Exosomal miR-146a-5p mediates macrophage polarization through TRAF6/NF-κB signaling in endometriosis

Exosomal miR-146a-5p mediates macrophage polarization through TRAF6/NF-κB signaling 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 Article Exosomal miR-146a-5p mediates macrophage polarization through TRAF6/NF-κB signaling in endometriosis Ming Yuan, Xingfei Lu, Yuxia Tang, Jiayi Li, Kaiqing Lin This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4988177/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Exosomes play significant roles in immune responses, neurogenesis, and angiogenesis, directly impacting the progression and symptomatic manifestations of endometriosis. This study aimed to investigate the role of exosomal miR-146a-5p in the pathogenesis of endometriosis. Through high-throughput RNA sequencing and qRT‒PCR, we revealed significant upregulation of miR-146a-5p in ectopic endothelial tissues, and Gene Ontology (GO) analysis revealed that miR-146a-5p has only one target, TNF receptor associated factor 6 (TRAF6). KEGG pathway analysis indicated that the NF-κB signaling pathway is the key signaling pathway involved. The study revealed that the upregulation of miR-146a-5p in macrophages is associated with an increase in M2 macrophages. In the U937 macrophage line, miR-146a-5p was capable of suppressing TRAF6 expression, which in turn decreased the phosphorylation level of NF-κB, whereas the overexpression of TRAF6 increased the activity of this pathway. Furthermore, we incorporated the NF-κB inhibitor EVP4593 into a macrophage culture, which revealed that blocking this pathway significantly induced both M1 and M2 macrophage polarization, particularly enhancing M2 polarization. In conclusion, exosome-derived miR-146a-5p promotes M2 polarization of macrophages by regulating the TRAF6/NF-κB pathway in endometriosis. Health sciences/Medical research/Biomarkers/Predictive markers Biological sciences/Molecular biology/Non coding rnas/Mirnas Health sciences/Diseases/Reproductive disorders Biological sciences/Immunology/Innate immune cells/Monocytes and macrophages Exosome Endometriosis miR-146a-5p Macrophage polarization Endometrial stromal cells Signaling pathway Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Endometriosis is an estrogen-dependent disease characterized by the presence of endometrial stromal cells (ESCs) and glands outside the uterine cavity, which can cause pelvic pain and infertility 1,2 . Although endometriosis is a benign disease, it is associated with malignant biological behaviors such as distant metastasis, adhesion, invasion, implantation, and recurrence 3 . This process is associated with genes related to pathways such as cell proliferation, angiogenesis, neurogenesis, and inflammation 4 . Recently, emerging evidence has suggested that genetic predispositions and associated immunological abnormalities are key to the pathogenesis of endometriosis 5 . Under normal conditions, peritoneal macrophages are capable of eliminating endometrial tissue that enters the peritoneal cavity via retrograde blood flow 6 . However, in patients with endometriosis, dysfunctional immune responses lead to a decrease in the phagocytic capacity of macrophages or alterations in the cytokine environment, often resulting in the ineffective surveillance and clearance of endometrial tissue deposited within the peritoneal cavity 7,8 . This defect in immunosurveillance allows the growth of endometriotic lesions and the persistence of disease symptoms 9 . The polarization state of macrophages is reversible, allowing them to change phenotypes according to the needs of the microenvironment 10,11 . Macrophages initially present as the M1 phenotype, secreting proinflammatory cytokines such as TNF-α, IL-1β, and IL-6 that trigger an inflammatory response, whereas M2-type macrophages secrete anti-inflammatory factors to suppress inflammation and maintain homeostasis 12–14 . The ability of macrophages to dynamically switch their phenotype and function in response to microenvironment signals makes them potential therapeutic targets. Notably, the role of microRNAs in regulating macrophage polarization has been well defined, with the overexpression of miRNA-155 shown to inhibit C/EBPβ protein expression and regulate the production of inflammatory cytokines in TAMs by targeting C/EBP 15 . Furthermore, numerous studies have investigated the function of microRNAs in endometriosis, with findings indicating that microRNAs are reliable biomarkers for this disease 16,17 . Therefore, we suspect that the dynamic switching of macrophage phenotypes may be influenced by microRNAs. Our preliminary experimental results suggested that exosomes derived from ectopic ESCs promote M2 macrophage polarization by delivering miR-146a-5p, which targets TRAF6, in the pathological process of endometriosis 15 . TRAF6 can activate multiple signaling pathways, such as the NF-κB pathway 18 . Additionally, research supports a close relationship between microRNAs and the NF-κB signaling pathway in certain diseases, including rheumatoid arthritis, cervical cancer, and colorectal cancer 19–23 . Thus, we hypothesized that miR-146a-5p may play an important role in the polarization process of endometriosis-related macrophages. Results Exosomes from ectopic ESCs enter macrophages. To investigate the impact of ESC-derived exosomes on macrophages, we isolated exosomes from eutopic and ectopic ESCs (vimentin positive, CK7 negative; Fig. 1 A). Transwell assays and exosome tracking experiments verified that the exosomes derived from the mesenchymal stromal cells of the donor could reach the recipient cells and be internalized, along with their contained cellular contents. The internalization of exosomes by U937 macrophages was visualized through Cy3 red fluorescence labeling (Fig. 1 B). Exosomes induce macrophage polarization toward the M2 phenotype by secreting miR-146a-5p. In our previous study, miR-146a-5p was silenced in inhibitor-transfected ectopic ESCs and overexpressed in mimic-transfected ESCs. Consequently, in the exosomes of these transfected ESCs, the expression of miR-146a-5p was also correspondingly silenced and overexpressed 24 , indicating that miR-146a-5p facilitates the polarization of M2 macrophages (data not shown). Flow cytometry analysis revealed that after treatment with the exosome inhibitor GW4869, ectopic ESCs significantly increased the expression of CD86 and iNOS (Fig. 2 A, B), whereas the proportions of M2 macrophages expressing CD163 and CD206 were reduced (Fig. 2 C, D). Flow cytometry plots of the control and GW4869 groups are shown. miR-146a-5p inhibits TRAF6 expression by targeting its 3′UTR in macrophages. U937 macrophages treated with exosomes from miR-146a-5p mimic- or inhibitor-transfected ectopic ESCs were used to detect miR-146a-5p and TRAF6 levels. Exosomes transfected with the miR-146a-5p inhibitor significantly increased TRAF6 expression, whereas exosomes transfected with the miR-146a-5p mimic significantly suppressed TRAF6 expression (Fig. 3 A, B). The exosomes transfected with the miR-146a-5p inhibitor significantly increased the protein expression of TRAF6 (Fig. 3 C, D), which is consistent with the changes observed at the mRNA level. Bioinformatics analysis revealed that