proposed as the molecular mechanism in rheumatoid arthritis ( 15 ) and intestinal inflammation ( 11 ), our data also imply a role of this pathway also in EMs formation. We confirmed the expression of SUCNR1 in ESCs and PMCs. FCM analysis highlighted the differences in SUCNR1 expression among various stromal cells, which may be reflected in their responses to succinate stimuli via SUCNR1. The FCM and real-time PCR results demonstrated that SUCNR1 expression was higher in primary ectopic ESCs than in normal ESCs, suggesting that the succinate-SUCNR1 signal might be a potential driver of EMs. Indeed, we confirmed that succinate-SUCNR1 signaling is involved in the survival and adhesion of ESCs, in a battery of important steps in ectopic endometrial lesion formation. Functional experiments verified that succinate promoted ESC survival and invasion, as well as that it conferred antiapoptotic effects. Interestingly, exogenous succinate increased the mRNA and protein expression of SUCNR1 in HMrSV5 cells, further secreted pro-inflammatory CCL2 recruiting macrophages, and induced remarkable adhesion between HMrSV5 cells and ESCs, which implyies that these changes among cells in the ectopic milieu trigger a vicious circle of EMs progression. In our study, succinate drove macrophages to polarize toward M1 phenotype, which is consistent with a previous study ( 34 ). The assessment of IL-8 via ELISA, and the SUCNR1membrane receptor and macrophage surface markers (CD80, CD86, CD163, and CD206) using FCM corroborated the proinflammatory potential of succinate stimulation, which was similar to that observed in LPS-stimulated macrophages. However, succinate at a certain range of concentrations endows macrophages with proinflammatory function, whereas excessively high concentrations of succinate (5mM) impair IL-8 production. Furthermore, extracellular succinate synergized with LPS during macrophage polarization toward to the M1 phenotype and proinflammatory functional transformation. Our findings highlight the importance of succinate-SUCNR1 signaling in macrophage polarization and suggest its role in immune regulation. 5. Conclusion In summary, our results show that patients with EMs present with high levels of succinate in the peritoneal fluid and with increased SUCNR1 expression in ESCs. This succinate-SUCNR1 axis exacerbates the inflammatory activity of macrophages and ESC activation and plays a role in endometrial lesion formation and peritoneal adhesion. Our study demonstrated, for the first time, the role of succinate and its receptors in EMs. We propose that, in the ectopic milieu of patients with EMs, SUCNR1 signaling exacerbates inflammation and benefits the invasion, survival, and adhesive growth of ectopic ESCs, indicating a possible target for EMs treatment. 6. Statistics Spearman’s correlation analysis was used to analyze the correlation between extracellular succinate levels and clinical symptoms in humans. The diagnostic performance of succinate was determined using the area under the receiver operating characteristic curve (AUROC) analysis to assess the overall discriminatory power of these assays in predicting EMs progression. The continuous variables are shown as mean ± SEM. Data from two groups were analyzed by the Student’s t-test, where data from multiple groups were analyzed by the one-way ANOVA using Tukey’s post-hoc test. Statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS Inc., Chicago, 26.0 version) and the Prism5.0 software(GraphPad Software Inc.). A P-value < 0.05 was considered statistically significant. Declarations Data Availability The metabolite data used and analysis during the current study are available from the corresponding author on reasonable request. Funding This research was supported by the Shanghai Shen Kang Hospital Development Center (SHDC12019106 and SHDC12019X27), National Natural Science Foundation of China (31600735),and Five-year Project Plan of the Whole Life Cycle Health Research Institute of Fudan University: Construction Project (DGF50107-037001) Conflict of Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements We thank the Shanghai Luming biological technology co., LTD (Shanghai, China) for their enthusiastic support of this metabolomics analysis, Xue-chun Dong, and Xiao-hong Zhou (Luming Biotech Co., Ltd, Shanghai, China) for providing metabolomics services. Ethics approval and consent to participate The research was approved by the Ethics Committee of the Obstetrics and Gynecology Hospital, Fudan University (2020-137). 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Wu JY, Huang TW, Hsieh YT, Wang YF, Yen CC, Lee GL, et al. Cancer-Derived Succinate Promotes Macrophage Polarization and Cancer Metastasis via Succinate Receptor. Mol Cell. 2020;77(2):213–27. e5. Mills EL, Kelly B, Logan A, Costa ASH, Varma M, Bryant CE, et al. Succinate Dehydrogenase Supports Metabolic Repurposing of Mitochondria to Drive Inflammatory Macrophages. Cell. 2016;167(2):457–70. e13. Wang T, Xu YQ, Yuan YX, Xu PW, Zhang C, Li F, et al. Succinate induces skeletal muscle fiber remodeling via SUNCR1 signaling. EMBO Rep. 2019;20(9):e47892. Zhang H, Zheng J, Lin J, Chen J, Yu Z, Chen C, et al. miR-758 mediates oxLDL-dependent vascular endothelial cell damage by suppressing the succinate receptor SUCNR1. Gene. 2018;663:8. Additional Declarations No competing interests reported. Supplementary Files Figure1AoriginaldataforOPLSDAanalysis.csv Figure1andFigure2DLM20228644Differenceresultorganicacidionpairinformation.xlsx SupplementaryFigure1.tif Figure 1. Succinate and SDHB expression in EMs milieu (A) Succinate accumulation in PF of patients with EMs was confirmed by ELISA. (B) SDHB expression in normal endometrium (n=5), and ectopic lesion (n=3) by immunohistochemistry. non-EMs: endometrium from patients without endometrioss; EMs: ectopic lesion from women with endometriosis. Original magnification: ×200. SupplementaryFigure2.tif Figure 2. Succinate Amplified the polarization of M1 phenotype (A-B, D-E) Under the initial stimulus condition, relative mRNA expression levels of M1 markers (CD80, CD86), M2 markers (CD206, CD163) in both vehicle and succinate (0, 1, 2, 2.5 and 5 mM) group. (C) CD 86 expression were assay via FCM in THP-1 cells stimulated with single succinate or LPS, as well as a combination of both for 24h. Points or bars in graphs represent mean ± SEM. Significant differences in relation to the vehicle group are shown by *P < 0.05, **P < 0.01, ***P < 0.001. (F-I) RT-PCR of IL-8, IL-1β, IL-6 in THP-1 derived macrophages treated with vehicle or succinate for 24 h. * p < 0.05, ** p < 0.01, *** p < 0.001, data are shown as mean±SEM (n = 3). (J) SUCNR1 expression were assay via FCM in THP-1 cells stimulated with succinate(0, 0.5, 1, 2.5 and 5 mM) for 48h. one-way ANOVA followed by Dunnett’s post hoc test , * p < 0.05, ** p < 0.01, *** p < 0.001, data are shown as mean ±SEM (n≥ 3). SupplementaryFigure3.png SupplementaryRawdataFig5ABphotographs.rar SupplementaryRawdataforFigure3.rar SupplementaryRawdataforFigure6F.rar SupplementaryTable1.docx SupplementaryTable2.docx Cite Share Download PDF Status: Published Journal Publication published 30 Jan, 2024 Read the published version in Cell Communication and Signaling → Version 1 posted Editorial decision: Major revision 12 Oct, 2023 Reviews received at journal 08 Oct, 2023 Reviewers agreed at journal 30 Sep, 2023 Reviewers invited by journal 14 Sep, 2023 Submission checks completed at journal 12 Sep, 2023 Editor assigned by journal 12 Sep, 2023 First submitted to journal 28 Aug, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3303001","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":229674239,"identity":"e9ea8a5a-3809-41d7-96fb-21a75a69af3d","order_by":0,"name":"Qi Tian","email":"","orcid":"","institution":"Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Qi","middleName":"","lastName":"Tian","suffix":""},{"id":229674240,"identity":"10406021-2ee6-493b-bb7d-85b7ea34eb8e","order_by":1,"name":"JingYao Ruan","email":"","orcid":"","institution":"Fudan University","correspondingAuthor":false,"prefix":"","firstName":"JingYao","middleName":"","lastName":"Ruan","suffix":""},{"id":229674241,"identity":"1a82a770-bf84-47ed-83dd-ff5bdc159380","order_by":2,"name":"Yuning Wang","email":"","orcid":"","institution":"Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Yuning","middleName":"","lastName":"Wang","suffix":""},{"id":229674242,"identity":"1305fc5e-6882-4b61-b413-11a7874cb264","order_by":3,"name":"Yinping Xiao","email":"","orcid":"","institution":"Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Yinping","middleName":"","lastName":"Xiao","suffix":""},{"id":229674243,"identity":"b4eca841-1b49-458d-a6e8-8490b38e8cf7","order_by":4,"name":"Qi Cheng","email":"","orcid":"","institution":"Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Qi","middleName":"","lastName":"Cheng","suffix":""},{"id":229674244,"identity":"e57fd44f-a13f-4c20-8a62-f401f8ab7744","order_by":5,"name":"Yun Chen","email":"","orcid":"","institution":"Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Yun","middleName":"","lastName":"Chen","suffix":""},{"id":229674245,"identity":"290aa873-5d04-453b-970a-69494871756f","order_by":6,"name":"Mingqing Li","email":"","orcid":"","institution":"Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Mingqing","middleName":"","lastName":"Li","suffix":""},{"id":229674246,"identity":"168a3a12-608a-43b8-b57d-9e9242a9ca59","order_by":7,"name":"Kaikai Chang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4UlEQVRIiWNgGAWjYDACCRBxIEGOvYHBAMRkbCBWizHPAVK1JPYQrYV/dvOzh1/OpKX3SCRv/FzAYCO74QDzswd4LblzzNxY5kZObo9EWrH0DIY04w0H2MwN8GkxkEgwk5b4UJG7XyLHQJqH4XDihgM8bBL4taR/A2lJ55HIMf7Nw/CfGC05ZpIfbuQkALWYAW05QFiLxI2cMmmGM2mGPTzPyqx5DJKNZx5mM8OrhX9G+jbJH8eS5XnYkzff5qmwk+073vwMrxYQYOZBuBPEJaQeCBh/EKFoFIyCUTAKRjAAAEhYR8zQBT7fAAAAAElFTkSuQmCC","orcid":"","institution":"Fudan University","correspondingAuthor":true,"prefix":"","firstName":"Kaikai","middleName":"","lastName":"Chang","suffix":""},{"id":229674247,"identity":"6f42f96f-6c61-443e-a647-bec983689d92","order_by":8,"name":"Xiaofang Yi","email":"","orcid":"","institution":"Fudan University","correspondingAuthor":false,"prefix":"","firstName":"Xiaofang","middleName":"","lastName":"Yi","suffix":""}],"badges":[],"createdAt":"2023-08-28 10:44:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3303001/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3303001/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12964-023-01415-7","type":"published","date":"2024-01-30T15:01:12+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":42777214,"identity":"50e4d37e-8d87-48c0-ad47-b3851debbd60","added_by":"auto","created_at":"2023-09-07 14:36:22","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":989371,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAltered metabolic profiles of peritoneal fluid in EMs compared with Healthy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) A PLS-DA score plot of EMs and Non-EMs in cohort. R2X = 0.24, R2Y = 0.231, Q2 =0.541.