{"paper_id":"a449faf8-f529-44c3-9fc8-18f8c536b519","body_text":"1\nScientific  RepoRts  |  (2017) 7:17903  | DOI:10.1038/s41598-017-18224-x\nwww.nature.com/scientificreports\nInterleukin-33 modulates \ninflammation in endometriosis\nJessica E. Miller1, Stephany P . Monsanto1, Soo Hyun Ahn1, Kasra Khalaj1, Asgerally T. Fazleabas2, \nSteven L. Young3, Bruce A. Lessey4, Madhuri Koti1 & Chandrakant Tayade1\nEndometriosis is a debilitating condition that is categorized by the abnormal growth of endometrial \ntissue outside the uterus. Although the pathogenesis of this disease remains unknown, it is well \nestablished that endometriosis patients exhibit immune dysfunction. Interleukin (IL)-33 is a danger \nsignal that is a critical regulator of chronic inflammation. Although plasma and peritoneal fluid levels \nof IL-33 have been associated with deep infiltrating endometriosis, its contribution to the disease \npathophysiology is unknown. We investigated the role of IL-33 in the pathology of endometriosis \nusing patient samples, cell lines and a syngeneic mouse model. We found that endometriotic lesions \nproduce significantly higher levels of IL-33 compared to the endometrium of healthy, fertile controls. \nIn vitro stimulation of endometrial epithelial, endothelial and endometriotic epithelial cells with IL-33 \nled to the production of pro-inflammatory and angiogenic cytokines. In a syngeneic mouse model of \nendometriosis, IL-33 injections caused systemic inflammation, which manifested as an increase in \nplasma pro-inflammatory cytokines compared to control mice. Furthermore, endometriotic lesions \nfrom IL-33 treated mice were highly vascularized and exhibited increased proliferation. Collectively, \nwe provide convincing evidence that IL-33 perpetuates inflammation, angiogenesis and lesion \nproliferation, which are critical events in the lesion survival and progression of endometriosis.\nEndometriosis is a chronic inflammatory, estrogen-dependent disease that affects 6–10% of reproductive-aged \nwomen. Despite this considerable prevalence, the cause of endometriosis remains unknown and a cure does not \nexist. Retrograde menstruation is a widely accepted theory to explain the pathogenesis. This theory suggests \nthat during menstruation, uterine contractions cause menstrual endometrial tissue to be refluxed into the ovi-\nducts and peritoneal cavity. It was shown that 76–90% of all women experience this reflux of menstrual debris\n1. \nHowever, only in endometriosis patients is this menstrual tissue able to adhere to peritoneal structures, develop \na blood supply and grow into an endometriotic lesion. Therefore, it is likely that the women who are developing \nendometriosis have genetic, biochemical or immune system dysfunction that does not allow the removal of the \ndebris but rather facilitates menstrual tissue adhesion to peritoneal structures and endometriotic lesion forma-\ntion\n2,3. Indeed, it is well established that women with endometriosis exhibit immune dysfunction in the form of \nheightened local and systemic inflammation2,4,5. More specifically, in the plasma and peritoneal fluid (PF), endo-\nmetriosis patients display aberrant numbers of immune cells and concentrations of cytokines and chemokines \nthat promote a chronic inflammatory environment compared to healthy women6–10. The chronic inflammatory \nenvironment has also been shown to contribute to the chronic pain and infertility experienced by endometriosis \npatients\n3,11.\nInterleukin(IL)-33 is an alarmin of the IL-1 family that acts upon both the innate and adaptive immune sys-\ntem and plays functional roles in both infectious and chronic inflammatory diseases12–15. IL-33 is constitutively \nexpressed in the nucleus of various cell types including endothelial cells and epithelial cells 16; however, upon \ntissue damage, necrosis or mechanical stress, functional IL-33 is released into the extracellular environment and \nspecifically binds to the ST2 receptor (suppressor of tumorigenicity 2)\n17,18. This receptor has two forms: a mem-\nbrane bound form that initiates signaling (ST2) and a soluble, decoy receptor (sST2). IL-33 has been shown to \nstimulate both myeloid and lymphoid immune cells through ST2 including but not limited to macrophages, mast \ncells, T cells, B cells, NK cells, neutrophils, and innate lymphoid cells\n12,19–22. Binding of IL-33 to ST2 induces \n1Department of Biomedical and Molecular Sciences, Queen’s University, Kingston, Ontario, K7L 3N6, Canada. \n2Department of Obstetrics, Gynecology and Reproductive Biology, Michigan State University College of Human \nMedicine, Grand Rapids, MI, 49503, USA. 3Department of Obstetrics and Gynecology, University of North Carolina, \nChapel Hill, North Carolina, NC, 27514, USA. 4Department of Obstetrics and Gynecology, Greenville Health Systems, \nGreenville, South Carolina, SC, 29605, USA. Correspondence and requests for materials should be addressed to C.T. \n(email: tayadec@queensu.ca)\nReceived: 21 July 2017\nAccepted: 7 December 2017\nPublished: xx xx xxxx\nOPEN\n\n\nwww.nature.com/scientificreports/\n2\nScientific  RepoRts  |  (2017) 7:17903  | DOI:10.1038/s41598-017-18224-x\nsignaling by recruiting MyD88 and transcription factors such as NF-KB through IRAK1/4 and TRAF6 kinases \nto perpetuate inflammation: primarily a type 2 immune response 23. Endometrial stromal cells produce IL-33 \nspecifically during decidualization suggesting an important role in female reproductive events such as embryo \nimplantation and early pregnancy\n24.