miR-146a-5p has several binding sites in the TRAF6 3′UTR (data not shown) 24 . To assess the binding of miR-146a-5p to the TRAF6 promoter, we used luciferase reporter gene plasmid transfection. Transfection with the wild-type TRAF6 promoter led to significant inhibition of TRAF6 transcription by the miR-146a-5p mimic and significant promotion by the miR-146a-5p inhibitor (Fig. 3 E). However, after transfection with the mutated TRAF6 promoter, the regulatory effects of the miR-146a-5p mimic and inhibitor on TRAF6 transcription vanished. To investigate the regulatory effects of miR-146a-5p on TRAF6 expression, we separately detected TRAF6 expression at the mRNA (Fig. 3 F) and protein (Fig. 3 G, H) levels. The data revealed that the siRNA promoted TRAF6 expression at both the mRNA and protein levels, whereas the mimics had the opposite effect. Inhibition of the NF- κ B pathway can induce macrophage polarization toward the M2 phenotype. To further investigate the role of TRAF6, we synthesized TRAF6 overexpression plasmids and interference fragments in vitro. After transfection, we assessed the efficiency of TRAF6 overexpression and interference by qRT‒PCR and Western blotting. The oeTRAF6 construct significantly increased TRAF6 expression at both the mRNA (Fig. 4 A) and protein (Fig. 4 B, C) levels, whereas siTRAF6 significantly decreased TRAF6 expression at the mRNA (Fig. 4 D) and protein (Fig. 4 E, F) levels. To examine the downstream regulatory pathways of TRAF6, following the transfection of the TRAF6 interference fragment and overexpression plasmids, we conducted Western blot analysis to assess and compare the NF-κB protein expression and phosphorylation levels among the overexpression group, the downregulation group, and their respective control groups, which revealed that TRAF6 increased NF-κB signaling pathway activation (Fig. 4 G, H). In the U937 macrophage line culture system, the NF-κB pathway inhibitor EVP4593 was introduced, and the results were compared with those of a blank control group. Flow cytometry at 0, 24, and 48 h posttreatment revealed increased expression levels of CD86, INOS, CD163, and CD206 (Fig. 5 A-D). The M1/M2 ratio graph revealed a significant increase in M2 cells at 48 h, and the line graph indicates that the inhibitor induced both M1 and M2 polarization, with increased M2 induction (Fig. 5 E). A flow cytometry plot at 48 h is shown (Fig. 5 F). The data revealed significant differences between type M1 macrophages (CD86 and iNOS) and type M2 macrophages (CD163 and CD206) after 48 h (Fig. 5 G). Discussion In our present study, through high-throughput sequencing and bioinformatics analyses, we identified miR-146a-5p for further study, and we showed that both miR-146a-5p in ectopic endometrial stromal cells and exosomal miR-146a-5p were highly expressed in endometriosis. miR-146a-5p is involved in inflammation and regulates the differentiation and function of immune cells 25–28 . Further GO analysis of miR-146a-5p was conducted to examine its gene functions in biological processes, cellular components, and molecular functions and to predict its target genes. The results showed that its target was exclusively TRAF6, a key signaling adaptor protein that plays roles primarily in immune and inflammatory responses. KEGG pathway analysis revealed key pathways, including the MAPK, JNK, and NF-κB signaling pathways. A previous study revealed a significant association between miR-146a and an increased incidence of endometriosis 29 , and the role of miRNAs in regulating macrophage polarization has been confirmed 30 . Thus, we speculate that miR-146a-5p plays a significant role in the pathogenesis of endometriosis by mediating TRAF6. This study utilizes the signaling function of exosomes to connect the pathogenicity of endometriosis with the functional transformation of macrophages, investigating the role and molecular mechanisms of macrophage functional transformation in endometriosis. GW4869 is a noncompetitive inhibitor of N-Smase (neutral sphingomyelinase) that can reduce the amount of exosomes released into conditioned medium 31 . By adding GW4689 to inhibit the secretion of exosomes and the release of miR-146a-5p from ectopic ESCs and detecting the levels of macrophage markers, we found that the polarization of macrophages toward the M1 phenotype was increased. Second, inhibitors and mimics were used to alter miR-146a-5p levels in ESCs and their exosomes, and we observed that the miR-146a-5p inhibitor significantly increased TRAF6 expression in macrophages. TRAF6 is an E3 ubiquitin ligase that is involved in numerous inflammatory diseases, and its primary function is focused on the regulation of the immune system 32 . Third, we overexpressed TRAF6 and transfected interference plasmids in U937 cells, and the results showed that an inhibitor of TRAF6 led to the suppression of the NF-κB pathway. This effect was similar to that of the NF-κB pathway-specific inhibitor EVP4593, resulting in an increased proportion of M2-type macrophages. The inhibition of this signaling cascade has been proven to suppress M1 polarization activity 33 . When the NF-κB pathway inhibitor EVP4593 was added to the U937 macrophage culture system, inhibition of the NF-κB pathway induced the polarization of macrophages toward the M2 phenotype. NF-κB is known to regulate immune responses and control the expression of inflammatory cytokines 34,35 , and NF-κB activation stimulates inflammation and cell proliferation and inhibits apoptosis, thereby promoting the development and maintenance of endometriosis 36 . The similarity between the invasive growth of ectopic endometrial tissue and the ease of cancer spread may be due to the active involvement of the stromal microenvironment in controlling invasion, whether by embryos or cancer cells 37,38 . Previous work has shown that the NF-κB signaling pathway is involved in the phenotypic conversion of M2-type macrophages to M1-type macrophages 39 . The switch from the M1 phenotype to the M2 phenotype mediates the immunosuppressive process, which is a marker of disease progression 40 . M1/M2 polarization imbalance plays a significant role in autoimmune diseases. Consequently, modulating the polarization of macrophages can ameliorate the pathogenesis of these conditions. Recent studies have shown that inhibiting the activation of NF-κB P65, thereby reducing M1 polarization, aids in the treatment of experimental autoimmune uveitis (EAU). Similar to these studies, our data showed that inhibition of the NF-κB pathway can induce macrophage polarization toward the M2 phenotype. The application of miR-146a-5p in endometriosis needs to be further verified through animal models and in vivo experiments. Moreover, the downstream JNK and MAPK pathways affected by TRAF6 are worthy of further investigation. In conclusion, our findings indicate that exosome-derived miR-146a-5p-mediated TRAF6 promotes the polarization of macrophages toward the M2 phenotype by regulating the activation of NF-κB. An increase in miR-146a-5p leads to a decrease in TRAF6 expression, which in turn reduces the phosphorylation of NF-κB. The present study may contribute to uncovering the pathogenesis of endometriosis and offer new insights for developing diagnostic and therapeutic