\u003c/p\u003e\n\u003cp\u003e(B, C) Volcano plot and bar graph of the differential metabolites in EMs and Non-EMs filtered by univariate analysis.\u003c/p\u003e\n\u003cp\u003e(D) Hierarchical clustering of the 22 differential organic acid metabolites (FC\u0026gt;1.2, p\u0026lt;0.05) in non-EMs and EMs group. Blue indicates a decreased level; red indicates an increased level.\u003c/p\u003e\n\u003cp\u003e(E) Bubble plot for quantitative enrichment analysis showing the metabolic pathway changes between EMs and non-EMs metabolomes in cohort.\u003c/p\u003e","description":"","filename":"OnlineFigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3303001/v1/750c8139c1a7b18e27819753.png"},{"id":42775551,"identity":"e27e36d8-4632-4f37-acca-f4285674b2d2","added_by":"auto","created_at":"2023-09-07 14:28:21","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":725876,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSuccinate accumulation of peritoneal fluid from EMs patients is associated with clinical relevance\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Level of succinate in peritoneal fluid from EMs patients with different stages. Succinate was assessed via targeting organic acid metabolomics analysis (EMs I-II stage, n = 12; III-IV stage, n = 24) (Student’s \u003cem\u003et\u003c/em\u003e-test).\u003c/p\u003e\n\u003cp\u003e(B) ROC curve for the assessment of EMs severity probability in cohort.\u003c/p\u003e\n\u003cp\u003e(C-E) The Pearson correlation coefficient was used for clinical correlation, and result showed the level of succinate was positively correlated with pain grade VAS (R2 = 0.0.46, P \u0026lt; 0.001, 95% confidence interval is 0.15–0.68) and rAFS stage (R2 = 0.38, P \u0026lt; 0.02, 95% confidence interval is 0.06–0.63), negative with EFI. ***P \u0026lt; 0.001.\u003c/p\u003e\n\u003cp\u003eVAS: Visual Analogue Scale; rAFS: revised American Fertility Society Scoring; EFI: endometriosis fertility index\u003c/p\u003e","description":"","filename":"OnlineFigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3303001/v1/b8ab25d57db66190ea05ab61.png"},{"id":42775549,"identity":"196caa8d-d7d4-4d27-b073-2176ab3ef9e8","added_by":"auto","created_at":"2023-09-07 14:28:21","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":50292,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePeritoneal mesothelial cells and M1-polarized Macrophage Mainly Produced Extracellular Succinate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Extracellular succinate secretion measured in different cell types, such as macrophage, HMrSV5 cells and ESCs.\u003c/p\u003e\n\u003cp\u003e(B) The highest level of succinate was in the co-cultured medium of M1-polarized macrophages after co-culture with HMrSV5 cells or ESCs.\u003c/p\u003e\n\u003cp\u003e(C) Naive macrophage state (M0) co-cultured with HMrSV5 cells or ESCs could trigger high level of succinate secretion in the cell medium, but not with IL-6 or CCL2 stimulation.\u003c/p\u003e\n\u003cp\u003e(D) Similarly, extracellular succinate secretion was increased under IL-6 exposure or in co-culture of M1 macrophages and HMrSV5 cells.\u003c/p\u003e\n\u003cp\u003eStatistical significance was assessed either with t test or \u003cem\u003eone-way ANOVA\u003c/em\u003e followed by \u003cem\u003eTukey’s or Holm-Sidak’s post-test\u003c/em\u003e. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"OnlineFigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3303001/v1/896f8dd84d85e77b5ed6353d.png"},{"id":42775552,"identity":"547a4906-8f84-409a-af89-0d1a7e844ced","added_by":"auto","created_at":"2023-09-07 14:28:22","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3283552,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe expression of SUCNR1 in cells from Ectopic milieu\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) SUCNR1 expression in female tissues from healthy human tissues (data are available from https://www.proteinatlas.org/)\u003c/p\u003e\n\u003cp\u003e(B-D) The level of SUCNR1\u003csup\u003e+\u003c/sup\u003e macrophages and peritoneal mesothelial cells (PMC) in peritoneal fluid from EMs patients was elevated and correlated with EMs stage. (E) The staining macroscopic observation of peritoneal mesothelial cells (PMC) in peritoneal fluid or intraperitoneal lavage (modified papanicolaou staining, 40X).\u003c/p\u003e\n\u003cp\u003e(F-G) FCM-based MFI assay and qT-PCR analysis designed to measure SUCNR1 expressions, and result showed primary ectopic stromal cell (hESC.D) expressed higher SUCNR1, compared with that of primary normal stromal cell (hESC.N) and human stromal cell line (hESC.N).\u003c/p\u003e\n\u003cp\u003e(H-I) FCM and qT-PCR assays showed the SUCNR1 expression of normal ESC stimulated with succiante with different concentrations. The optimal concentration of succinate is 2.5mM.\u003c/p\u003e\n\u003cp\u003eSignificance was assessed either with t test or \u003cem\u003eone-way ANOVA\u003c/em\u003e followed by \u003cem\u003eTukey’s or Holm-Sidak’s post-test.\u003c/em\u003e *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"OnlineFigure4.png","url":"https://assets-eu.researchsquare.com/files/rs-3303001/v1/77f97128c7f968f62ce69f97.png"},{"id":42775557,"identity":"5f7a0116-7fc3-4c88-a322-e74832434d1d","added_by":"auto","created_at":"2023-09-07 14:28:22","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":7405818,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSuccinate induces the invasion, wound healing and survival of ESCs\u003c/strong\u003e (A) Invasion assays of ESCs Invasion assay was measured after exposure of ESC to different concentrations of succinate for 48h, and CCL2 stimulation was used as the positive control (scale bar-200um)(n = 3). \u003cem\u003eOne-way ANOV\u003c/em\u003eA , \u003cem\u003eDunnett’s post hoc test\u003c/em\u003e , *P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001 (B) Scrath assays of ESC Scrath assay was conducted after PRE treatment in ESCs with different concentration of succinate for 48h. Results are from 3 independent trials (n ≥ 3 for mimic) and data depicted as column mean graphs with error bars showing confidence intervals (scale bar-200um) \u003cem\u003eOne-way ANOV\u003c/em\u003eA , \u003cem\u003eDunnett’s post hoc test\u003c/em\u003e, *P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001. (C-D) Cell viability of ESCs and HMrSV5 cells was determined by CCK8 assay ESCs or HMrSV5 cells were respectively incubated with different concentrations of succinate for 24 or 48 hours. and then cell viability was determined by CCK8 assay. Data represent mean ± SEM (n = 3). *P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001. (E-F) Succinate does not affect the apoptosis of ESCs and HMrSV5 cells. ESCs or HMrSV5 cells were cultureed with different concentration of succinate for 48h, and the proportion of 7AAD\u003csup\u003e+\u003c/sup\u003e and Annexin V\u003csup\u003e+\u003c/sup\u003e via FCM was shown in HMrSV5 cells or ESC cells via FCM(n = 3). \u003cem\u003eOne-way ANOVA\u003c/em\u003e, \u003cem\u003eDunnett’s post hoc test\u003c/em\u003e , *P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"OnlineFigure5.png","url":"https://assets-eu.researchsquare.com/files/rs-3303001/v1/d3904fc67629ae74017310b2.png"},{"id":42775565,"identity":"1691b9f4-393c-4409-8669-565ce51f0968","added_by":"auto","created_at":"2023-09-07 14:28:22","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":696408,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSuccinate Enhances the Effect of Peritoneal mesothelial cells on Macrophage Recruitment and ESCs Adhesion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-B) RT-PCR was used to analyzed the CCL2 mRNA in HMrSV5 cells treated with vehicle or succinate (0, 1 and 2.5 mM) for 48h, and ELISA was used to detected protein level of CCL2 (n = 3). All were analyzed by \u003cem\u003eone-way ANOVA\u003c/em\u003e followed by \u003cem\u003eDunnett’s post hoc test\u003c/em\u003e, * p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001, data are shown as mean ±SEM (CCL-2 group was positive control). (C) ESCs were seeded in upper chamber and HMrSV5 cells were treated with 2.5mM succinate in lower chamber for 48h, and then the ESCs was replaced with THP-1 cells for another 12h. THP-1 cells were prelabeled with red fluorescent reagent CellTracker red. The chemotaxis assay for THP-1 cells was calculated as cell number per field (scale bar-200µm) (red tracer staining THP-1 cell)(n = 3). \u003cem\u003eOne-way ANOVA \u003c/em\u003efollowed by \u003cem\u003eDunnett’s post hoc test\u003c/em\u003e, * p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001, data are shown as mean ±SEM (CCL-2 group was positive control) . (D) Adhesion assays show ESCs adhere to HMrSV5 cells pretreated with different concentration of succinate for 48h. ESCs(1×10\u003csup\u003e5\u003c/sup\u003e cells/well) were prelabeled with green fluorescent reagent CellTracker green and seeded in the HMrSV5-well. Results are from 3 independent trials (n ≥ 3 for mimic) and data depicted as column mean graphs with error bars showing confidence intervals (scale bar-100um). (E) RT-PCR of ICAM-1 in HMrSV5 cells treated with vehicle or succinate (1,2.5 and 5 mM) for 48h and analyzed by \u003cem\u003eone-way ANOVA\u003c/em\u003e followed by \u003cem\u003eDunnett’s post hoc test \u003c/em\u003e(n = 3)\u003cem\u003e,\u003c/em\u003e * p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001, data are shown as mean ± SEM . (F) WB of ICAM-1 in HMrSV5 cells treated with different concentrations of succinate for 48h. (G) Transwell migration assay of ESC in the presence of HMrSV5 cells with vehicle or 2.5mM succinate treatment for 48h. Media with CCL2 100ng/ml. (H) RT-PCR of MMP2, MMP9 and MMP11 in HMrSV5 cells treated with succinate (0, 1, 2.5 and 5 mM) for 48h and analyzed by \u003cem\u003eone-way ANOVA\u003c/em\u003e followed by \u003cem\u003eDunnett’s post hoc test \u003c/em\u003e(n = 3), * p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001, data are shown as mean ± SEM\u003c/p\u003e","description":"","filename":"OnlineFigure6.png","url":"https://assets-eu.researchsquare.com/files/rs-3303001/v1/8752336914facfdaac2c6f24.png"},{"id":42775558,"identity":"e76c341c-346f-4f77-808d-feaa06df05e6","added_by":"auto","created_at":"2023-09-07 14:28:22","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":6415619,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSuccinate Induce the Enrichment of SUCNR1\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e Macrophages and Ectopic Lesion Formation \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e The wight change of mouse EMs model during the two weeks. Ctrl: PBS treatment; M: Model group; M+S: Model with succinate treatment group. The data are expressed as the mean±SEM. (One-way ANOVA) *P\u0026lt;0.05, **P\u0026lt;0.01 and ***P\u0026lt;0.001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B) \u003c/strong\u003eThe macroscopic observation of the morphology of endometriosis-like lesions from endometriosis mouse models. M: Model group, M+S: Model with succinate treatment group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C-D) \u003c/strong\u003eThe number and wight of EMs lesions was measured after the treatment of succinate (100 mg/kg) or PBS. M: Model group, M+S: Model with succinate treatment group. (\u003cem\u003eStudent's t-test\u003c/em\u003e) *P\u0026lt;0.05, **P\u0026lt;0.01 and ***P\u0026lt;0.001.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(E-I) \u003c/strong\u003eThe levels of CD80, CD163 and SUCNR1 on peritoneal CD11b(+)F4/80(+) macrophages from mouse models were analyzed by using flow cytometry. MFI of SUCNR1 on M1 and M2 macrophages obtained were shown respectively. Ctrl: marked in red; M: marked in blue; M+S: marked in orange. (\u003cem\u003eOne-way ANOVA\u003c/em\u003e) *P\u0026lt;0.05, **P\u0026lt;0.01 and ***P\u0026lt;0.001\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(J) \u003c/strong\u003eMMP9 and ICAM-1 expression in endometriosis-like lesion from model mice (M) and succinate exposing mice (M+S) by immunohistochemistry. M: Model group; M+S: Model with succinate treatment group. Original magnification: ×200.\u003c/p\u003e","description":"","filename":"OnlineFigure7.png","url":"https://assets-eu.researchsquare.com/files/rs-3303001/v1/9450ceb9c002eaf670adf2f7.png"},{"id":42777215,"identity":"c2bf1c40-c9d5-4bf9-b27c-3b58adcd9005","added_by":"auto","created_at":"2023-09-07 14:36:22","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":21391,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe schematic roles of Succinate from macrophage and peritoneal mesothelial cells in the progression of endometriosis by inducing inflammation and promoting ectopic growth.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the microenvironment of ectopic foci, exposure to inflammatory cytokines or contaction with ESCs obviously triggered succinate secretion from M1 polarized macrophages and peritoneal mesothelial cells, which are main producers of succinate. Interestingly, compared with that of normal endometrium, ESCs in ectopic tissues express high levels of SUCNR1. For peritoneal mesothelial cells, succinate promotes the auto-secretion of succinate and expression of SUCNR1 in an autocrine amplification manner. In addition to promoting succinate accumulation, the interactions among macrophages, mesothelial cells and ESCs in ectopic milieu amplify the regulatory effect on cellular function of succinate via SUCNR1 signal. Succinate promoted the survival, adhesion, invasion and deep infiltration of ESCs via SUCNR1 signaling, participating in the formation of an ectopic lesion in endometriosis. In conclusion, succinate in ectopic milieu synergizes with polarized macrophage to exacerbate inflammation and facilitate the progress of endometriosis via a succinate-SUCNR1-dependent mechanisms.\u003c/p\u003e","description":"","filename":"OnlineFigure8.png","url":"https://assets-eu.researchsquare.com/files/rs-3303001/v1/6ab54f8c12795f0413fea21d.png"},{"id":50673952,"identity":"b04529ef-165d-4969-9386-fc162fb8d5c7","added_by":"auto","created_at":"2024-02-05 15:08:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3524706,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3303001/v1/4c3075aa-1337-4431-bce2-472e1055f01f.pdf"},{"id":42775548,"identity":"2bcab923-247d-46a3-bf9f-43efe6f91c5c","added_by":"auto","created_at":"2023-09-07 14:28:21","extension":"csv","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4036,"visible":true,"origin":"","legend":"","description":"","filename":"Figure1AoriginaldataforOPLSDAanalysis.csv","url":"https://assets-eu.researchsquare.com/files/rs-3303001/v1/5e02cb8750e112f847864b33.csv"},{"id":42777213,"identity":"9ee385c7-bbe4-44f4-9b26-8dc13332b62f","added_by":"auto","created_at":"2023-09-07 14:36:21","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":10294,"visible":true,"origin":"","legend":"","description":"","filename":"Figure1andFigure2DLM20228644Differenceresultorganicacidionpairinformation.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-3303001/v1/6a19b85eb6968a87b9d76a6b.xlsx"},{"id":42775554,"identity":"f44b8b53-42cd-499b-be7b-746a50c00e45","added_by":"auto","created_at":"2023-09-07 14:28:22","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":4681252,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 1. Succinate and SDHB expression in EMs milieu\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Succinate accumulation in PF of patients with EMs was confirmed by ELISA.\u003c/p\u003e\n\u003cp\u003e(B) SDHB expression in normal endometrium (n=5), and ectopic lesion (n=3) by immunohistochemistry. non-EMs: endometrium from patients without endometrioss; EMs: ectopic lesion from women with endometriosis. Original magnification: ×200.\u003c/p\u003e","description":"","filename":"SupplementaryFigure1.tif","url":"https://assets-eu.researchsquare.com/files/rs-3303001/v1/e1f5b133a2fa9152de30b938.tif"},{"id":42775582,"identity":"053af6ef-1efd-4403-a8d2-6ff11324a96d","added_by":"auto","created_at":"2023-09-07 14:28:23","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":26438060,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 2. Succinate Amplified the polarization of M1 phenotype\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-B, D-E) Under the initial stimulus condition, relative mRNA expression levels of M1 markers (CD80, CD86), M2 markers (CD206, CD163) in both vehicle and succinate (0, 1, 2, 2.5 and 5 mM) group.\u003c/p\u003e\n\u003cp\u003e(C) CD 86 expression were assay via FCM in THP-1 cells stimulated with single succinate or LPS, as well as a combination of both for 24h. Points or bars in graphs represent mean ± SEM. Significant differences in relation to the vehicle group are shown by *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001.\u003c/p\u003e\n\u003cp\u003e(F-I) RT-PCR of IL-8, IL-1β, IL-6 in THP-1 derived macrophages treated with vehicle or succinate for 24 h. * p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001, data are shown as mean±SEM (n = 3).\u003c/p\u003e\n\u003cp\u003e(J) SUCNR1 expression were assay via FCM in THP-1 cells stimulated with succinate(0, 0.5, 1, 2.5 and 5 mM) for 48h. \u003cem\u003eone-way ANOVA\u003c/em\u003e followed by \u003cem\u003eDunnett’s post hoc test\u003c/em\u003e, * p \u0026lt; 0.05, ** p \u0026lt; 0.01, *** p \u0026lt; 0.001, data are shown as mean ±SEM (n≥ 3).\u003c/p\u003e","description":"","filename":"SupplementaryFigure2.tif","url":"https://assets-eu.researchsquare.com/files/rs-3303001/v1/582eb7aefa74ee8fe9da3ec2.tif"},{"id":42777216,"identity":"bd30a218-e87a-40db-8328-1db7426f5fe6","added_by":"auto","created_at":"2023-09-07 14:36:22","extension":"png","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":2777109,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3303001/v1/3c67b872bd5e8ce26555287d.png"},{"id":42775576,"identity":"4f252f03-9504-49b6-9799-808c062364fe","added_by":"auto","created_at":"2023-09-07 14:28:22","extension":"rar","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":11449438,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryRawdataFig5ABphotographs.rar","url":"https://assets-eu.researchsquare.com/files/rs-3303001/v1/1db980148f4c08a00991475f.rar"},{"id":42775571,"identity":"dd9d5556-bdc3-4efa-952d-f9d6573b7902","added_by":"auto","created_at":"2023-09-07 14:28:22","extension":"rar","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":6562344,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryRawdataforFigure3.rar","url":"https://assets-eu.researchsquare.com/files/rs-3303001/v1/93e0804e5858be05d1f2bbf0.rar"},{"id":42777949,"identity":"5776129f-727e-429d-bb58-7589ec4a6756","added_by":"auto","created_at":"2023-09-07 14:44:22","extension":"rar","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":6737,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryRawdataforFigure6F.rar","url":"https://assets-eu.researchsquare.com/files/rs-3303001/v1/6b2da4f592d66fcfb87df1cc.rar"},{"id":42775578,"identity":"86dfba83-01aa-4ccd-8b70-01ad090d3135","added_by":"auto","created_at":"2023-09-07 14:28:22","extension":"docx","order_by":9,"title":"","display":"","copyAsset":false,"role":"supplement","size":27295,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable1.docx","url":"https://assets-eu.researchsquare.com/files/rs-3303001/v1/07d8c6d11bda661657f68192.docx"},{"id":42775560,"identity":"57e5a04a-d8ff-4160-9331-bdf1d2857b41","added_by":"auto","created_at":"2023-09-07 14:28:22","extension":"docx","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":28322,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTable2.docx","url":"https://assets-eu.researchsquare.com/files/rs-3303001/v1/8af3b3d588043d645c89b94b.