\nIn the literature surrounding endometriosis and IL-33, there are only two reports so far. In a study conducted \nin 532 endometriosis patients and 130 women without endometriosis, IL-33, in serum and PF samples, was sig-\nnificantly higher in patients with a deep infiltrating endometriosis phenotype compared to control women25. In \nanother study conducted in 30 endometriosis patients and 20 control women from Tunisia, IL-33 was reported \nto be higher in the serum and PF of women with endometriosis\n26. Furthermore, sST2 concentrations were sig-\nnificantly higher in PF from endometriosis patients compared to controls26. Additionally, they show that IL-33 \nmRNA expression was higher in cells obtained from PF from endometriosis patients compared to healthy con-\ntrols\n26. These reports suggest that women endometriosis, specifically with deep infiltrating endometriosis, have \nhigh levels of IL-33 in their PF and plasma but they do not suggest whether IL-33 is playing an active role in the \ndisease progression or if it is a bystander. Interestingly, IL-33 is a critical regulator of number of processes includ-\ning inflammation, vascularization, hypernociception, and fibrosis\n27–30, which are known to be involved in the \npathophysiology of endometriosis. Therefore, it is possible that IL-33 plays a role in the progression of endome-\ntriosis; however, a significant knowledge gap exists with regards to how IL-33 contributes to the disease pathology \nand which tissues and cells are producing IL-33. In the present study, we show for the first time that endometriotic \nlesions contribute to the production of IL-33 and that levels of IL-33 are significantly higher in the endometriotic \nlesions of advanced staged patients compared to the endometrium of healthy, fertile controls. We also show that \nendometriotic lesions express the ST2 receptor. Using a series of in vitro studies in cell lines, we documented the \neffects of human recombinant IL-33 (rIL-33) on the proliferation, angiogenesis and cytokine profile. Finally, we \nshow that mouse rIL-33 induces local and systemic inflammation, increases the lesion size and increases vascu-\nlarization within the lesion itself in a syngeneic endometriotic mouse model.\nResults\nEndometriotic (ectopic) lesions produce IL33 and express the ST2 receptor.  Previous reports \nindicated high levels of IL-33 in the serum and PF of deep infiltrating endometriosis patients, the source of IL-33 \nwas not clear. We show for the first time that the endometriotic lesions from stages III and IV patients produce \nsignificantly higher levels of IL-33 compared to endometrial tissue from healthy, fertile controls and that the \nendometriotic lesions from these advanced staged patients produce significantly more IL-33 than matched tissue \nfrom their endometrium (Fig. 1A). We also report that endometriotic lesions express the ST2 receptor (Fig. 1B). \nNo significant differences were found in the expression of ST2 between endometriosis patient tissue and tissue \nfrom healthy, fertile controls (Fig. 1B). Additionally, we did not find any significant difference in the levels of sST2 \nin the plasma of endometriosis patients compared to healthy fertile controls (Fig. 1C).\nHuman rIL-33 stimulates angiogenic and proinflammatory cytokines production.  First, we \nconfirmed using RT-PCR and sequencing that all three cell lines used in the study (EECC, HUVECs and 12Zs) \nexpress the ST2 receptor. Then, we sought to understand the effect of IL-33 signalling on the proliferation and \ncytokine production in these cell lines. Using IncuCyte, live cell analysis platform, we found that treating EECCs, \nHUVECs and 12Zs with varying concentrations of human rIL-33 (1 ng/mL, 10 ng/mL, 50 ng/mL and 100 ng/mL) \ndid not have any effect on their proliferation (Supplemental Fig. 1). To assess the cytokine production of each \ncell line in response to human rIL-33, we screened the cell supernatants from EECCs, HUVECs and 12Zs treated \nwith varying concentrations of rIL-33. Supernatants from EEECs revealed significantly higher (P < 0.005) levels \nof angiogenic cytokines including VEGF and PDGF-AA and significantly lower expression of TGF-β compared \nto PBS-treated cells (Fig. 2A–C). Treating HUVECs with human rIL-33 led to significantly higher (P < 0.05) con-\ncentration of IL-1α and TNF-α compared to PBS-treated cells (Fig. 2D,E). Treatment of the endometriotic epithe-\nlial cell line, 12Z, with human rIL-33 stimulated significantly higher (P < 0.05) production of pro-inflammatory \ncytokines such as CXCL1, IL-6, GM-CSF , and IL-15 compared to PBS treated cells (Fig.  2F–J). These results \nfurther confirm that IL-33 is a potent pro-inflammatory and angiogenic cytokine.