strategies for this disease. Materials and Methods Ethical approval. The use of human samples was approved by the Women’s Hospital of Hangzhou Normal University. All the subjects signed a written informed consent. The experimental protocols and procedures related to humans were approved by the Ethics Committee of the Women’s Hospital of Hangzhou Normal University (No. 2022-A-03). All methods were performed in accordance with the relevant guidelines and regulations. Clinical samples. A total of 5 patients with endometriosis were recruited to provide ectopic and eutopic endometrial tissue when they underwent laparoscopy/hysteroscopy treatment at the Women’s Hospital of Hangzhou Normal University. These samples were obtained during the proliferative phase of the menstrual cycle, which was determined based on preoperative medical history and histological examination. The inclusion and exclusion criteria were as follows: regular menstruation for a period of 28–32 days; and patients who had received hormone therapy and contraception. Coculture of macrophages and ESCs. Primary ESCs of ectopic and eutopic endometria were cultured as described in previous studies 24 . The human histiocytic lymphoma cell line U937 was obtained from the cell bank of the Shanghai Academy of Biological Sciences. The medium consisted of RPMI 1640 (Millipore, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, USA) and a 1% dual-antibiotic penicillin‒streptomycin mixture (PS) (Solarbio, China). The ectopic ESC culture conditions were 10% FBS + 1% PS + DMEM/F12 (HyClone, USA). All the cells were cultured in a humidified incubator containing 5% CO 2 at 37 °C. Exosome extraction and characterization. Specimens of ovarian endometrial tissue were collected, and ectopic ESCs were isolated and cultured with exosome-free serum in primary culture for 48 h. Exosomes were isolated with an exosome isolation kit (Invitrogen, USA). The isolated exosomes were stored at −80 °C as a backup. The purity of the cells was determined by immunofluorescence using vimentin and C-cell keratin factor (CK7). Transwell assays. Transwell inserts (Corning, 3413) were used to detect cell migration and invasion. The isolated ectopic endothelial mesenchymal stromal cells were cocultured with the U937 macrophage line. Cy3-labeled miR-146a-5p molecules were transfected into mesenchymal stromal cells, which were then cultured in the upper chamber. U937 macrophages that did not express Cy3 were placed in the lower chamber, and red fluorescent Cy3-miR-146a-5p molecules were detected in the lower chamber after 12 hours. Plasmid construction and cell transfection. The miR-146a-5p mimics (5’-UGAGAACUGAAUUCCAUGGGUU-3′), the miR-146a-5p siRNA (5′- AACCCAUGGAAUUCAGUUCUCA-3′), the miRNA negative control (NC) (5′-GUACGCCAAAAGUUAAACC-3′), the TRAF6 siRNA sequence (5’-GGUGAAAUGUCCAAAUGAAGGUUCAUUUGGACAUUUCACCAU-3ʹ), and the siNC (5’- UUGUACUACACAAAAGUACUG-3’) were obtained from Beyotime (Beijing, China). Cell transfection was performed with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Quantitative real-time PCR (qRT‒PCR) . mRNA levels were detected by qRT‒PCR. Total RNA from cells and tissues was isolated using TRIzol Reagent (Invitrogen, USA) and then converted to cDNA using the Hifair® II 1st Strand cDNA Synthesis Kit (Yeasen Biotechnology, Shanghai) following the manufacturer’s instructions. Quantitative real-time PCR was then performed using Hieff® qPCR SYBR Master Mix (Yeasen Biotechnology, Shanghai) on an ABI 7500 real-time PCR system (Applied Biosystems, Foster, USA) according to the manufacturer’s instructions. qRT‒PCR for miRNA was performed using a stem‒loop RT primer and the Hifair® miRNA 1st Strand cDNA Synthesis Kit (Yeasen Biotechnology, Shanghai) following the manufacturer’s protocols. The 2-ΔΔCt method was used to calculate the relative expression levels of the targeted genes. The internal controls for mRNA and miRNA were GAPDH and U6, respectively. The primers used are shown in Tables S1 and S2. Western blot analysis. Protein levels were detected by Western blot analysis. Protein samples to be measured were isolated from whole-cell lysates or exosomes using RIPA lysis buffer (Millipore, USA). The total protein content was quantified using a Pierce™ BCA protein quantification kit (Thermo Fisher Scientific, USA). Briefly, the lysates were separated by gel electrophoresis. Proteins were then transferred onto polyvinylidene fluoride membranes and blocked with 5% nonfat milk. The membranes were then sequentially incubated with optimally diluted primary and secondary antibodies. Immunoreactivity signals of the targeted proteins were visualized in a chemiluminescent assay with an enhanced chemiluminescence (ECL) chromogenic substrate (Bio-Rad Laboratories, USA). The antibodies used for Western blotting are listed in Table S3. Exosome blockade. The ectopic ESCs cultured in vitro were supplemented with the exosome inhibitor GW4689 for culture as the exosome inhibition group, and a blank control group was set up to obtain 40 ml of supernatant and isolate the exosomes. The obtained exosomes were added to each group of macrophages for 24 h, and the macrophages were subjected to RNA extraction and subsequent experiments. Exosome regulation. Upregulated or downregulated miR-146a-5p molecules were transfected into ectopic ESCs cultured in vitro using mimics/siRNAs to obtain 40 ml of supernatant, which was subsequently isolated to obtain exosomes. In the U937 macrophage culture system, 50 μg of exosomes from each of the above groups were added separately and cultured for 24 h. Macrophage RNA and proteins were extracted for subsequent experiments. FCM analysis. After treatment with the exosome inhibitor GW4869 or the NF-κB pathway inhibitor EVP4593 at different time points, U937 cells were collected at 48, 24, and 0 h for FCM analysis of macrophage polarization. The markers CD86 and iNOS were used to detect M1 macrophages, and CD163 and CD206 were used to detect M2 macrophages 41 . The cells were washed twice with PBS, and then, the cell pellet was collected by centrifugation and incubated with iNOS monoclonal antibody (CXNFT), Alexa Fluor 488 (53-5920-80, eBioscience, USA), CD86 monoclonal antibody (PO3), PE (MA516921, eBioscience, USA), CD163 monoclonal antibody (MAC 2-158), Alexa Fluor 488(53-1637-42, eBioscience, USA), CD206 monoclonal antibody (685641), and PE (MA5-23594, eBioscience, USA) at 4 °C for 15–30 min in the dark. After being washed with precooled PBS, the cells were resuspended in PBS for FCM analysis. After incubation, the cells were washed and placed in a Beckman flow cytometer. The data were analyzed with FlowJo software (FlowJo LLC, USA). Luciferase assay. Luciferase gene reporter gene analysis was used to detect the posttranscriptional regulation of TRAF6 by miR-146a-5p. The luciferase reporter vector pcDNA3.1(+) (Addgene, USA) was used to construct TRAF6 wild-type (WT) or mutant 3'-UTRs. Recombinant reporter vectors with miR-146a-5p mimics or inhibitors were combined with Lipofectamine 2000 Reagent to transduce U937 cells. Luciferase activity was assayed using a Luciferase Assay Kit (Promega, USA). Statistical analysis. The data were statistically analyzed using GraphPad Prism software (version 9.0.0, USA). At least three independent experiments were performed for each result. The data are presented as the means ± SDs. One-way ANOVA and Student’s t test were applied for the comparisons of mean values. P < 0.05 was regarded as statistically significant. Declarations Acknowledgments This study was supported by grants from the National Nature Science Foundation of China (82171641, 81873825). Author contributions Y.M. performed the experiments, analyzed the data and wrote and edited the manuscript. L.X., T.Y. and L.J. oversaw collection of and provided human samples and performed the experiments. L.K. conceived the ideas and designed the experiments. 