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Extracellular Succinate Derived From Ectopic Milieu Drives Adhesion and Implantation Growth of Endometrial stromal cells via the SUCNR1 signal in endometriosis","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eEndometriosis (EMs) is a heterogeneous clinical syndrome characterized by a chronic inflammatory process that is strongly linked to peritoneal adhesion, infertility, dysmenorrhea, and chronic pelvic pain (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Endometrial stromal cells (ESCs) and epithelial cells within the retrograde menstrual endometrium commonly attached to the pelvic peritoneum. Once an ectopic lesion is formed, ectopic tissue with periodic bleeding is exposed to immune surveillance, leading to chronic inflammation and repeated tissue repair. The presence of cytokines and shifts in circulating immune cell populations creates a widespread inflammatory environment and peritoneal adhesions (PA) extending outside the pelvis (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Monolayers of peritoneal-mesothelial cells and macrophages form major cell populations in the peritoneal fluid, which may play a central role in lesion establishment and maintenance by driving chronic inflammation and tissue remodeling. Although the pathogenesis of EMs remains unclear, genetics and the microenvironment are its key drivers (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSuccinate occupies an extremely vital position in the metabolism because of its direct connection to the Krebs cycle and the mitochondrial respiratory chain (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). The final stage of the Krebs cycle involves the regeneration of oxaloacetate, involves a process in which succinate is oxidated to fumarate via succinate dehydrogenase (SDH) (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Dynamic changes in SDH under physiological or pathological metabolic conditions are associated with succinate accumulation (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Over the past 10 years, the new roles of extracellular succinate have expanded beyond metabolism and into signaling. Beyond its metabolic role in conditions of stress and damage, an increasing body of evidence points to additional immunological functions, especially in subacute inflammatory conditions, such as Inflammatory Bowel Disease (IBD), Crohn\u0026rsquo;s disease(CD) (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e), gestational diabetes (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e), and non-alcoholic fatty liver disease (NAFLD) (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Succinate accumulation could be followed by succinate release from cells, which would then act on other cell types via the SUCNR1, driving inflammation or type 2 immunity (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e).These multiple functions indicate that succinate plays an extensive role in cellular activation and inflammation. Succinate triggers macrophage polarization and subsequent inflammation. However, the role of succinate-SUCNR1 signal in EMs, which is characterized by chronic inflammation, remains unclear.\u003c/p\u003e \u003cp\u003eOn this basis, our study hypothesized that stimulation with cytokines, such as IL-6, or contact with endometrial stromal cells during retrograde menstruation triggers succinate release from peritoneal mesothelial cells and macrophages. Extracellular succinate polarizes macrophages into the M1-like type and continuously recruits macrophages via CCL2 secretion from peritoneal mesothelial cells. Thus, the crosstalk between these cells leads to massive succinate accumulation and an inflammatory microenvironment. Eventually, accumulated succinate enhances the survival, adhesion and deep infiltration of ESCs via SUCNR1 signaling, leading to the acceleration of EMs progress.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Patients and Tissues collection\u003c/h2\u003e \u003cp\u003ePremenopausal women diagnosed with EMs or benign gynecological diseases (such as fibroids and benign teratomas) underwent laparoscopic surgery at the Obstetrics and Gynecology Hospital of Fudan University between January 2020 and December 2022. Normal endometrial samples were obtained from 6 patients with EMs who underwent combined laparoscopy and hysteroscopy for tubal infertility. EMs was diagnosed based on clinical symptoms and imaging findings. Symptoms related to EMs include pelvic masses, chronic pelvic pain, dysmenorrhea, dyspareunia, infertility, and cyclical alterations in bowel and urinary habits that occur only during menstruation. A total of 2\u0026ndash;10 ml of undiluted peritoneal fluid was drawn at the beginning of the laparoscopy, and biopsies from ectopic lesions were obtained from each patient. Finally, according to the rAFS (ASRM, 1997), patients with pathologically confirmed EMs were grouped according to disease stage(stages I-II, n\u0026thinsp;=\u0026thinsp;12; stages III-IV, n\u0026thinsp;=\u0026thinsp;24). Thirty, patients without EMs were included in the control group, and peritoneal fluid was collected during laparoscopic surgery.\u003c/p\u003e \u003cp\u003eThe enrolled patients were free of hormonal medication for at least 6 months. Patients with acute and subacute inflammatory diseases, autoimmune disorders, pregnancy, or malignant tumors were excluded from this study. The clinical and demographic characteristics of all the participants are shown in Supplementary Table\u0026nbsp;1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Peritoneal Fluid Isolation\u003c/h2\u003e \u003cp\u003ePeritoneal fluid was placed on ice at acquisition and then transferred to the laboratory within 30 minutes for further experiments. Fresh peritoneal fluid was centrifuged at 1500 rpm(4℃) for 5 minutes. The pellet was resuspended and the erythrocyte lysis solution (1\u0026times;) added according to the instructions. After centrifugation (1000 rpm) for three times, the fresh cell pellets were immediately prepared for flow cytometry. The supernatant of the PF cells and debris was packaged in 1.5mL centrifugal tubes and store in a refrigerator at \u0026minus;\u0026thinsp;80\u0026deg;C until the metabolomics detection and ELISA analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Targeting Organic Acid Metabolomics Analysis\u003c/h2\u003e \u003cp\u003eShanghai Lu-Ming Biotech Company Limited (Shanghai, China) provided an experimental platform and assistance for the target organic acid metabolomics analysis. Briefly, a mixture of acetonitrile and methanol (2:1, V/V, containing seven isotopes internal standards) was used to collect 0.1ml per sample. After shaking and centrifugation, 100\u0026micro;l of supernatant per sample was freeze-dried. Finally, a mixture of BSTFA and n-hexane (4:1 by volume) was added to the sample, vortexed vigorously for 2 min, and derivatized at 70\u0026deg; C for 60 min. The samples were analyzed using a gas chromatography system Trace1310 coupled to a TSQ9000 Mass spectrometer equipped with an Electron ionization (EI) source (Thermo Fisher Scientific, USA).\u003c/p\u003e \u003cp\u003eThe raw data exported by UPLC-MS/MS were processed using the QuanMET software (v1.0, Metabo-Profile, Shanghai, China). The concentrations and peak areas of the standards were used to construct a standard curve and calculate the sample concentration. The calculated concentrations of bile acids in all samples were imported into the SIMCA-P\u0026thinsp;+\u0026thinsp;software (v. 14.1, Umetrics, Sweden) for multivariate analysis, including principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis (OPLS-DA). An independent sample non-parametric test judgment was used to test for significant differences between the groups (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and the variables of importance (VIP) values in the OPLS-DA model were used to identify potential biomarkers.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Cell Culture and Treatments\u003c/h2\u003e \u003cp\u003ePrimary Human Endometrial stromal cells(hESCs) from the endometrium of patients with or without EMs were isolated using collagenase digestion, as described previously(\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e), and cultured in DMEM F12 supplemented with 10% fetal bovine serum (FBS) for FCM analysis.\u003c/p\u003e \u003cp\u003eThe cell line used in this study, ESCs, was a gifted from Professor Ming-qing Li of the Reproductive Immunology Laboratory of Obstetrics and Gynecology Hospital, Fudan University. ESCs were cultured in DMEM F12(10% FBS) for the co-culture system and other assays.\u003c/p\u003e \u003cp\u003eHMrSV5 and THP-1 cells were obtained from the National Collection of Authenticated Cell Cultures of China. HMrSV5 cells cultured in DMEM F12(10% FBS) and THP-1 cells cultured in RPMI-1640(10%FBS) were incubated with or without various concentrations of drugs for the predefined times before each experiment, according to the cell experiment protocol.\u003c/p\u003e \u003cp\u003eFor macrophage polarization, THP-1 cells were differentiated and polarized by using 100 ng/ml phorbol 12-myristate13-acetate (PMA; Sigma-Aldrich) for 48 h to obtain M0, and M0 were transformed into M1 through LPS (Peprotech) (100ng/ml) and IFN-γ (PeproTech)(20ng/ml) stimulation or M2 through IL-4 (PeproTech) (20ng/ml) and IL-13 (PeproTech) (20ng/ml) stimulation for 48h. FCM was performed to verify successful induction via CD80, CD86, CD163, and CD206. Non-adherent macrophages were cleaned using phosphate-buffered saline (PBS), and adherent cells were cultured in fresh RPMI-1640 medium.\u003c/p\u003e \u003cp\u003eThe details of cytokines used in this study are as follows: IFN-γ Peprotech Cat#300-02 lot#091927; LPS Peprotech Cat#M9524; IL-4 Peprotech Cat#200-04 lot#051914; IL-10 Peprotech Cat#200\u0026thinsp;\u0026minus;\u0026thinsp;10 lot#11021; IL-6 Peprotech Cat#200-06; IL-13 Peprotech Cat#200\u0026thinsp;\u0026minus;\u0026thinsp;13 lot#102123.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Cell Viability Assays\u003c/h2\u003e \u003cp\u003eThe cell viability was measured using the CCK-8 assay. ESCs or HMrSV5 cells (four replicates per group) were seeded in 96-well plates (Corning) with 100ml medium (10% FBS) and then incubated at 37℃ overnight. The cell supernatants were removed after stimulation with succinate for48h. Into each well the CCK-8 solution (10 \u0026micro;l) and culture medium(100 \u0026micro;l) were added. After incubation for another 1h at 37℃, the plates were measured via a microplate reader at an absorbance of 450 nm (Bio-Rad 680, Bio-Rad, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Apoptosis Assays\u003c/h2\u003e \u003cp\u003eFor apoptosis assay, ESCs or HMrSV5 cells were respectively seeded in 24-well plates and cultured with LPS or succinate for 48 h. Cells were then co-stained with Annexin V-PE (BD Pharmingen, Heidelberg, Germany) and 7AAD. Flow cytometer (Beckman) was performed to obtain data on apoptosis, and analyzed using the FlowJo software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Scratch Wound Assay\u003c/h2\u003e \u003cp\u003eHuman ESCs (1\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/well, four replicates per group) were seeded into a 12-well plate. After reaching confluence, the cells were scratched using a sterile tip to mimic the shape of a wound. An FBS-free medium was used to wash and remove loose cells. ESCs were then treated with succinate (0, 1, 2.5, or 5 mM) and photographed at 0, 24, and 48 h by using a light microscope. The closure area of wound was calculated as follows: Wound Closure (%) = ((Primary wound size-Final wound size)*100%/ Primary wound size.