\nHuman rIL-33 stimulates tubulogenesis in Human Umbilical Venule Endothelial Cells. To con-\nfirm previous reports showing the angiogenic properties of IL-33 31, we conducted a tubulogenesis assay using \nHUVECs in matrigel. HUVECs were stimulated with varying concentrations of human rIL-33 (10 ng/mL, 50 ng/\nmL, and 100 ng/mL) and total tubule branch length was measured. PBS was used as a negative control and VEGF \nwas used as a positive control (Supplemental Fig. 2F).\nRecombinant IL-33 treatment in mouse model of endometriosis perpetuates systemic inflam-\nmation. To understand whether the elevated levels of IL-33 in the PF of deep infiltrating endometriosis \npatients25 contributes to the disease, we recreated heightened IL-33 environment in a syngeneic mouse model \nby injecting mouse rIL-33 into the peritoneal cavity. Because inflammation has been speculated to contribute \nto both the pathophysiology and symptoms of the disease, we used the plasma cytokine levels as an indicator to \nunderstand the effect of mouse rIL-33 on systemic inflammation. The plasma cytokine levels were analyzed at \nthree different time points (day 7, 18 and 25) using a mouse cytokine multiplex assay (Fig. 3A). On day 7, before \nthe start of injections, both treatment and control mice showed similar levels of plasma cytokines. Following \ninjections of mouse rIL-33, we observed significantly higher levels of CXCL1, IL-6, GM-CSF , Eotaxin, IL-5, IL-7 \nand IL-33 compared to control mice treated with PBS (Fig. 3B–F).\nWe also evaluated whether rIL-33 would modulate inflammation in mice in the absence of endometriotic \nlesions. The plasma samples obtained from C57Bl6 mice treated with rIL-33 had elevated levels of CXCL1, IL-6, \n\nwww.nature.com/scientificreports/\n3\nScientific  RepoRts  |  (2017) 7:17903  | DOI:10.1038/s41598-017-18224-x\nGM-CSF , IL-7, IL-5 and Eotaxin (supplemental Fig. 3). These results further confirm that mouse rIL-33 can pro-\nmote systemic inflammation.\nRecombinant IL-33 treatment in mouse model of endometriosis stimulated proliferation and \nvascularization of the endometriotic lesions. As mentioned previously, hallmark features of endome-\ntriosis include the ability of the endometriotic lesions to grow and establish a blood supply. After treatment with \nmouse rIL-33, treated mice had qualitatively larger lesions and enhanced vascularization supplying the lesion \ncompared to control (Fig. 4A,B). Endometriotic lesions obtained from both rIL-33 treated and control mice were \nevaluated using an immunohistochemistry for Ki67, the well-established cell proliferation marker, and CD31, a \nknown marker of endothelial cells. Semi quantitative analysis of Ki67 showed that mice treated with rIL-33 had \nsignificantly increased staining compared to control mice (Fig. 5A–G). Additionally, Ki67 staining appeared to \nbe abundant and widely dispersed in the lesions from rIL-33 treated mice; however, Ki67 staining in the lesions \nfrom PBS treatment mice appeared to be localized to certain areas. Quantitative analysis of CD31 indicated that \nmice treated with rIL-33 had a trend of increased CD31 compared to control; however, this was not statistically \nsignificant (p = 0.09, Fig. 5H–N).\nDiscussion\nIL-33 has emerged as a critical regulator of several chronic inflammatory diseases, autoimmune diseases and \nfibrotic disorders including asthma, rheumatoid arthritis, ulcerative colitis, lung fibrosis and cardiovascular dis-\nease\n18,30,32–34. However, the role of IL-33 in the progression of endometriosis is not well described. In this study, \nwe set out to understand whether endometriotic lesions from patients produce IL-33 and express its receptor \nST2 and to understand the mechanistic basis of their involvement in the pathophysiology of endometriosis using \nrepresentative cell lines and a syngeneic mouse model.\nFrom previous reports, we know that PF cells had significantly higher IL-33 mRNA\n26 suggesting that a variety \nof immune cells are producing IL-33 and potentially contributing to the high levels observed in PF 26. To our \nknowledge, we show for the first time that endometriotic lesions from advanced stage patients had significantly \nhigher levels of IL-33 compared to endometrial samples from healthy, fertile controls. This finding supports an \nearlier report showing higher concentrations of IL-33 in peritoneal and sera samples of subsets of patients with \nFigure 1. Protein levels of IL-33, ST2 and sST2 in plasma and tissue samples from healthy, fertile volunteers \nand endometriosis patients. (A) Levels of IL-33 are significantly higher in the ectopic tissue from advanced \nstaged endometriosis patients (n = 5) compared to healthy fertile controls (n = 11). No significant differences \nwere found between healthy, fertile controls compared to early staged patients (n = 14) or eutopic tissue from \nadvanced staged patients (n = 5). (B) The ST2 receptor is expressed on ectopic, endometriotic tissue. No \nsignificant differences were found between levels of ST2 in matched tissue from endometriosis patients (n = 7) \ncompared to healthy, fertile controls (n = 11). (C) No significant differences were found between levels of \nsST2 in the plasma of endometriosis patients (n = 24) compared to healthy, fertile controls (n = 16) *p < 0.05 \n**p < 0.01 ***p < 0.0001.