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Additional Declarations No competing interests reported. Supplementary Files Table.docx Cite Share Download PDF Status: Posted Version 1 posted 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. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-4988177","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":362742964,"identity":"4fb86574-8715-47dc-8933-265e5ae132bd","order_by":0,"name":"Ming Yuan","email":"","orcid":"","institution":"Medical School, Hangzhou Normal University, Hangzhou, Zhejiang","correspondingAuthor":false,"prefix":"","firstName":"Ming","middleName":"","lastName":"Yuan","suffix":""},{"id":362742965,"identity":"4838236e-f4fa-45fd-a788-dd7c28f4d2b1","order_by":1,"name":"Xingfei Lu","email":"","orcid":"","institution":"The Second Affiliated School of Zhejiang Chinese Medical University, Hangzhou","correspondingAuthor":false,"prefix":"","firstName":"Xingfei","middleName":"","lastName":"Lu","suffix":""},{"id":362742966,"identity":"7579c1eb-93a2-4146-8692-bb6b0cd6498a","order_by":2,"name":"Yuxia Tang","email":"","orcid":"","institution":"The Second Affiliated School of Zhejiang Chinese Medical University, Hangzhou","correspondingAuthor":false,"prefix":"","firstName":"Yuxia","middleName":"","lastName":"Tang","suffix":""},{"id":362742967,"identity":"2c85570c-e182-4661-adad-22356d706d58","order_by":3,"name":"Jiayi Li","email":"","orcid":"","institution":"The Second Affiliated School of Zhejiang Chinese Medical University, Hangzhou","correspondingAuthor":false,"prefix":"","firstName":"Jiayi","middleName":"","lastName":"Li","suffix":""},{"id":362742968,"identity":"cb43e7a4-8f62-44ea-8a60-6ead64360d5b","order_by":4,"name":"Kaiqing Lin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6ElEQVRIie3RMQrCMBSA4VcCdYntmqLoFZ5kkFLBq0Q6uDq5KghOHkDv4NAjRDq4VF0zKq4KQhcHB2MU3NqOgvmXQMhHEh6AzfaDeQQIAAIFIBLYa0uWEPdLXFGRwIuYKL7XUlKrp3gfRc2uv85v4RxanhJOPip8mBcPFjik4fKaYDAHHihBGstCQrmkmFJUu0RoMkiU0JslZPMwJDtKTSaVSGxuOSycqSYCqxDe1H9B5XJge9ZZZadZo4j4fsaDyyPq4yE952zca3vbeJMXkU96NkwAYWaYzrQcGOJLcG5VztpsNtvf9QRyJkGD+6z6igAAAABJRU5ErkJggg==","orcid":"","institution":"Department of Gynecology and Obstetrics, Integrated Chinese and Western Medicine Hospital of Zhejiang Province, Hangzhou","correspondingAuthor":true,"prefix":"","firstName":"Kaiqing","middleName":"","lastName":"Lin","suffix":""}],"badges":[],"createdAt":"2024-08-28 05:06:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4988177/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4988177/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":66153981,"identity":"9094320c-e92e-4a48-af63-712892ae1b34","added_by":"auto","created_at":"2024-10-08 08:20:32","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1364840,"visible":true,"origin":"","legend":"\u003cp\u003eExosomes from ectopic ESCs enter macrophages. (A) Identification of eutopic and ectopic ESCs (vimentin positive, CK7 negative) (scale bar, 50 μm). (B) Cy3-labeled exosomes, which emit red fluorescence, were engulfed by U937 macrophages. (scale bar, 50 μm).\u003c/p\u003e","description":"","filename":"OnlineFigure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4988177/v1/fe4b0e2b4d9ae1f3ca8d8ea2.png"},{"id":66152525,"identity":"7e826f9d-8158-4ae9-8f80-223ca511d0f5","added_by":"auto","created_at":"2024-10-08 08:04:32","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":946520,"visible":true,"origin":"","legend":"\u003cp\u003eExosomes induce macrophage polarization toward the M2 phenotype by secreting miR-146a-5p. (A-D) Ectopic ESCs were exposed to the exosome inhibitor GW4869 and cocultured with U937 macrophages, and the expression levels of CD86, INOS, CD163 and CD206 were examined by flow cytometry. *P\u0026lt;0.05 compared with the control.\u003c/p\u003e","description":"","filename":"OnlineFigure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4988177/v1/07ae05dda1f4321917d49da9.png"},{"id":66153663,"identity":"1d20fb42-a1f2-4e21-af0f-75cb1be0d133","added_by":"auto","created_at":"2024-10-08 08:12:32","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":204113,"visible":true,"origin":"","legend":"\u003cp\u003emiR-146a-5p negatively regulates TRAF6 expression by targeting its 3′UTR in U937 macrophages. (A-B) qRT‒PCR was performed to detect the expression levels of TRAF6 mRNA (A) and the relative miR-146a-5p levels (B) in each group. (C-D) Western blot analysis was performed to assess the protein expression levels of TRAF6 (C) in each group, the grouping of gels/blots cropped from different parts of the same gel (D). (E-H) The binding of miR-146a-5p to the TRAF6 3′UTR was investigated using the luciferase reporter gene method (E). U937 cells transfected with the miR-146a-5p mimic or inhibitor were used to measure the expression levels of TRAF6 mRNA (F) and protein (G). Western blot analysis was used to determine the TRAF6 protein levels in each group, the grouping of gels/blots cropped from different parts of the same gel (H). \u0026nbsp;**P\u0026lt;0.01, ***P\u0026lt;0.001 compared with NC-exos; \u003csup\u003e\u0026amp;\u0026amp;\u003c/sup\u003eP\u0026lt;0.01, \u003csup\u003e\u0026amp;\u0026amp;\u0026amp;\u003c/sup\u003eP\u0026lt;0.001 compared with WT+NC; \u003csup\u003e##\u003c/sup\u003eP\u0026lt;0.01, \u003csup\u003e###\u003c/sup\u003eP\u0026lt;0.001 compared with NC.\u003c/p\u003e","description":"","filename":"OnlineFigure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4988177/v1/8fb403d7a35381bd346d4a50.png"},{"id":66153662,"identity":"97eab645-f5a5-4f0b-b964-a6a9e10822dd","added_by":"auto","created_at":"2024-10-08 08:12:32","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":275180,"visible":true,"origin":"","legend":"\u003cp\u003ePlasmid overexpression of TRAF6 significantly increased the mRNA and protein levels of TRAF6, and interference with TRAF6 significantly inhibited the phosphorylation level of NF-κB. U937 macrophages were transfected with oeTRAF6 or siTRAF6. (A-C) Macrophages were collected for determinations of TRAF6 mRNA (A) and protein (B, C) expression. (D-F) Macrophages were collected for assessments of TRAF6 expression at the mRNA (D) and protein levels (E, F). (G-H) Western blot analysis was used to compare the protein (G) and phosphorylation (H) levels of NF-κB in the different TRAF6 expression groups. The grouping of gels/blots cropped from different parts of the same gel. **P\u0026lt;0.01, ***P\u0026lt;0.001 compared with the Vector group; \u003csup\u003e#\u003c/sup\u003eP\u0026lt;0.05, \u003csup\u003e##\u003c/sup\u003eP\u0026lt;0.01, compared with the siNC group.