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Matrigel Invasion, Chemotaxis and Adhesion Assays,\u003c/h2\u003e \u003cp\u003eFor the transwell assay, ESCs or HMrSV5 (1\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/well, three replicates per group) were seeded into the upper chamber of 24-well transwell plates (8 \u0026micro;m pore filters) (Corning, USA). The lower chamber was supplemented with medium (10% FBS) with or without succinate (0, 1, 2.5, 5mM) or CCL-2 (Abclone Cat#RP01411) (100ng/ml). After 48 h, the migrated cells on the lower surface were stained and observed under a microscope.\u003c/p\u003e \u003cp\u003eFor chemotaxis assay, ESCs (1\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/well, three replicates per group) were seeded in the upper chamber of 24-well transwell plates(8\u0026micro;m pore filters) (Corning, USA), and HMrSV5 cells (1\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/well) stimulated with succinate (0, 2.5mM) or CCL2 (100 ng/ml) were put in lower chamber. CCL2 treatment was used as the positive control. After 48 hours, ESCs were replaced with THP-1 cells which were pre-labeled with CellTracker red (DiO, Beyotime, C1995S, China), and then continuously incubated for another 12 hours with 3 \u0026micro;m pore filters. Finally, the traced THP-1 cells were collected and calculated as cell number per field (scale bar-100 \u0026micro;m) (red tracer staining THP-1 cell).\u003c/p\u003e \u003cp\u003eFor the adhesion assay, HMrSV5 cells (2\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/well, three replicates per group) were treated with succinate(0, 1, 2.5, 5mM) and seeded in 6-well plates. ESCs(1\u0026times;10\u003csup\u003e4\u003c/sup\u003e cells/well, three replicates per group) pre-labeled with CellTracker green (DiO, Beyotime, C1993S, China) were further seeded into each HMrSV5-well. Finally, the traced ESCs were calculated as cell number per field by using a fluorescence microscope (scale bar-200\u0026micro;m) (green tracer staining ESCs).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Flow cytometry\u003c/h2\u003e \u003cp\u003ePeritoneal cell pellets collected from the PE were suspended in PBS and stained with the following antibodies: anti-human CD14 -PerCy5.5 (BioLegend,325621), anti-human CD45 APC-Cy7 (BioLegend, 368515) and anti-human GPR91/SUCNR1 FITC (Alomone, ASR-090-F). After staining for half an hour, the cells were washed and prepared for FCM (Beckman Coulter). Data were analyzed using the FlowJo(v10) software.\u003c/p\u003e \u003cp\u003eFlow cytometry was also performed to analyze the expression of CD80, CD86, CD163, and CD206 in macrophages \u003cem\u003ein vivo\u003c/em\u003e or in THP-1 cells \u003cem\u003ein vitro\u003c/em\u003e, as well as SUCNR1 levels in ESCs. The FCM antibodies used were as follows: anti-human CD86 Percp/cy5.5 (BioLegend, 305419); anti-human/Mouse GPR91/SUCNR1 FITC (Alomone, ASR-090-F); anti-mouse CD45 percp (BioLegend, 103129); anti-human/mouse GPR91/SUCNR1 FITC (Alomone, ASR-090-F); anti-ouse CD80 PE (BioLegend, 104707), anti-mouse CD86 ALexa fluor 700 (BioLegend, 105024); anti-mouse CD163 BV421 (BioLegend, 155309); anti-mouse CD206 BV605 (BioLegend, 141721), anti-mouse CD11b PCy 7(BioLegend, 101215); anti-mouse F4/80 APC (BioLegend, 123116).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 ELISA Assay\u003c/h2\u003e \u003cp\u003eThe level of succinate in the PF or cell supernatants were measured using ELISA assay. Briefly, HMrSV5 cells, ESCs, and macrophages polarized from THP-1 cells were seeded into 6-well plates. After culturing for 48 h, the cells were treated with BMDM alone, IL-6 (100 ng/ml) or CCL2 (50 ng/ml) for another 48 h. The cell supernatant was centrifuged at 1000 rpm (4\u0026deg; C) for 10 minutes. Peritoneal fluid was collected as described above. PF was put on ice at acquisition and then was centrifuged twice at 1500 rpm for 5 minutes at 4\u0026deg; C to remove the cells. All the samples were diluted twice and analyzed according to the specifications of the ELISA kit (Abcam, ab204718).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Quantitative Real-time PCR (RT-qPCR) Analysis\u003c/h2\u003e \u003cp\u003eTotal RNA was extracted via RNA Purification Kit (EZBioscience, USA) and reverse-transcribed into cDNA by using Hifair\u0026reg;II 1st Strand cDNA Synthesis SuperMix for qPCR (Yeasen, Shanghai, China). The quantitative PCR (RT-qPCR) was performed according to the protocol (Hieff UNICON\u0026reg; Universal Blue qPCR SYBR Green Master Mix, Yeasen). Data analysis was repeated three times and analyzed using 2\u003csup\u003e\u0026minus;ΔΔ\u003c/sup\u003eCt method. The primer sequences used in this study are listed in \u003cb\u003eSupplementar\u003c/b\u003ey \u003cb\u003eTable\u0026nbsp;2.\u003c/b\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12 Immunohistochemistry (IHC)\u003c/h2\u003e \u003cp\u003eImmunohistochemistry for SDHB was performed according to the manufacturer\u0026rsquo;s instructions. In brief, after dewaxing and antigen repair, the primary antibody for SDHB (Abcam, ab178423) was incubated overnight(4\u0026deg; C) at a 1:150 dilution. Subsequently, the membranes were incubated with the secondary antibody at room temperature for half an hour. Sections were stained with hematoxylin and photographed under a microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.13 Western Blotting Assay\u003c/h2\u003e \u003cp\u003eHMrSV5 cells (2\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/well) were seeded in 6-well plates and treated with different concentrations of succinate (0, 0.5, 1, 2.5 and 5 mM) for 48 h. Proteins were extracted by cell lysis. The protein sample (15 \u0026micro;g/lane) was evaluated with electrophoresis and transfected into 0.45 \u0026micro;m polyvinylidene fluoride (PVDF) which was stained with ECL after incubating with the primary antibody anti-ICAM1 (Abcam, ab53013) (1:1000) at 4\u0026deg; C overnight and with a secondary antibody(1:5000) at 24\u0026deg;C for 2 h. The total gray scale of each strip was quantified using ImageJ software with the values normalized based on housekeeping proteins (i.e., \u003cem\u003eβ\u003c/em\u003e-actin).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.14 Mouse Model of EMs\u003c/h2\u003e \u003cp\u003eThirty adult C57BL/6 female mice (6\u0026ndash;8 weeks, weight 20\u0026thinsp;\u0026plusmn;\u0026thinsp;2g) were purchased from the Laboratory Animal Facility of Fudan University and used for this study. The animal protocols were approved by the Ethics Committee of the Obstetrics and Gynecology Hospital, Fudan University. All the mice were randomly assigned to one of the three groups. Intraperitoneal EMs-like lesions were surgically induced by injecting fragments of uterine tissue into the peritoneal cavity. 17-β-Estradiol-3-benzoate (30 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\mu\\)\u003c/span\u003e\u003c/span\u003eg/kg, Sigma) was administered to each postoperative mouse every 3 days for 14 days. Three days after surgery, each mouse in the experimental group received succinate (100 mg/kg, Sigma) intraperitoneally every 3 days for 14 days. PBS was used instead of succinate for the sham-operated group. In the control group, no surgery was performed and PBS was used instead of succinate. Fourteen days after the operation, endometrial-like lesions were established, the mice were sacrificed, and peritoneal lavage fluids and ectopic lesions were harvested. SUCNR1 and M1/M2 macrophage markers were measured and analyzed via FCM. MMP9 and ICAM-1 of lesions were detected via IHC.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Succinate Accumulation in Peritoneal Fluid and Clinical Relevance in EMs\u003c/h2\u003e \u003cp\u003eIn order to assess the level of metabolites in the peritoneal fluid(PF), we applied targeted organic acid metabolomic analysis to study the differences in organic acid profiles in healthy and EMs subjects. The results showed significantly difference in the peritoneal fluid between EMs and non-EMs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). As found by assessing metabolomics results of PF, four organic acid metabolites, including 5-hydroxymethy-2-furancarbosylic acid, 2-hydroxyhippuric acid, succinic acid, and 2-hydroxy-3-methylpentanoic acid, were clearly elevated in EMs patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eB-D). Specifically, succinate was significantly increased in EMs patients (328.65\u0026thinsp;\u0026plusmn;\u0026thinsp;105.4ng/ml) in comparison with those in the non-EMs group (244.27\u0026thinsp;\u0026plusmn;\u0026thinsp;43.76 ng/ml), which corresponded with the ELISA results \u003cb\u003e(Supplementary Fig.\u0026nbsp;1A)\u003c/b\u003e. Succinate dehydrogenase (SDH), with unique characteristic of oxidation of succinate to fumarate, is a classic mitochondrial enzyme. Immunohistochemical staining revealed a decreased expression of SDHB in ectopic lesions when compared to that in the normal endometrium \u003cb\u003e(Supplementary Fig.\u0026nbsp;1B)\u003c/b\u003e. Metabolic pathway enrichment analysis showed that pathways were differentially regulated between EMs and non-EMs, oxidative phosphorylation and citrate cycle (TCA cycle) pathways were significantly upregulated in EMs group.