\n\nwww.nature.com/scientificreports/\n4\nScientific  RepoRts  |  (2017) 7:17903  | DOI:10.1038/s41598-017-18224-x\na deep infiltrating endometriosis phenotype 25. Future studies are required to address the specific cell types in \nthe endometriotic lesions as well as in the peritoneal cavity that are contributing to the elevated IL-33 levels in \nendometriosis patients. Additionally, we show that endometriotic lesions express ST2, suggesting that endome-\ntriotic lesions may respond to IL-33 signaling in an autocrine fashion. Previous reports found that endometriosis \npatients had significantly higher levels of sST2 in the PF compared to healthy controls\n26. Because we did not find \nsignificant differences between the levels of plasma sST2 in endometriosis patients compared to controls, we spec-\nulate that perhaps high levels of sST2 are localized to the peritoneal microenvironment in attempt to modulate \nthe local, elevated levels of IL-33.\nTo gain further insights into IL-33 signaling in the progression of endometriosis, we studied the effect of \nhuman rIL-33 stimulation of endometrial and endometriotic epithelial and endothelial cell lines. Our results \nshow that human rIL33 stimulated the production of pro-inflammatory and angiogenic cytokines and promoted \nFigure 2. Cell supernatant cytokine profile in EECC, HUVEC and 12Z upon stimulation with varying \nconcentrations of human rIL-33 (10, 50 and 100 ng/mL). All experiments were conducted in triplicates. Cell \nsupernatant was analyzed using a human multiplex assay and non-significant cytokines are not shown.  \n(A–C) Endometrial epithelial carcinoma cells (EECC) produced significantly higher levels of VEGF and \nPDGF-AA and a significant reduction in the level of TGF-b. (C,D) Human umbilical vein endothelial cells \n(HUVEC) produced significantly higher levels of IL-1a and TNF-a. (F–J) Endometriotic epithelial cells (12Z) \nproduced significantly higher levels of CXCL1, IL-15, GM-CSF and IL-6 and significantly lower levels of \nPDGF-AA. *p < 0.05 **p < 0.01 ***p < 0.0001.\n\nwww.nature.com/scientificreports/\n5\nScientific  RepoRts  |  (2017) 7:17903  | DOI:10.1038/s41598-017-18224-x\ntubulogenesis. This further confirms IL-33’s established function as a pro-inflammatory and angiogenic \ncytokine29,31,35. As mentioned previously, a pro-inflammatory response is critical for disease progression because \nthe inflammation in the peritoneal cavity of endometriosis patients has been linked to chronic pain and infertility \nFigure 3. Plasma cytokine profile in mice with endometriosis and treated with PBS (control) (n = 5) or mouse \nrIL-33 (treated) (n = 5). Plasma was analyzed using a mouse multiplex assay and non-significant cytokines are \nnot shown. (A) An outline of mouse experiment. (B–G) Plasma cytokines revealed significantly higher levels of \nEotaxin, GM-CSF , IL-6, IL-7, IL-5, CXCL1, and IL-33. *p < 0.05 **p < 0.01 ***p < 0.0001.\nFigure 4. Endometriotic lesions harvested from PBS (control) and mouse rIL-33 (treated) mice. Endometriotic \nlesions harvested from mouse r-IL33 mice (n = 5) appear qualitatively larger with enhanced vasculature  \n(B) compared to PBS treated (n = 5) mice (A). Black triangles indicate the endometriotic lesion.\n\nwww.nature.com/scientificreports/\n6\nScientific  RepoRts  |  (2017) 7:17903  | DOI:10.1038/s41598-017-18224-x\nexperienced by these patients 3,11. Also, the ability for the endometriotic lesion to establish a blood supply is an \nimportant step in endometriosis development, as this allows the lesion to grow and develop after its initial adhe-\nsion36. Together, our data generated from in vitro studies suggests that IL-33 promotes inflammation and angio-\ngenesis, which likely impacts the inflammatory mileu and disease progression in endometriosis patients.\nOur patient data and in vitro  data, along with previous reports, provide convincing evidence to suggest a \npotential role of IL-33 in endometriosis. Therefore, we used a syngeneic immunocompetent mouse model to \nunderstand the impact of an artificially elevated IL-33 microenvironment on the progression of endometriosis. \nMice treated with mouse rIL-33 had significantly elevated levels of CXCL1, IL-6, GM-CSF , Eotaxin, IL-5, IL-7 \nand IL-33 in the plasma (Fig.  3) suggesting that IL-33 causes systemic inflammation in mice. In another study \nusing a mouse model of allergic-induced inflammation, treatment with mouse rIL-33 polarized CD4 + T cells \nFigure 5. Immunohistochemistry with a proliferation marker, Ki67 (A–F) and vasculature marker CD31 (H–M) \nrevealed intense immunostaining. Semi-quantitative analysis of Ki67 (G) showed significant increase in % \npositive Ki67 cells in the mouse rIL-33 treated mice compared to control. Semi-quantitative analysis of CD31 (N) \nimmunostaining shows a higher trend in rIL33 treated mice compared to controls. Black arrows indicate positive \nstaining. *p < 0.05 **p < 0.01 ***p < 0.0001.