\u003c/p\u003e","description":"","filename":"OnlineFigure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4988177/v1/c82b02675e8bd7101351e507.png"},{"id":66152527,"identity":"d20c3820-5857-45be-bca7-acebffcaada3","added_by":"auto","created_at":"2024-10-08 08:04:32","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1681952,"visible":true,"origin":"","legend":"\u003cp\u003eInhibition of the NF-κB pathway led to greater percentages of M2-type macrophages after 24 and 48 hours. (A-D) Flow cytometry was used to assess the expression of macrophage polarization markers and the proportions of M1-polarized CD86 (A), iNOS (B), and M2-polarized CD163 (C) and CD206 (D) cells. (E) Treatment with EVP4953 altered the M1/M2 cell ratio. (F) Comparisons between groups at 48 h. (A-D) *P\u0026lt;0.05, **P\u0026lt;0.01, compared with the same treatment at 0 hours. (F)****P\u0026lt;0.0001, compared between groups; ns, not significant.\u003c/p\u003e","description":"","filename":"OnlineFigure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4988177/v1/83437e360d4a86fe7a299a90.png"},{"id":67624266,"identity":"d4ed21d0-f81e-4acf-89a2-5120a9ca85a1","added_by":"auto","created_at":"2024-10-28 07:32:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1977997,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4988177/v1/ddc6d6dd-ed15-4bb0-b21b-07d84f194b17.pdf"},{"id":66152521,"identity":"fc14c5ca-48d5-4a8c-b99e-b678a418470b","added_by":"auto","created_at":"2024-10-08 08:04:32","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":18200,"visible":true,"origin":"","legend":"","description":"","filename":"Table.docx","url":"https://assets-eu.researchsquare.com/files/rs-4988177/v1/185b49fbed923ee2ec5f7af0.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Exosomal miR-146a-5p mediates macrophage polarization through TRAF6/NF-κB signaling in endometriosis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEndometriosis is an estrogen-dependent disease characterized by the presence of endometrial stromal cells (ESCs) and glands outside the uterine cavity, which can cause pelvic pain and infertility\u003csup\u003e1,2\u003c/sup\u003e. Although endometriosis is a benign disease, it is associated with malignant biological behaviors such as distant metastasis, adhesion, invasion, implantation, and recurrence\u003csup\u003e3\u003c/sup\u003e. This process is associated with genes related to pathways such as cell proliferation, angiogenesis, neurogenesis, and inflammation\u003csup\u003e4\u003c/sup\u003e. Recently, emerging evidence has suggested that genetic predispositions and associated immunological abnormalities are key to the pathogenesis of endometriosis\u003csup\u003e5\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eUnder normal conditions, peritoneal macrophages are capable of eliminating endometrial tissue that enters the peritoneal cavity via retrograde blood flow\u003csup\u003e6\u003c/sup\u003e. However, in patients with endometriosis, dysfunctional immune responses lead to a decrease in the phagocytic capacity of macrophages or alterations in the cytokine environment, often resulting in the ineffective surveillance and clearance of endometrial tissue deposited within the peritoneal cavity\u003csup\u003e7,8\u003c/sup\u003e. This defect in immunosurveillance allows the growth of endometriotic lesions and the persistence of disease symptoms\u003csup\u003e9\u003c/sup\u003e. The polarization state of macrophages is reversible, allowing them to change phenotypes according to the needs of the microenvironment\u003csup\u003e10,11\u003c/sup\u003e. Macrophages initially present as the M1 phenotype, secreting proinflammatory cytokines such as TNF-α, IL-1β, and IL-6 that trigger an inflammatory response, whereas M2-type macrophages secrete anti-inflammatory factors to suppress inflammation and maintain homeostasis\u003csup\u003e12\u0026ndash;14\u003c/sup\u003e. The ability of macrophages to dynamically switch their phenotype and function in response to microenvironment signals makes them potential therapeutic targets.\u003c/p\u003e \u003cp\u003eNotably, the role of microRNAs in regulating macrophage polarization has been well defined, with the overexpression of miRNA-155 shown to inhibit C/EBPβ protein expression and regulate the production of inflammatory cytokines in TAMs by targeting C/EBP\u003csup\u003e15\u003c/sup\u003e. Furthermore, numerous studies have investigated the function of microRNAs in endometriosis, with findings indicating that microRNAs are reliable biomarkers for this disease\u003csup\u003e16,17\u003c/sup\u003e. Therefore, we suspect that the dynamic switching of macrophage phenotypes may be influenced by microRNAs.\u003c/p\u003e \u003cp\u003eOur preliminary experimental results suggested that exosomes derived from ectopic ESCs promote M2 macrophage polarization by delivering miR-146a-5p, which targets TRAF6, in the pathological process of endometriosis\u003csup\u003e15\u003c/sup\u003e. TRAF6 can activate multiple signaling pathways, such as the NF-κB pathway\u003csup\u003e18\u003c/sup\u003e. Additionally, research supports a close relationship between microRNAs and the NF-κB signaling pathway in certain diseases, including rheumatoid arthritis, cervical cancer, and colorectal cancer\u003csup\u003e19\u0026ndash;23\u003c/sup\u003e. Thus, we hypothesized that miR-146a-5p may play an important role in the polarization process of endometriosis-related macrophages.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eExosomes from ectopic ESCs enter macrophages.\u003c/b\u003e To investigate the impact of ESC-derived exosomes on macrophages, we isolated exosomes from eutopic and ectopic ESCs (vimentin positive, CK7 negative; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Transwell assays and exosome tracking experiments verified that the exosomes derived from the mesenchymal stromal cells of the donor could reach the recipient cells and be internalized, along with their contained cellular contents. The internalization of exosomes by U937 macrophages was visualized through Cy3 red fluorescence labeling (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eExosomes induce macrophage polarization toward the M2 phenotype by secreting miR-146a-5p.\u003c/b\u003e In our previous study, miR-146a-5p was silenced in inhibitor-transfected ectopic ESCs and overexpressed in mimic-transfected ESCs. Consequently, in the exosomes of these transfected ESCs, the expression of miR-146a-5p was also correspondingly silenced and overexpressed\u003csup\u003e24\u003c/sup\u003e, indicating that miR-146a-5p facilitates the polarization of M2 macrophages (data not shown). Flow cytometry analysis revealed that after treatment with the exosome inhibitor GW4869, ectopic ESCs significantly increased the expression of CD86 and iNOS (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, B), whereas the proportions of M2 macrophages expressing CD163 and CD206 were reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC, D). Flow cytometry plots of the control and GW4869 groups are shown.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003emiR-146a-5p inhibits TRAF6 expression by targeting its 3\u0026prime;UTR in macrophages.