\u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e1\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSuccinate is an important intracellular metabolic intermediate. Although normally regarded as an intermediate, succinate accumulates under certain pathophysiological conditions, especially at the sites of inflammation and metabolic stress (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Numerous studies have shown that succinate is not simply an inert byproduct of metabolism, but that it also plays an active role in downstream cellular responses and can have tissue-specific and systemic effects as a proinflammatory mediator (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Similarly, we found that higher levels of succinate accumulate in the peritoneal fluid of patients with severe EMs (stages Ⅲ-Ⅳ) than in those with mild EMs (stages Ⅰ-Ⅱ)(Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Therefore, we speculated that succinate levels may reflect disease severity. To some extent, succinate has potential clinical value in reflecting EM severity (AUC\u0026thinsp;=\u0026thinsp;0.951) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Furthermore, Pearson's correlation analysis was conducted on the EMs clinical data, and the results revealed a linear correlation between succinate and clinical symptoms/indicators, such as pain (VAS, R2\u0026thinsp;=\u0026thinsp;0.46, P\u0026thinsp;\u0026lt;\u0026thinsp;0.001, 95% confidence interval: 0.15\u0026ndash;0.68), EMs stage according to the revised American Fertility Society (rAFS,1985) score (rAFS, R2\u0026thinsp;=\u0026thinsp;0.38, P\u0026thinsp;=\u0026thinsp;0.02, 95% confidence interval: 0.06\u0026ndash;0.63), and fertility prediction after EMs surgical staging (EFI, R2 = -0.44, P\u0026thinsp;\u0026lt;\u0026thinsp;0.01, 95% confidence interval: -0.67\u0026ndash; -0.13)(Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-E). \u003cb\u003eSupplementary Table\u0026nbsp;1\u003c/b\u003e presents the clinical parameters of patients with EMs and non-EMs subjects.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Succinate is Prone to Polarize M1-Like Macrophages in the Endometriotic Milieu\u003c/h2\u003e \u003cp\u003eAs succinate triggers inflammatory changes in macrophages, changes in gene expression triggered by succinate in M0 macrophages derived from THP-1 cells were compared with the marker gene expression observed in human M1 and M2 macrophages. By using a real-time quantitative polymerase chain reaction (PCR) analysis of macrophages exposed to succinate (0, 0.5, 1, 2.5, and 5 mM) for 48h, we compared the gene expression changes of M1 markers (CD80, CD86) with M2 markers (CD206, CD163) triggered in M0 macrophages by succinate. As illustrated in \u003cb\u003eSupplementary Fig.\u0026nbsp;2A-E\u003c/b\u003e, in resting M0 macrophages without lipopolysaccharide (LPS) stimulation exposed to succinate, genes that were preferentially expressed by M1 macrophages were upregulated by succinate, and genes that were preferentially expressed in M2 macrophages were downregulated by succinate. Thus, succinate exposure causes polarization of na\u0026iuml;ve macrophages toward M1. As previously reported(\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e), the effects of extracellular succinate and LPS can be superimposed, and can especially augment the LPS-driven M1 phenotype (\u003cb\u003eSupplementary Fig.\u0026nbsp;2C).\u003c/b\u003e Additionally, we found that succinate also markedly induced the proinflammatory cytokine IL-8, IL-1β, and IL-6 transcription in macrophages, however, a higher concentration of succinate (5 mM) created an opposite effect in IL-8 and IL-6 (Supplementary Fig.\u0026nbsp;2F-I).\u003c/p\u003e \u003cp\u003eTo evaluate the expression of SUCNR1 in M0 macrophages exposed to succinate, we chose to to detect the transcription of SUCNR1 via real-time PCR. The results showed a dose-dependent expression of \u003cem\u003eSUCNR1\u003c/em\u003e mRNA in M0 macrophages exposed to succinate as compared to that in the vehicle (\u003cb\u003eSupplementary Fig.\u0026nbsp;2J\u003c/b\u003e). Succinate appears to activate inflammatory pathways and the switch to the M1-like phenotype, at least in part, via SUCNR1. These data demonstrate that exposing monocyte-derived macrophages to relevant concentrations of extracellular succinate unequivocally regulates the expression of immune function genes, resulting in the polarization of the M1-like phenotype or synergism with LPS.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Extracellular Succinate is Mainly Released From M1-Polarized Macrophages and Peritoneal Mesothelial Cells\u003c/h2\u003e \u003cp\u003eMacrophage were the most common population (~\u0026thinsp;60%) of PF leukocytes in patients with EMs, and the peritoneal mesothelial cell (PMC) monolayer that lines the abdominal cavity is the first barrier encountered by menstrual fragments. To understand the capacity of extracellular succinate secretion among these cell types, THP-1 cells were respectively polarized into the M1 phenotype with LPS and IFNγ, or into the M2 phenotype with IL-4 and IL-13 as previously described (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). Macrophages in different polarizing states were cultured for 48 h, and the levels of extracellular succinate in the cell supernatant were measured by ELISA. Consistently with previous reports that M1 polarized macrophages are major producers of succinate, our study showed that the production of succinate in M1 polarized macrophages and peritoneal mesothelial cells was higher than that in na\u0026iuml;ve and M2-polarized macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo identify the main source of succinate in the endometriotic milieu, a co-culture model with ESC, peritoneal mesothelial cell line HMrSV5, or THP-1 cells was constructed to imitate the ectopic immune microenvironment of EMs. High levels of succinate were observed in the culture supernatants of M1-polarized macrophages after being co-cultured with HMrSV5 cells or ESCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). As reported in our previous studies, some inflammatory cytokines (such as IL-6 and CCL-2) are significantly elevated in the peritoneal fluid of patients with EMs (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). Interestingly, co-culture of resting M0 cells with HMrSV5 cells or ESCs also triggered succinate release in macrophages, but not when stimulated with IL-6 or CCL2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eC), showing that contact with ESCs or HMrSV5 cells, rather than the M0 phenotype, determines succinate secretion. In parallel, MCP-1 from the endometriotic milieu failed to induce succinate secretion in HMrSV5 cells; however, IL-6 stimulation or interaction with ESCs significantly increased succinate production (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Based on these findings, such changes in the ectopic milieu establish a vicious circle, with each condition promoting the other and accelerating succinate accumulation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.4 SUCNR1 is Elevated in Ectopic ESCs and Macrophages From EMs Patients\u003c/h2\u003e \u003cp\u003eTo analyze the expression and distribution of SUCNR1 in human tissues, we found, via the Human Protein Atlas/Dataset (data available from proteinatlas.org), that SUCNR1 expression in normal female tissues was low under physiological homeostasis but high in their immune system and gastrointestinal tract (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo analyze whether the accumulation of succinate participates in EMs progression, we further measured SUCNR1 expression in CD45\u003csup\u003e+\u003c/sup\u003eCD14\u003csup\u003e+\u003c/sup\u003e macrophages, PMCs, and ESCs from patients with or without EMs via FCM. Serosal healing involves free-floating mesothelial cells (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e), and our results showed the macroscopic shed mesothelial cells float in clumps in peritoneal fluid (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eE), and found a 16-fold increase of SUCNR1 expression in mesothelial cell mass of EMs patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eB-C). Macrophages from the PF of EMs patients underwent significant phenotypic changes, with a 1.8 to 3.9-fold increase of SUCNR1 expression in macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, D), and a parallel 2-fold increase in SUCNR1 expression in ectopic ESCs (hESC.D) compared with ESC cell line (ESC.L) and primary normal ESCs (hESC.N) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eF-G). Accordingly, we evaluated the effect of succinate on SUCNR1 expression in HMrSV5 cells. Increasing the succinate concentrations induced a dose-dependent increase in SUCNR1 expression in HMrSV5 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e4\u003c/span\u003eH-I), indicating an important role of succinate in mesothelial cell function during EMs lesion formation. Using this assay, the optimal concentration of 2.5mM was determined.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Succinate Enhances ESCs Survival and Implantation Capacity via the SUCNR1\u003c/h2\u003e \u003cp\u003eGiven the importance of extracellular succinate as an immunometabolic signal in the ectopic milieu, we investigated whether succinate released by type 1 proinflammatory macrophages could regulate the activity of surrounding PMCs in the peritoneal cavity, including that of refluxed and colonized ESCs. Considering that endometriotic lesion formation is a multi-step process that includes not only endometrial tissue proliferation but also antiapoptosis and invasion, we investigated whether succinate might acts as a chemoattractant for ESCs by using a transwell migration assay. The average number of ESCs per field that migrated toward succinate after 48 h was significantly higher than that in the vehicle. This invasive effect was dose-dependent and equivalent to that of CCL2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Next, we analyzed the migration of ESCs into the wounded cell-free areas using a scratch assay. Time-lapse imaging revealed the ability of succinate to promote wound healing in ESCs in a concentration dependent manner, reaching statistical significance upon treatment with 1 mM of succinate relative to the vehicle, up to the maximal effect at the optimal concentration (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Furthermore, we performed cell viability assay on ESCs and HMrSV5 cells treated with succinate for 24 and 48 h. The OD450 analysis showed a significantly higher survival capacity of ESCs treated with succinate when compared to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eC), similarly to the effect found in HMrSV5 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Finally, we performed the AnnexinV/propidium iodide staining of ESCs and HMrSV5 cells treated with a vehicle or succinate for 48 h. We evaluated the percentages of apoptotic and live cells and observed no differences with succinate compared to the vehicle (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e5\u003c/span\u003eE-F).