\n\nwww.nature.com/scientificreports/\n7\nScientific  RepoRts  |  (2017) 7:17903  | DOI:10.1038/s41598-017-18224-x\nto produce IL-5 and facilitated the disease pathology by promoting a type 2 immune response and perpetuated \nairway inflammation37. Due to the upregulation of IL-5 in the plasma of the mice treated with mouse rIL-33, we \ncan speculate that these mice are similarly exhibiting a type 2 immune response. Overall, mouse rIL-33 led to \nsystemic upregulation of cytokines known to be involved in the progression of endometriosis\n5,38; however, we did \nnot pin point which specific immune cell subsets or other cells were responding to the excess of rIL-33. When \nmice without endometriosis were injected with mouse rIL-33 or PBS, the plasma of mice treated with mouse \nrIL-33 had elevated levels of CXCL1, IL-6, GM-CSF , IL-7, IL-5 and Eotaxin, further suggesting that IL-33 likely \nperpetuates inflammation by interacting with immune cells rather than interacting with the endometriotic lesion \nitself. From previous study using other disease models, we can speculate that immune cells such as macrophages, \nT cells, innate lymphoid cells, and mast cells were likely involved in the progression of inflammation in response \nto rIL-33\n19,32,39.\nOne of the most important observations of our study was the localized effect of IL-33 on the proliferation and \ngrowth of the endometriotic lesion. Mice treated with mouse rIL-33 had larger and highly vascularized lesions \ncompared to controls (Fig. 4). While the growth and size of the lesion is not always correlated with the symptoms \nof endometriosis, the goal of curing endometriosis would be to restrict growth and development of the lesion to \nthe point where it ceased to exist. Contrary to our in vivo observation of increased proliferation of endometriotic \nlesions in response to IL-33 treatment, our in vitro data did not show any proliferative effects in the representative \ncell lines (endometrial epithelial, endothelial and endometriotic epithelial cell lines). This could simply be because \nof the complex composition of endometriotic lesions and perhaps a cross talk is present between cells within \nthe lesion microenvironment causing increased proliferation in vivo . Additionally, the endometriotic lesions \nfrom mice treated with mouse rIL-33 had increased lymphocyte infiltration, suggesting a localized inflammatory \nresponse.\nInterestingly, CXCL1, IL-6 and GM-CSF , were elevated in both the supernatant from endometriotic cell line \n(12Z) and in the plasma of mice treated with mouse rIL-33, which provides further validity to the effect of IL-33 \nin two different disease models. CXCL1, IL-6 and GM-CSF have been shown to be upregulated in the tissue \nand plasma of women with endometriosis compared to healthy fertile controls and suggests that IL-33 could be \nindirectly attracting neutrophils and macrophages to the microenvironment, through the upregulation of these \ncytokines and chemokines\n5,40. As mentioned earlier, with the ability to stimulate several innate and adaptive \nimmune cells, IL-33 likely contributes to the progression of an inflammatory milieu  through the activation of \nimmune cells. However, these mechanisms need to be further studied in the context of endometriosis. The limita-\ntions of this study are common to studies that investigate endometriosis in general. First, the classification system \nof endometriosis is rudimentary due to the heterogeneity and complexity of the disease. Additionally, collecting \nendometriosis patient samples can be difficult due to the current diagnostic delay of 6–11 years and the inva-\nsive procedure required to obtain samples. Therefore, regardless of the prevalence of the disease, gathering well \nstratified samples from patients is an evolving challenge. Additionally, we used the syngeneic immunocompetent \nmouse model of endometriosis, which may not truly represent the human condition, but it provided us with the \nopportunity to gain mechanistic insights on the disease progression in an artificially created IL-33 dominant \nmicroenvironment. Additionally, we did not design experiments in the present study to account for the influence \nof intrinsic estrogen on IL-33 pathway. Estrogen has been shown to modulate levels of IL-33\n41. Despite these lim-\nitations, we have shown that human endometriotic lesions produce IL-33, express ST2 and that levels of IL-33 are \nsignificantly elevated in the endometriotic tissue of advanced staged patients compared to healthy, fertile controls. \nAdditionally, we confirm previous reports that human rIL-33 initiates tube formation in endothelial cells and we \nshow that human rIL-33 stimulates the production of angiogenic and pro-inflammatory cytokines. Finally, we \nshow that in a syngeneic mouse model, elevated levels of mouse rIL-33 initiates local and systemic inflamma-\ntion, stimulates the proliferation of the endometriotic lesion and induces angiogenesis. Overall, the present study \nprovides evidence to suggest that IL-33 is likely one of the important players contributing towards inflammation \nobserved in advanced staged endometriosis patients and the progression of the disease.\nMethods\nEthics statement. Ethics was approved for this study by the Greenville Health System (South Carolina) and \nthe University of North Carolina from Chapel Hill, USA. Human ectopic, eutopic endometrial tissue samples \nfrom endometriosis patients and control samples from fertile women were collected as per institutional approved \nprotocols and guidelines. Written, informed consent was acquired before patient sample collection and storage. \nThe Health Sciences Research Ethics Board, Queen’s University, Kingston approved ethics for this study. All meth-\nods were performed as per institutional approved guidelines.