\u003c/b\u003e U937 macrophages treated with exosomes from miR-146a-5p mimic- or inhibitor-transfected ectopic ESCs were used to detect miR-146a-5p and TRAF6 levels. Exosomes transfected with the miR-146a-5p inhibitor significantly increased TRAF6 expression, whereas exosomes transfected with the miR-146a-5p mimic significantly suppressed TRAF6 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B). The exosomes transfected with the miR-146a-5p inhibitor significantly increased the protein expression of TRAF6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, D), which is consistent with the changes observed at the mRNA level. Bioinformatics analysis revealed that miR-146a-5p has several binding sites in the TRAF6 3\u0026prime;UTR (data not shown)\u003csup\u003e24\u003c/sup\u003e. To assess the binding of miR-146a-5p to the TRAF6 promoter, we used luciferase reporter gene plasmid transfection. Transfection with the wild-type TRAF6 promoter led to significant inhibition of TRAF6 transcription by the miR-146a-5p mimic and significant promotion by the miR-146a-5p inhibitor (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). However, after transfection with the mutated TRAF6 promoter, the regulatory effects of the miR-146a-5p mimic and inhibitor on TRAF6 transcription vanished. To investigate the regulatory effects of miR-146a-5p on TRAF6 expression, we separately detected TRAF6 expression at the mRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF) and protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG, H) levels. The data revealed that the siRNA promoted TRAF6 expression at both the mRNA and protein levels, whereas the mimics had the opposite effect.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eInhibition of the NF-\u003c/b\u003eκ\u003cb\u003eB pathway can induce macrophage polarization toward the M2 phenotype.\u003c/b\u003e To further investigate the role of TRAF6, we synthesized TRAF6 overexpression plasmids and interference fragments in vitro. After transfection, we assessed the efficiency of TRAF6 overexpression and interference by qRT‒PCR and Western blotting. The oeTRAF6 construct significantly increased TRAF6 expression at both the mRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA) and protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB, C) levels, whereas siTRAF6 significantly decreased TRAF6 expression at the mRNA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD) and protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, F) levels. To examine the downstream regulatory pathways of TRAF6, following the transfection of the TRAF6 interference fragment and overexpression plasmids, we conducted Western blot analysis to assess and compare the NF-κB protein expression and phosphorylation levels among the overexpression group, the downregulation group, and their respective control groups, which revealed that TRAF6 increased NF-κB signaling pathway activation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG, H).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the U937 macrophage line culture system, the NF-κB pathway inhibitor EVP4593 was introduced, and the results were compared with those of a blank control group. Flow cytometry at 0, 24, and 48 h posttreatment revealed increased expression levels of CD86, INOS, CD163, and CD206 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-D). The M1/M2 ratio graph revealed a significant increase in M2 cells at 48 h, and the line graph indicates that the inhibitor induced both M1 and M2 polarization, with increased M2 induction (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). A flow cytometry plot at 48 h is shown (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). The data revealed significant differences between type M1 macrophages (CD86 and iNOS) and type M2 macrophages (CD163 and CD206) after 48 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn our present study, through high-throughput sequencing and bioinformatics analyses, we identified miR-146a-5p for further study, and we showed that both miR-146a-5p in ectopic endometrial stromal cells and exosomal miR-146a-5p were highly expressed in endometriosis. miR-146a-5p is involved in inflammation and regulates the differentiation and function of immune cells\u003csup\u003e25\u0026ndash;28\u003c/sup\u003e. Further GO analysis of miR-146a-5p was conducted to examine its gene functions in biological processes, cellular components, and molecular functions and to predict its target genes. The results showed that its target was exclusively TRAF6, a key signaling adaptor protein that plays roles primarily in immune and inflammatory responses. KEGG pathway analysis revealed key pathways, including the MAPK, JNK, and NF-κB signaling pathways. A previous study revealed a significant association between miR-146a and an increased incidence of endometriosis\u003csup\u003e29\u003c/sup\u003e, and the role of miRNAs in regulating macrophage polarization has been confirmed\u003csup\u003e30\u003c/sup\u003e. Thus, we speculate that miR-146a-5p plays a significant role in the pathogenesis of endometriosis by mediating TRAF6. This study utilizes the signaling function of exosomes to connect the pathogenicity of endometriosis with the functional transformation of macrophages, investigating the role and molecular mechanisms of macrophage functional transformation in endometriosis.\u003c/p\u003e \u003cp\u003eGW4869 is a noncompetitive inhibitor of N-Smase (neutral sphingomyelinase) that can reduce the amount of exosomes released into conditioned medium\u003csup\u003e31\u003c/sup\u003e. By adding GW4689 to inhibit the secretion of exosomes and the release of miR-146a-5p from ectopic ESCs and detecting the levels of macrophage markers, we found that the polarization of macrophages toward the M1 phenotype was increased. Second, inhibitors and mimics were used to alter miR-146a-5p levels in ESCs and their exosomes, and we observed that the miR-146a-5p inhibitor significantly increased TRAF6 expression in macrophages. TRAF6 is an E3 ubiquitin ligase that is involved in numerous inflammatory diseases, and its primary function is focused on the regulation of the immune system\u003csup\u003e32\u003c/sup\u003e. Third, we overexpressed TRAF6 and transfected interference plasmids in U937 cells, and the results showed that an inhibitor of TRAF6 led to the suppression of the NF-κB pathway. This effect was similar to that of the NF-κB pathway-specific inhibitor EVP4593, resulting in an increased proportion of M2-type macrophages. The inhibition of this signaling cascade has been proven to suppress M1 polarization activity\u003csup\u003e33\u003c/sup\u003e. When the NF-κB pathway inhibitor EVP4593 was added to the U937 macrophage culture system, inhibition of the NF-κB pathway induced the polarization of macrophages toward the M2 phenotype. NF-κB is known to regulate immune responses and control the expression of inflammatory cytokines\u003csup\u003e34,35\u003c/sup\u003e, and NF-κB activation stimulates inflammation and cell proliferation and inhibits apoptosis, thereby promoting the development and maintenance of endometriosis\u003csup\u003e36\u003c/sup\u003e. The similarity between the invasive growth of ectopic endometrial tissue and the ease of cancer spread may be due to the active involvement of the stromal microenvironment in controlling invasion, whether by embryos or cancer cells\u003csup\u003e37,38\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003ePrevious work has shown that the NF-κB signaling pathway is involved in the phenotypic conversion of M2-type macrophages to M1-type macrophages\u003csup\u003e39\u003c/sup\u003e. The switch from the M1 phenotype to the M2 phenotype mediates the immunosuppressive process, which is a marker of disease progression\u003csup\u003e40\u003c/sup\u003e. M1/M2 polarization imbalance plays a significant role in autoimmune diseases. Consequently, modulating the polarization of macrophages can ameliorate the pathogenesis of these conditions. Recent studies have shown that inhibiting the activation of NF-κB P65, thereby reducing M1 polarization, aids in the treatment of experimental autoimmune uveitis (EAU). Similar to these studies, our data showed that inhibition of the NF-κB pathway can induce macrophage polarization toward the M2 phenotype.