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e3.6 Succinate-Stimulated Peritoneal Mesothelial Cells Recruit Macrophages and Boost Ectopic Growth and Implantation of ESCs\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eBased on the above assay, we showed that succinate promotes various endometriotic processes in ESCs, including survival, migration, adhesion and invasion. As macrophages are the principal mediators of pathological EMs, we considered whether succinate could recruit macrophages and promote the adhesion of ESCs to HMrSV5 cells. As we previously reported that endometrial stromal cells from the ectopic milieu continuously recruit monocytes and is beneficial to expansion of monocyte-derived CCR2\u003csup\u003e+\u003c/sup\u003e macrophages (\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e), we then tested the ability of HMrSV5 cells to attract macrophages after succinate stimulation. Our results revealed that treatment of HMrSV5 cells with succinate (0, 1, and 2.5 mM) for 48 h remarkably induced CCL2 gene expression and protein secretion (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-B). Next, in a co-culture system of ESCs in the upper chamber and HMrSV5 cells exposed to 2.5mM succinate in the lower chamber for 48 h, we then replaced ESCs with THP-1 cells that were was pre-labeled with the CellTracker red and cultured them for another 12h. The chemotaxis assay showed that HMrSV5 cells exposed to succinate readily attracted the THP-1 (CCL2 treatment was the positive control) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e, suggesting that this chemotactic effect on monocytes/macrophages might be mediated by the CCL2 which was secreted by mesothelial cells after stress.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAlthough the theory of retrograde menstruation and immune disorders is helpful for understanding EMs, the mechanisms underlying the pathological factors and their roles in ectopic implantation and aggressive growth of ESCs are still poorly understood. In this study, we analyzed the effect of succinate on the adhesion of ESCs to peritoneal mesothelial cells. The adhesion assay showed that succinate triggered the adhesion of ESCs to HMrSV5 cells more strongly than the vehicle, and that enhanced adhesion occurred when HMrSV5 cells were exposed to 2.5mM succinate \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e. Further investigations showed that succinate-driven adhesion of ESCs to HMrSV5 cells might depend on the expression of ICAM-1 because succinate induced a concentration-dependent increase of ICAM-1 expression and of the adhesion capacity of HMrSV5 cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003eE-F\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eThus, succinate may play a pivotal role in the implantation growth of ESCs. Next, we investigated whether succinate derived from the ectopic milieu could induce the invasion and migration of ESCs in the presence of HMrSV5 cells. This effect was evaluated using a Matrigel invasion assay. ESCs in the presence of HMrSV5 cells were stimulated with a vehicle or with 2.5 mM succinate for 48 h, and CCL2 treatment was used as a positive control. Indeed, we observed that 2.5 mM succinate strongly induced invasion of ESCs in the presence of HMrSV5 cells \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003eG\u003cb\u003e)\u003c/b\u003e. Matrix metalloproteinases (MMPs) are vital regulators of invasion and extracellular matrix remodeling. We also demonstrated that succinate (0.5\u0026ndash;2.5 mM) stimulation induced the expression of MMPs in HMrSV5 cells, such as MMP2, MMP9 and MMP11 \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e6\u003c/span\u003eH\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eFurther \u003cem\u003ein vivo\u003c/em\u003e analysis of the role of succinate in EMs progression was performed using an EMs allograft model. The body weight of the mice and the number and weight of lesions were recorded after treatment with either succinate (100 mg/kg) or PBS \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-B\u003cb\u003e)\u003c/b\u003e. Our results showed that the intraperitoneal injection of succinate did not affect the body weight of BALB/c mice \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e7\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e or the ectopic lesion weight \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e7\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e. Interestingly, succinate-treated mice showed more ectopic lesions \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e7\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e. To analyze the expression of SUCNR1 in M1 or M2 macrophages, SUCNR1 and CD80/CD163 on CD11b(+)F4/80(+) macrophages were assessed for mean fluorescence intensity (MFI) using Flow cytometry (FCM). The results revealed that SUCNR1 expression was higher in both on M1-like or M2-like peritoneal macrophages in the succinate exposed group than in the control and model groups \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e7\u003c/span\u003eE-I\u003cb\u003e)\u003c/b\u003e. Similarly, succinate-exposure increaded the expression of ICAM-1) and MMP9 in ectopic lesions in compare with that of model group (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e7\u003c/span\u003eJ).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn summary, these data indicate that succinate accumulation induced by polarized macrophages and peritoneal mesothelial cells obviously enhances the aggressive implantation of ectopic ESCs and the adhesion between ESCs and peritoneal mesothelial cells, promoting the progression of EMs via SUCNR1 signaling.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eMetabolites have been considered as the vital players in metabolism over the past few decades, and have been recently proven to have key immune regulatory functions in recent years. Among these, succinate stands out for its multifaceted roles in immune and metabolic function regulation (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e), dynamic changes, and selective cell release(\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Notably, several pathogenic states, such as obesity (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e), diabetes (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e), hypertension (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e), and various inflammatory conditions (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e), have been associated with elevated levels of extracellular succinate in the body.\u003c/p\u003e \u003cp\u003eOur study identified an important axis that comprises succinate and its cognate receptor, SUCNR1, as the potential drivers of ectopic endometrial survival and adhesion in the EMs milieu. As EMs progresses, succinate accumulates in the PF of EMs patients. Hypoactivity/deficiency or overproduction of SDH may cause succinate accumulation in the ectopic milieu and is associated with a range of clinical symptoms in EMs, such as dysmenorrhea, and with the revised American Fertility Society (rAFS) classification and the Endometriosis Fertility Index (EFI).\u003c/p\u003e \u003cp\u003eIn the present study, we investigated the main sources of succinate in the extracellular fluid of patients with EMs. It has been previously revealed that under the stimulation or polarization with LPS, macrophages changed from oxidative phosphorylation to glycolysis, which was accompanied by the elevated level of intracellular and extracellular succinate (\u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). Our data revealed that succinate was mainly from the type 1 polarized macrophages. Surprisingly, peritoneal mesothelial cells produced high levels of succinate comparable to those produced by M1 macrophages. Conditions that trigger succinate secretion include stimulation by the inflammatory cytokine IL-6 and exposure of stromal cells. The elevations of succinate in the cell supernatant partly depended on the direct physical contact between the mesothelial cells/macrophages and the ESCs. However, mechanistic insights into the effects of dysregulated succinate levels on ectopic cellular functions in this pathology remain unavailable.\u003c/p\u003e \u003cp\u003eSUCNR1 is widely and heterogeneously expressed in various cell types throughout the body. The most well-studied SUCNR1-expressing cells are monocytes and macrophages (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). In addition, many non-immune tissues, including the intestine (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e), placenta (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e), skeletal muscle satellite (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e), and endothelial (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e), express SUCNR1 and respond to paracrine signaling in the form of succinate secretion in response to local pathologies. It has been speculated that the inflammation and fibrosis associated with these pathologies are, at least in part, attributable to chronic SUCNR1 agonism in tissue-resident cell populations. Whereas succinate-SUCNR1 interplay has been proposed as the molecular mechanism in rheumatoid arthritis (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e) and intestinal inflammation (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e), our data also imply a role of this pathway also in EMs formation. We confirmed the expression of SUCNR1 in ESCs and PMCs. FCM analysis highlighted the differences in SUCNR1 expression among various stromal cells, which may be reflected in their responses to succinate stimuli via SUCNR1. The FCM and real-time PCR results demonstrated that SUCNR1 expression was higher in primary ectopic ESCs than in normal ESCs, suggesting that the succinate-SUCNR1 signal might be a potential driver of EMs. Indeed, we confirmed that succinate-SUCNR1 signaling is involved in the survival and adhesion of ESCs, in a battery of important steps in ectopic endometrial lesion formation. Functional experiments verified that succinate promoted ESC survival and invasion, as well as that it conferred antiapoptotic effects. Interestingly, exogenous succinate increased the mRNA and protein expression of SUCNR1 in HMrSV5 cells, further secreted pro-inflammatory CCL2 recruiting macrophages, and induced remarkable adhesion between HMrSV5 cells and ESCs, which implyies that these changes among cells in the ectopic milieu trigger a vicious circle of EMs progression.