\nAnalysis of sST2 in human plasma samples using an ELISA. Blood was collected in an \nEDTA-containing vacuum tube from 24 patients before undergoing surgery to ablate endometriotic lesions. \nSimilarly, plasma was collected from 16 healthy, fertile controls free from the disease at the University of North \nCarolina School of Medicine. None of the healthy volunteers had signs or symptoms of endometriosis or infer-\ntility. Plasma samples were stored at − 80 °C. Levels of ST2 were analyzed using an ST2 ELISA (R&D Systems; \nDST200). All reagents were prepared per the manufacturer’s protocol. In a 96 well microplate, 100uL of assay \ndiluent was added and 50uL of the protein normalized sample or standard was added to each well and incubated \nfor 2 hours at room temperature. Following aspiration and four washes, 200 uL of Human ST2 conjugate was \nadded to each well and incubated for 2 hours at room temperature. Following washes, 200uL of substrate solution \nwas added to each well and incubated for 30 mins at room temperature. After 30 mins, 50uL of stop solution was \nadded and plates were read using a microplate reader set to 450 nm.\n\nwww.nature.com/scientificreports/\n8\nScientific  RepoRts  |  (2017) 7:17903  | DOI:10.1038/s41598-017-18224-x\nHuman endometrium (eutopic) and endometriosis (ectopic) tissue samples.  Tissue sam -\nples were obtained from 20 endometriosis patients that underwent resection of endometriotic lesions due to \nendometriosis-associated infertility at Greenville Hospital after informed consent. All the patients enrolled in \nthe study were free of any form of hormone therapy at least 3 months prior to surgery. Patients were grouped \ninto disease stage (stage I–IV) as per American Society for Reproductive Medicine criteria. Patients were group \nas either early staged (stage I and II, n  = 14) or advanced staged patients (Stage III and IV , n = 5). Endometrial \nsamples (eutopic) were collected from endometriosis patients using pipelle biopsy. Similarly, endometrial biopsies \nwere collected from 11 healthy, fertile controls free from the disease at the University of North Carolina School of \nMedicine. None of the healthy volunteers had signs or symptoms of endometriosis or infertility. Tissue samples \nwere stored at −80 °C.\nProtein extraction from tissue samples and analysis of IL-33 and ST2 in tissue using a multiplex \ncytokine array and an ELISA. Total protein was extracted from eutopic and ectopic tissue (from endome-\ntriosis patients) and endometrial tissue samples (from healthy, fertile controls) Samples were placed in microcen-\ntrifuge tubes containing 500uL of tissue extraction reagent I (FNN0071; Thermo Fisher), 5 uL protease inhibitor \n(Sigma-Aldrich, St. Louis, MO, USA) and homogenized using a rotor-stator homogenizer on ice. The samples \nwere centrifuged at 18000 RPM at 4 °C and the supernatants were collected. Protein concentrations were meas-\nured using a bicinchoninic acid (BCA) assay (Biorad, Mississauga, ON, CA). All samples were normalized. Levels \nof IL-33 were analyzed using a commercially available human multiplex cytokine assay from Eve Technologies, \nCalgary, AL, Canada. Levels of ST2 were analyzed using an ST2 ELISA (R&D Systems; DST200). All reagents \nwere prepared per the manufacturer’s protocol, as explained above.\nCell culture. Endometrial epithelial carcinoma cells (EECC, CRL-1671; American Type Culture Collection, \nManassas, V A), human umbilical vein endothelial cells (HUVEC, 200–05 f; Cell Applications, San Diego, CA) \nand epithelial, endometriotic 12Zs (generously provided Professor Anna Starzinski-Powitz) were incubated at \n37 °C and 5% CO\n2. EECCs were maintained in Dulbecco’s Modified Eagle’s Medium (D6429; Sigma Aldrich) sup-\nplemented with 10% Fetal Bovine Serum (FBS) and 1% penicillin and streptomycin (Sigma Aldrich). HUVECs \nwere maintained in Endothelial Cell Growth Medium (211–500; Cell Application). 12Zs were maintained in \nDMEM/F-12 (11330; Thermo Fischer Scientific) supplemented with 10% FBS, 1% penicillin and streptomycin \nand 1x sodium pyruvate.\nRNA extraction and RT-PCR. Total RNA from the three cell lines (EECC, HUVEC and 12Z) was extracted \nusing RNA extraction kit (Norgen Biotek, CA) using manufacturer’s instructions. Total RNA was reverse tran-\nscribed using First-strand cDNA synthesis kit (GE Healthcare Life Sciences, Canada) as per the manufactur -\ner’s protocol. Primers were designed using Primer3 software (http://frodo.wi.mit.edu/primer3/) from human \nsequences available on NCBI’s Nucleotide. Real-time PCR was performed using plate-based LC-480 (Roche \nDiagnostics, Montreal, Canada)\n42. Relative quantification was performed using ACTB as a housekeeping control \ngene. Samples were run in triplicates. The run protocol for both genes used was: Denaturation: 95 °C, 15 min; \nAmplification: 45 cycles: 95 °C for 15 s, 55 °C for 30 s, 70 °C for 30 s; Melting Curve: 70–95 °C, at a rate of 0.1 °C per \nsecond. Data was analyzed using the ∆∆Ct method.