\u003c/p\u003e \u003cp\u003eThe application of miR-146a-5p in endometriosis needs to be further verified through animal models and in vivo experiments. Moreover, the downstream JNK and MAPK pathways affected by TRAF6 are worthy of further investigation.\u003c/p\u003e \u003cp\u003eIn conclusion, our findings indicate that exosome-derived miR-146a-5p-mediated TRAF6 promotes the polarization of macrophages toward the M2 phenotype by regulating the activation of NF-κB. An increase in miR-146a-5p leads to a decrease in TRAF6 expression, which in turn reduces the phosphorylation of NF-κB. The present study may contribute to uncovering the pathogenesis of endometriosis and offer new insights for developing diagnostic and therapeutic strategies for this disease.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eEthical approval.\u003c/strong\u003e The use of human samples was approved by the Women\u0026rsquo;s Hospital of Hangzhou Normal University. All the subjects signed a written informed consent. The experimental protocols and procedures related to humans were approved by the Ethics Committee of the Women\u0026rsquo;s Hospital of Hangzhou Normal University (No. 2022-A-03). All methods were performed in accordance with the relevant guidelines and regulations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical samples.\u003c/strong\u003e A total of 5 patients with endometriosis were recruited to provide ectopic and eutopic endometrial tissue when they underwent laparoscopy/hysteroscopy treatment at the Women\u0026rsquo;s Hospital of Hangzhou Normal University. These samples were obtained during the proliferative phase of the menstrual cycle, which was determined based on preoperative medical history and histological examination.\u0026nbsp;The inclusion and exclusion criteria were as follows: regular menstruation for a period of 28\u0026ndash;32 days; and patients who had received hormone therapy and contraception.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCoculture of macrophages and ESCs.\u003c/strong\u003e Primary ESCs of ectopic and eutopic endometria were cultured as described in previous studies\u003csup\u003e24\u003c/sup\u003e.\u0026nbsp;The human histiocytic lymphoma cell line U937 was obtained from the cell bank of the Shanghai Academy of Biological Sciences. The medium consisted of RPMI 1640 (Millipore, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, USA) and a 1% dual-antibiotic penicillin‒streptomycin mixture (PS) (Solarbio, China). The ectopic ESC culture conditions were 10% FBS + 1% PS + DMEM/F12 (HyClone, USA). All the cells were cultured in a humidified incubator containing 5% CO\u003csub\u003e2\u003c/sub\u003e at 37 \u0026deg;C.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExosome extraction and characterization.\u003c/strong\u003e Specimens of ovarian endometrial tissue were collected, and ectopic ESCs were isolated and cultured with exosome-free serum in primary culture for 48 h. Exosomes were isolated with an exosome isolation kit (Invitrogen, USA). The isolated exosomes were stored at \u0026minus;80 \u0026deg;C as a backup. The purity of the cells was determined by immunofluorescence using vimentin and C-cell keratin factor (CK7).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTranswell assays.\u0026nbsp;\u003c/strong\u003eTranswell inserts (Corning, 3413) were used to detect cell migration and invasion. The isolated ectopic endothelial mesenchymal stromal cells were cocultured with the U937 macrophage line. Cy3-labeled miR-146a-5p molecules were transfected into mesenchymal stromal cells, which were then cultured in the upper chamber. U937 macrophages that did not express Cy3 were placed in the lower chamber, and red fluorescent Cy3-miR-146a-5p molecules were detected in the lower chamber after 12 hours.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlasmid construction and cell transfection.\u0026nbsp;\u003c/strong\u003eThe miR-146a-5p mimics (5\u0026rsquo;-UGAGAACUGAAUUCCAUGGGUU-3\u0026prime;), the miR-146a-5p siRNA (5\u0026prime;-\u0026nbsp;AACCCAUGGAAUUCAGUUCUCA-3\u0026prime;), the miRNA negative control (NC) (5\u0026prime;-GUACGCCAAAAGUUAAACC-3\u0026prime;), the TRAF6 siRNA sequence (5\u0026rsquo;-GGUGAAAUGUCCAAAUGAAGGUUCAUUUGGACAUUUCACCAU-3ʹ), and the siNC\u0026nbsp;(5\u0026rsquo;-\u0026nbsp;UUGUACUACACAAAAGUACUG-3\u0026rsquo;)\u0026nbsp;were obtained from Beyotime (Beijing, China).\u0026nbsp;Cell transfection was performed with Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantitative real-time PCR (qRT‒PCR)\u003c/strong\u003e. mRNA levels were detected by qRT‒PCR. Total RNA from cells and tissues was isolated using TRIzol Reagent (Invitrogen, USA) and then converted to cDNA using the Hifair\u0026reg; II 1st Strand cDNA Synthesis Kit (Yeasen Biotechnology, Shanghai) following the manufacturer\u0026rsquo;s instructions. Quantitative real-time PCR was then performed using Hieff\u0026reg; qPCR SYBR Master Mix (Yeasen Biotechnology, Shanghai) on an ABI 7500 real-time PCR system (Applied Biosystems, Foster, USA) according to the manufacturer\u0026rsquo;s instructions. qRT‒PCR for miRNA was performed using a stem‒loop RT primer and the Hifair\u0026reg; miRNA 1st Strand cDNA Synthesis Kit (Yeasen Biotechnology, Shanghai) following the manufacturer\u0026rsquo;s protocols. The 2-\u0026Delta;\u0026Delta;Ct method was used to calculate the relative expression levels of the targeted genes. The internal controls for mRNA and miRNA were GAPDH and U6, respectively. The primers used are shown in Tables S1 and S2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blot analysis.\u003c/strong\u003e Protein levels were detected by Western blot analysis. Protein samples to be measured were isolated from whole-cell lysates or exosomes using RIPA lysis buffer (Millipore, USA). The total protein content was quantified using a Pierce\u0026trade; BCA protein quantification kit (Thermo Fisher Scientific, USA). Briefly, the lysates were separated by gel electrophoresis. Proteins were then transferred onto polyvinylidene fluoride membranes and blocked with 5% nonfat milk. The membranes were then sequentially incubated with optimally diluted primary and secondary antibodies. Immunoreactivity signals of the targeted proteins were visualized in a chemiluminescent assay with an enhanced chemiluminescence (ECL) chromogenic substrate (Bio-Rad Laboratories, USA). The antibodies used for Western blotting are listed in Table S3.