\u003c/p\u003e \u003cp\u003eIn our study, succinate drove macrophages to polarize toward M1 phenotype, which is consistent with a previous study (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). The assessment of IL-8 via ELISA, and the SUCNR1membrane receptor and macrophage surface markers (CD80, CD86, CD163, and CD206) using FCM corroborated the proinflammatory potential of succinate stimulation, which was similar to that observed in LPS-stimulated macrophages. However, succinate at a certain range of concentrations endows macrophages with proinflammatory function, whereas excessively high concentrations of succinate (5mM) impair IL-8 production. Furthermore, extracellular succinate synergized with LPS during macrophage polarization toward to the M1 phenotype and proinflammatory functional transformation. Our findings highlight the importance of succinate-SUCNR1 signaling in macrophage polarization and suggest its role in immune regulation.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn summary, our results show that patients with EMs present with high levels of succinate in the peritoneal fluid and with increased SUCNR1 expression in ESCs. This succinate-SUCNR1 axis exacerbates the inflammatory activity of macrophages and ESC activation and plays a role in endometrial lesion formation and peritoneal adhesion. Our study demonstrated, for the first time, the role of succinate and its receptors in EMs. We propose that, in the ectopic milieu of patients with EMs, SUCNR1 signaling exacerbates inflammation and benefits the invasion, survival, and adhesive growth of ectopic ESCs, indicating a possible target for EMs treatment.\u003c/p\u003e"},{"header":"6. Statistics","content":"\u003cp\u003eSpearman\u0026rsquo;s correlation analysis was used to analyze the correlation between extracellular succinate levels and clinical symptoms in humans. The diagnostic performance of succinate was determined using the area under the receiver operating characteristic curve (AUROC) analysis to assess the overall discriminatory power of these assays in predicting EMs progression. The continuous variables are shown as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Data from two groups were analyzed by the Student\u0026rsquo;s t-test, where data from multiple groups were analyzed by the one-way ANOVA using Tukey\u0026rsquo;s post-hoc test. Statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS Inc., Chicago, 26.0 version) and the Prism5.0 software(GraphPad Software Inc.). A P-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe metabolite data used and analysis during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by the Shanghai Shen Kang Hospital Development Center (SHDC12019106 and SHDC12019X27), National Natural Science Foundation of China (31600735),and Five-year Project Plan of the Whole Life Cycle Health Research Institute of Fudan University: Construction Project (DGF50107-037001)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the Shanghai Luming biological technology co., LTD (Shanghai, China) for their enthusiastic support of this metabolomics analysis, Xue-chun Dong, and Xiao-hong Zhou (Luming Biotech Co., Ltd, Shanghai, China) for providing metabolomics services.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe research was approved by the Ethics Committee of the Obstetrics and Gynecology Hospital, Fudan University (2020-137). All participants provided written informed consent, and the permissions for obtain data.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eAuthor Contribution\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQ.T. and J.Y.R. are joint first authors. Conception and design: K.K.C, X.F.Y. Development of methodology: Q.T., J.Y.R, Y.N.W, Q.C, Y.C and Y.P.X. Data curation: Q.T. and J.Y.R. Writing, review and/or revision of the manuscript: K.K.C., Q.T., M.Q.L and X.F.Y. Study supervision: X.F.Y. All authors read and approved the fnal manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003e\u003cspan\u003eFernandez-Veledo S, Ceperuelo-Mallafre V, Vendrell J. Rethinking succinate: an unexpected hormone-like metabolite in energy homeostasis. Trends Endocrinol Metab. 2021;32(9):680\u0026ndash;92.\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eAdamson GD, Pasta DJ. 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Gene. 2018;663:8.\u003c/span\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-communication-and-signaling","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ccas","sideBox":"Learn more about [Cell Communication and Signaling](http://biosignaling.biomedcentral.com/)","snPcode":"12964","submissionUrl":"https://submission.nature.com/new-submission/12964/3","title":"Cell Communication and Signaling","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Endometriosis, Succinate, Peritoneal Mesothelial cell, Endometrial stromal cells (ESCs), Macrophage, SUCNR1","lastPublishedDoi":"10.21203/rs.3.rs-3303001/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3303001/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eAs 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. Meanwhile, extracellular succinate released into the microenvironment is considered an inflammatory alarm that can be sensed by its membrane receptor SUCNR1, boosts proinflammatory responses and acts akin to classical hormones and cytokines. Succinate has been reported to play an important role in inflammatory disease. It is worth exploring whether succinate can facilitate the progress of endometriosis (EMs), which is characterized by chronic inflammation and peritoneal adhesion.\u003c/p\u003e\u003ch2\u003eObjective\u003c/h2\u003e \u003cp\u003eTo evaluate the main source and potential role of succinate in endometriosis, we mimics the ectopic milieu \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. The molecular and functional effects of succinate on macrophages and peritoneal mesothelial cells in peritoneal cavity were assessed. The succinate/SUCNR1 signal acting on ectopic endometrial stromal cells (ESCs) was further explored in this study.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eIn this study, we used targeted organic acid metabolomics analysis and \u003cem\u003ein vitro\u003c/em\u003e assay to assess whether there was an obvious accumulation of succinate in the peritoneal fluid of EMs patients and its correlated with disease severity, Visual Analogue Scale (VAS), and the Endometriosis Fertility Index (EFI). Flow cytometry, Enzyme linked immunosorbent assay (ELISA), western-blot assay, and quantitative real-time PCR, and other molecular biology techniques were used for exploring the potential mechanisms.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eBy mimicking the ectopic milieu, we constructed an \u003cem\u003ein vitro\u003c/em\u003e co-culture system and found that M1 polarized macrophages and the peritoneal mesothelial cell line (HMrSV5) mainly released succinate into their microenvironment and activated the succinate receptor (SUCNR1) signal, which further polarizes macrophages and significantly enhances the invasive survival of ESCs, and the adhesion with peritoneum. We further investigated the pathological effect of extracellular succinate \u003cem\u003ein vivo\u003c/em\u003e using xenograft mouse models of endometriosis.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eCollectively, the succinate-SUCNR1 signal facilitates in creating the inflammatory nice and plays a vital role in EMs progression and peritoneal adhesion. Our work on the molecular mechanism of succinate accumulation and function will be helpful to elucidate the phenotypic mystery of pain and infertility in EMs.\u003c/p\u003e","manuscriptTitle":"Extracellular Succinate Derived From Ectopic Milieu Drives Adhesion and Implantation Growth of Endometrial stromal cells via the SUCNR1 signal in endometriosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-09-07 14:28:16","doi":"10.21203/rs.3.rs-3303001/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2023-10-12T14:17:44+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2023-10-09T01:18:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"873f978f-466d-4b06-9d39-89f4552469a5","date":"2023-09-30T10:25:51+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-09-14T22:35:31+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2023-09-12T13:18:34+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-09-12T13:18:34+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell Communication and Signaling","date":"2023-08-28T10:35:28+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-communication-and-signaling","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ccas","sideBox":"Learn more about [Cell Communication and Signaling](http://biosignaling.biomedcentral.com/)","snPcode":"12964","submissionUrl":"https://submission.nature.com/new-submission/12964/3","title":"Cell Communication and Signaling","twitterHandle":"@bmc","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"1faf8594-1b80-40bd-8581-285192345446","owner":[],"postedDate":"September 7th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-02-05T15:03:37+00:00","versionOfRecord":{"articleIdentity":"rs-3303001","link":"https://doi.org/10.1186/s12964-023-01415-7","journal":{"identity":"cell-communication-and-signaling","isVorOnly":false,"title":"Cell Communication and Signaling"},"publishedOn":"2024-01-30 15:01:12","publishedOnDateReadable":"January 30th, 2024"},"versionCreatedAt":"2023-09-07 14:28:16","video":"","vorDoi":"10.1186/s12964-023-01415-7","vorDoiUrl":"https://doi.org/10.1186/s12964-023-01415-7","workflowStages":[]},"version":"v1","identity":"rs-3303001","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3303001","identity":"rs-3303001","version":["v1"]},"buildId":"B-jG_2CBjPDmsCi4Wdhf-","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}