\nCell proliferation using IncuCyte Cell Confluence Assay.  Cell proliferation was evaluated using the \nIncuCyte cell confluence proliferation assay methodology (IncuCyte ZOOM 2016A; Essen Biosciences Inc.), \nas conducted previously by our group43. EECC, HUVECs and 12Zs were harvested with 1x Trypsin-EDTA and \n2.0 × 104 cells/well were seeded onto a 24-well tissue-culture plate (Sarsted Newton), followed by human rIL-33 \nstimulation at 1, 10, 50, and 100 ng/ml (3625-IL-010; R&D Systems, Minneapolis, MN) in triplicates. PBS was \nused as a control. Cell confluence was measured over 48 hours.\nCell culture supernatant cytokine analysis using multiplex array. EECCs, HUVECs and 12Zs were \nharvested with 1x Trypsin-EDTA and seeded at 105 cells/well onto a six-well plate (Sarstedt Newton) and stimu-\nlated with 10, 50, and 100 ng/ml of human rIL-33 (3625-IL-010; R&D Systems, Minneapolis, MN) in triplicates. \nConcentrations of rIL-33 were selected based on the levels of IL-33 in the PF found previously 25. PBS was used \nas control. The cells were incubated for 24 h at 37 °C with 5% CO 2. The conditioned media was collected and \nanalyzed using a commercially available human multiplex cytokine assay from Eve Technologies, Calgary, AL, \nCanada.\nAngiogenesis (tubulogenesis) Assay. The angiogenesis assay was performed as per manual instructions \n(R&D Systems; 3470–096-K). 50uL of Cultrex ® RGF BME was aliquoted into each well of a 96 well plate and \nincubated at 37 °C for 30 minutes to allow the BME to gel. Then, HUVECs were harvested with 1x Trypsin-EDTA \nand seeded at 104 cells/well and varying concentrations (10, 50, and 100 ng/mL) of human rIL-33 (3625-IL-010; \nR&D Systems, Minneapolis, MN) in triplicates. Cells were incubated for 6 hours in a CO 2 incubator at 37 °C. \nImages were taken using IncuCyte ZOOM (2016A; Essen Biosciences Inc) and tube formation was evaluated and \nquantified using NIH ImageJ with the angiogenesis analyzer plugin\n44.\nSyngeneic mouse model of endometriosis. All animal experiments were performed under protocols \napproved by Queen’s University Institutional Animal Care Committee as per Canadian Council of Animal Care \nguidelines. Mature (8–10-week-old) C57Bl/6 mice (n = 15, Charles River, USA) were housed in cages of 3–4. \nAfter euthanasia, the uterine horns were harvested from donor mice (n = 5), and placed in a petri dish containing \nPBS. The endometrium was cut into 2 mm\n3 fragments using an epidermal puncher and kept on ice until they were \nsurgically explanted into the recipient mice (n = 10). Under 4% isofluorane vaporizer anesthesia, small incisions \n\nwww.nature.com/scientificreports/\n9\nScientific  RepoRts  |  (2017) 7:17903  | DOI:10.1038/s41598-017-18224-x\nwere made in the abdomen of recipient mice and the donor endometrial fragments were adhered to the right \nside of the peritoneal cavity using bonding agent. Following surgery, the animals were rested for 14 days. Then, \nmice in the treatment group (n = 5) received intraperitoneal injections of mouse rIL-33 at 1ug/mouse in 100uL \nvolume (Ebioscience; 14-8332-80) two times a week for two weeks. Mice from the control group (n = 5) received \ntwo intraperitoneal injections of PBS for two weeks. Blood was collected from all the experimental mice at day 7, \nday 18 and day 25, where day 0 indicates the date of surgery and day 15 indicates the onset of PBS or mouse rIL-\n33 treatment (Fig. 3A). Mice were sacrificed on day 25 and ectopic lesions, uterus, ovaries, spleen, liver, kidneys \nand heart were harvested, fixed using 4% paraformaldehyde, and stored in 70% ethanol prior to tissue processing.\nTo understand the effect of rIL-33 on mice without endometriosis, we injected 8–10 weeks old C57Bl/6 mice \n(n = 6, Charles River, USA) with mouse rIL-33. These healthy mice without endometriosis underwent biweekly \nintraperitoneal injections for one week with either PBS (n  = 3) or mouse rIL-33 (1ug/mouse) in 100uL volume \n(n = 3). After one week, the mice were sacrificed and blood was collected by cardiac puncture. Plasma samples \nwere analyzed using multi-plex cytokine assay from Eve technologies as explained below.\nCytokine analysis in plasma samples from experimental mice using a multiplex array. 100 uL \nof blood was collected from the submandibular vein at three time points (day 7, day 18 and day 25) in tubes \ncoated with EDTA (K\n2EDTA; 365974) and immediately placed on ice. The blood was centrifuged for 15 mins at \n3,000 rpm at 4 C, plasma collected and aliquoted for storage at − 80C. Aliquots were diluted in PBS to a dilution \nfactor of 2 and were analyzed using a mouse multiplex cytokine analysis from Eve Technologies, Calgary, AL, \nCanada as described previously\n5. Cytokines included in the multiplex panel were: Eotaxin, G-CSF , GM-CSF , IFN \nγ, IL-1a, IL-1b, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-17A, IP-10, \nKC, LIF , LIX, MCP-1, M-CSF , MIG, MIP-1alpha, MIP-1beta, MIP-2, RANTES, TNFalpha, VEGF , IL-17E, IL-17F , \nIL-21, IL-22, IL-23, IL-27, IL-28B, IL-31, IL-33, and MIP-3a. Blood was collected from mice without endometri-\nosis in tubes coated with EDTA (K 2EDTA; 365974) and immediately placed on ice. Plasma was collected and \nstored at − 80C. Aliquots were diluted in PBS to a dilution factor of 2 and were analyzed using the same mouse \nmultiplex cytokine analysis from Eve Technologies, Calgary, AL, Canada.