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExosome blockade.\u003c/strong\u003e The ectopic ESCs cultured in vitro were supplemented with the exosome inhibitor GW4689 for culture as the exosome inhibition group, and a blank control group was set up to obtain 40 ml of supernatant and isolate the exosomes. The obtained exosomes were added to each group of macrophages for 24 h, and the macrophages were subjected to RNA extraction and subsequent experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eExosome regulation.\u003c/strong\u003e Upregulated or downregulated miR-146a-5p molecules were transfected into ectopic ESCs cultured in vitro using mimics/siRNAs to obtain 40 ml of supernatant, which was subsequently isolated to obtain exosomes. In the U937 macrophage culture system, 50 \u0026mu;g of exosomes from each of the above groups were added separately and cultured for 24 h. Macrophage RNA and proteins were extracted for subsequent experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFCM analysis.\u003c/strong\u003e After treatment with the exosome inhibitor GW4869 or the NF-\u0026kappa;B pathway inhibitor EVP4593 at different time points, U937 cells were collected at 48, 24, and 0 h for FCM analysis of macrophage polarization.\u0026nbsp;The markers CD86 and iNOS were used to detect M1 macrophages, and CD163 and CD206 were used to detect M2 macrophages\u003csup\u003e41\u003c/sup\u003e.\u0026nbsp;The cells were washed twice with PBS, and then, the cell pellet was collected by centrifugation and incubated with iNOS monoclonal antibody (CXNFT), Alexa Fluor 488 (53-5920-80, eBioscience, USA), CD86 monoclonal antibody (PO3), PE (MA516921, eBioscience, USA), CD163 monoclonal antibody (MAC 2-158), Alexa Fluor 488(53-1637-42, eBioscience, USA), CD206 monoclonal antibody (685641), and PE (MA5-23594, eBioscience, USA) at 4 \u0026deg;C for 15\u0026ndash;30 min in the dark. After being washed with precooled PBS, the cells were resuspended in PBS for FCM analysis. After incubation, the cells were washed and placed in a Beckman flow cytometer. The data were analyzed with FlowJo software (FlowJo LLC, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLuciferase assay.\u003c/strong\u003e Luciferase gene reporter gene analysis was used to detect the posttranscriptional regulation of TRAF6 by miR-146a-5p. The luciferase reporter vector pcDNA3.1(+) (Addgene, USA) was used to construct TRAF6 wild-type (WT) or mutant 3\u0026apos;-UTRs. Recombinant reporter vectors with miR-146a-5p mimics or inhibitors were combined with Lipofectamine 2000 Reagent to transduce U937 cells. Luciferase activity was assayed using a Luciferase Assay Kit (Promega, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis.\u003c/strong\u003e The data were statistically analyzed using GraphPad Prism software (version 9.0.0, USA). At least three independent experiments were performed for each result. The data are presented as the means \u0026plusmn; SDs. One-way ANOVA and Student\u0026rsquo;s t test were applied for the comparisons of mean values. P \u0026lt; 0.05 was regarded as statistically significant.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by grants from the National Nature Science Foundation of China (82171641, 81873825).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eY.M. performed the experiments, analyzed the data and wrote and edited the manuscript.\u003c/p\u003e\n\u003cp\u003eL.X., T.Y. and L.J. oversaw collection of and provided human samples and performed the experiments. L.K. conceived the ideas and designed the experiments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData is provided within the manuscript or supplementary information files.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003e\u003cspan\u003eZondervan, K. T., Becker, C. M. \u0026amp; Missmer, S. A. Endometriosis. \u003cem\u003eN Engl J Med\u003c/em\u003e \u003cstrong\u003e382\u003c/strong\u003e, 1244\u0026ndash;1256 (2020).\u003c/span\u003e\u003c/li\u003e\n \u003cli\u003e\u003cspan\u003eChen, S. \u003cem\u003eet al.\u003c/em\u003e Peritoneal immune microenvironment of endometriosis: Role and therapeutic perspectives. \u003cem\u003eFront. 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Exosomal miR-214 from endometrial stromal cells inhibits endometriosis fibrosis. \u003cem\u003eMHR: Basic science of reproductive medicine\u003c/em\u003e (2018) doi:10.1093/molehr/gay019.\u003c/span\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Exosome, Endometriosis, miR-146a-5p, Macrophage polarization, Endometrial stromal cells, Signaling pathway","lastPublishedDoi":"10.21203/rs.3.rs-4988177/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4988177/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eExosomes play significant roles in immune responses, neurogenesis, and angiogenesis, directly impacting the progression and symptomatic manifestations of endometriosis. This study aimed to investigate the role of exosomal miR-146a-5p in the pathogenesis of endometriosis. Through high-throughput RNA sequencing and qRT‒PCR, we revealed significant upregulation of miR-146a-5p in ectopic endothelial tissues, and Gene Ontology (GO) analysis revealed that miR-146a-5p has only one target, TNF receptor associated factor 6 (TRAF6). KEGG pathway analysis indicated that the NF-κB signaling pathway is the key signaling pathway involved. The study revealed that the upregulation of miR-146a-5p in macrophages is associated with an increase in M2 macrophages. In the U937 macrophage line, miR-146a-5p was capable of suppressing TRAF6 expression, which in turn decreased the phosphorylation level of NF-κB, whereas the overexpression of TRAF6 increased the activity of this pathway. Furthermore, we incorporated the NF-κB inhibitor EVP4593 into a macrophage culture, which revealed that blocking this pathway significantly induced both M1 and M2 macrophage polarization, particularly enhancing M2 polarization. In conclusion, exosome-derived miR-146a-5p promotes M2 polarization of macrophages by regulating the TRAF6/NF-κB pathway in endometriosis.\u003c/p\u003e","manuscriptTitle":"Exosomal miR-146a-5p mediates macrophage polarization through TRAF6/NF-κB signaling in endometriosis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-08 08:04:27","doi":"10.21203/rs.3.rs-4988177/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"602a7823-8b3a-46a7-b03c-32d5192c47f8","owner":[],"postedDate":"October 8th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":38587096,"name":"Health sciences/Medical research/Biomarkers/Predictive markers"},{"id":38587097,"name":"Biological sciences/Molecular biology/Non coding rnas/Mirnas"},{"id":38587098,"name":"Health sciences/Diseases/Reproductive disorders"},{"id":38587099,"name":"Biological sciences/Immunology/Innate immune cells/Monocytes and macrophages"}],"tags":[],"updatedAt":"2024-10-28T07:24:34+00:00","versionOfRecord":[],"versionCreatedAt":"2024-10-08 08:04:27","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4988177","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4988177","identity":"rs-4988177","version":["v1"]},"buildId":"B-jG_2CBjPDmsCi4Wdhf-","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}