\nCD31 and Ki67 Immunohistochemistry.  Paraformaldehyde fixed, paraffin embedded endometriotic \nlesions from mice were sectioned at 5 μm thickness. After deparafinization in series of graded alcohols and citro-\nsolve solution, sections were immunostained using a referral service with automated stainer in the Department \nof Pathology and Molecular Medicine at Queen’s University (BenchMark XT automated stainer from Ventana \nMedical System Inc., Tucson, AZ, USA). Following antigen retrieval with cell conditioning 1 for 60 min (Ventana \nMedical System Inc), sections were incubated with primary anti-CD31 (ab28364, Abcam 1:5000) and anti-Ki67 \nantibodies (ab16667, Abcam 1:1000). The sections were the stained with secondary antibodies for 60 min and \nUltrablue DAB detection kit was used for color development (Ventana Medical System Inc). Finally, sections \nwere counterstained with haematoxylin and bluing reagent for 4 min before adding the coverslips. For CD31 and \nKi67 staining, differential cell counting analyses, image analysis software was conducted using NIH ImageJ as per \npreviously published protocol from our group\n45. Briefly, 100 cells were manually counted from three areas from \nthree different slides in each experimental group. All images had a total of 2014 × 1536 pixels each and were all \nfrom the same magnification (200 X).\nStatistical analysis. The matched eutopic and ectopic tissue from the same patient was analyzed using a \npaired t-test. Protein levels from patients compared to normal controls were analyzed using a one-way analysis of \nvariance (ANOV A). The data collected the angiogenesis and the cell supernatant experiments was analyzed using \na one-way analysis of variance (ANOV A). The data collected from the IncuCyte cell confluence proliferation assay \nand blood cytokine levels were analyzed using a two-way analysis of variance (ANOV A) with repeat measure. \nDifferential cell counting experiments (CD31, Ki67) were compared using non-parametric student’s t-test to \ncompare between treated and control groups. A p < 0.05 was considered as statistically significant. All statistical \nanalysis was conducted and graphs were generated using Graphpad Prism 7.0 software (GraphPad Software Inc., \nCalifornia, USA).\nReferences\n 1. Halme, J., Hammond, M. G., Hulka, J. F ., Raj, S. G. & Talbert, L. M. Retrograde menstruation in healthy women and in patients with \nendometriosis. Obstet. 64, 151–154 (1984).\n 2. Ahn, S. H. et al. Pathophysiology and immune dysfunction in endometriosis. BioMed Research International 2015 (2015).\n 3. Miller, J. E. et al. Implications of immune dysfunction on endometriosis associated infertility. Oncotarget. https://doi.org/10.18632/\noncotarget.12577 (2016).\n 4. Kyama, C. M., Debrock, S., Mwenda, J. M. & D’Hooghe, T. M. Potential involvement of the immune system in the development of \nendometriosis. Reprod. Biol. Endocrinol. 1, 123 (2003).\n 5. Monsanto, S. P . et al. Surgical removal of endometriotic lesions alters local and systemic proinflammatory cytokines in endometriosis \npatients. Fertil. 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CXCL10 alters the tumour immune microenvironment and disease progression in a syngeneic murine model of high-\ngrade serous ovarian cancer. Gynecol. Oncol. 145, 436–445 (2017).\n 44. Gilles Carpentier. Angiogenesis Analyzer for ImageJ. (2012). Available at: http://image.bio.methods.free.fr/ImageJ/?Angiogenesis-\nAnalyzer-for-ImageJ (Accessed: 4th July 2017).\n 45. Khalaj, K. et al. RNA binding protein, tristetraprolin in a murine model of recurrent pregnancy loss. Oncotarget . https://doi.\norg/10.18632/oncotarget.12539 (2016).\nAcknowledgements\nThe authors would like to acknowledge financial support by National Institutes of Health (NIH – A.T.F ., S.L.Y & \nB.A.L), and Canadian Institutes of Health Research (CIHR – C.T. M.K).\nAuthor Contributions\nJ.E.M. and C.T. conceived the experiments; S.L.Y ., B.A.L., S.S.S. collected patient samples; A.T.F ., C.T. contributed \nreagents; J.E.M., S.M., S.H.A., K.K., conducted the experiments. J.E.M. analyzed the results; J.E.M. wrote the \nmanuscript; C.T., M.K. supervised data analysis and provided critical suggestions for manuscript writing. All \nauthors reviewed the manuscript.\nAdditional Information\nSupplementary information accompanies this paper at https://doi.org/10.1038/s41598-017-18224-x.\nCompeting Interests: The authors declare that they have no competing interests.\n\nwww.nature.com/scientificreports/\n11\nScientific  RepoRts  |  (2017) 7:17903  | DOI:10.1038/s41598-017-18224-x\nPublisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and \ninstitutional affiliations.\nOpen Access This article is licensed under a Creative Commons Attribution 4.0 International \nLicense, which permits use, sharing, adaptation, distribution and reproduction in any medium or \nformat, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre-\native Commons license, and indicate if changes were made. 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