{"paper_id":"7e52898d-b84d-4de1-be72-48194edce38e","body_text":"All animal studies were conducted in accordance with the National Institutes of\nHealth Guidelines for Humane Use and Care of Animals and with approved National\nInstitute of Environmental Health Sciences (NIEHS) animal protocol. Mice\n( α ERKO) containing a deletion of exon 3 of the\n Esr1  (ER α ) gene were generated as\ndescribed previously ( 36 ). Adult female\nC57/BL6 mice were purchased from Charles River Laboratories (Raleigh, NC), adult\nfemale IL-6KO mice (B6.129S2- IL6 tm1/Kopf /J) were\npurchased from The Jackson Laboratory (Bar Harbor, ME),  α ERKO\nmice were generated from the NIEHS  α ERKO colonies at Charles\nRiver Laboratories (Wilmington, MA) or were generated by in-house breeding at NIEHS.\nMice used were female and aged between 2 and 6 months. Mice were in a controlled\ntemperature range (22°C to 23°C) on a 12-hour light, 12-hour dark\ncycle. Mice were given food and water  ad libitum .\nRecipient mice of various genotypes depending on the experimental design were\novariectomized through two 0.5-cm dorsolateral skin incisions, and endogenous\nhormones were allowed to clear for 7 to 10 days. Mice were then randomly divided into\ntwo treatment groups, E 2  valerate (2.5 µg/mouse/wk; Sigma-Aldrich,\nSt. Louis, MO) in corn oil or corn oil vehicle (n = total of 6 to 12 mice per group\nwith experimental replicates). Mice were dosed subcutaneously once prior to\nexperimental endometriosis induction and then once weekly for the duration of the\nstudy for long-term studies. Donor mice were primed 41 hours prior to uterus removal\nwith pregnant mare serum gonadotropin 5 IU intraperitoneal ( 9 ). The donor uterus was removed en bloc after euthanasia, cleaned\nof excess tissue, and the outer myometrium was peeled away. The tissue was then\nwashed thrice in sterile phosphate-buffered saline (PBS). In a glass 60-mm dish, the\nuterus was slit with a linear incision longitudinally and minced (≤1.5 mm).\nRecipient mice were anesthetized using isoflurane/oxygen and given buprenorphine (0.1\nmg/kg) for pain management. A 0.5-cm right dorsolateral incision was made in the\nrecipient abdomen while the donor uterus was minced. The minced donor uterine tissue\nwas suspended in 500  µ L of PBS, was injected into the\nperitoneal cavity of the recipient using a p1000 tip, and the peritoneal wall was\noverlapped to close the cavity, the outer skin was closed with 9-mm clips, and a\ngentle massage was given to disperse the tissue throughout the peritoneal cavity. An\nequivalent amount (∼100 mg) of minced tissue was transferred into all\nrecipients (WT and IL-6KO donors were used at a one donor uterus: one host ratio,\nwhereas the  α ERKO donors, with hypoplastic uteri, were used\nat a five donor uteri: one host ratio). Sham mice received the same operations as\nexperimental mice but were injected with PBS alone. Mice were treated up to an\nadditional 3 weeks with E 2  valerate  ( International Union of\nPure and Applied Chemistry:\n[(8R,9S,13S,14S,17S)-3-hydroxy-13-methyl-6,7,8,9,11,12,14,15,16,17-decahydrocyclopenta[a]phenanthren-17-yl]\npentanoate )  or vehicle ( Fig.\n1A ). Groups were designated in the following manner: donor to host: WT to WT,\nWT to  α ERKO, and  α ERKO to WT. For\nanti-IL-6 experiments, mice were injected subcutaneously with 200 µg/mouse\nanti-IL-6 (Bio X Cell, West Lebanon, NH; clone MP5-20F3) every third day starting 3\ndays prior to tissue injection. Mice per group are based on sample size calculations\ndone using preliminary data to compare sham vs endometriosis or treated vs control\nand were conducted using SAS Proc Power (SAS Institute, Cary, NC; 2008).\nEndometriosis lesion macroscopic and microscopic appear similar 72 hours after\ndisease initiation. (A–F) Gross appearance of lesions (×7.5) in\nWT to WT (left column), WT to  α ERKO (middle column),\nand  α ERKO to WT (right column). Lesions in A–C\nare localized to the injection site. Lesions in D–F are localized\nthroughout the peritoneal cavity. Lesions are outlined by dotted white line.\n(G–I) Histological evaluation (hematoxylin and eosin) of lesion tissue\n(×100). Representative examples are E 2  treated.\nAfter 24, 48, or 72 hours or 3 weeks ( Supplemental Fig. 1 ), mice were euthanized with\nCO 2 . Peritoneal wash was performed by injecting 1 mL of PBS + 0.5%\nbovine serum albumin (BSA) + 2 mM EDTA into the peritoneal cavity. The cavity was\ngently massaged, a small incision was made into the inner skin lining the peritoneal\ncavity, and the fluid was gently removed not drawing organs into the syringe. The\nperitoneal wash was immediately spun at 800 g  for 5 minutes, the\nsupernatant was snap-frozen on dry ice and stored at −80°C until use,\nand the cell pellet was resuspended in PBS + 0.5% BSA + 2 mM EDTA and kept cold on\nice until antibody staining. To assess the effects of genotype on ectopic uterine\ntissue, ectopic lesions were photographed to document  in situ  images\nof endometriosis-like lesions (Leica dissecting microscope MZ16FA and Leica camera\nDFC490, Germany). Endometriosis-like lesions were visualized, dissected, measured,\nweighed, and then removed and either fixed in 10% formalin or snap frozen on dry ice\nand stored at −80°C until use. Resuspended cell pellets containing red\nblood cells (RBCs) were lysed one to two times with RBC lysis buffer for 15 to 30\nseconds and 10 times volume of PBS was immediately added. Cells were resuspended for\ncell counting, cytospin, and flow cytometry analysis. A hemocytometer was used for\ncell counting, and 150,000 cells were used for differentials and the remaining for\nfluorescence-activated cell sorting (FACS; see later).\nThe fixed tissues were routinely processed for paraffin embedding. Five micron\nsections were cut and the slides were used for hematoxylin and eosin (Sigma-Aldrich)\nstaining. All the slides were deparaffinized and hydrated through descending grades\nof alcohol, stained, dehydrated, and cover slipped.\nDifferentials were stained with modified Giemsa (Hema 3 according to\nmanufacturer’s protocol).\nFrozen endometriosis-like lesions from the mice were pulverized under liquid nitrogen\nand RNA was isolated using TRIzol as per manufacturer’s instructions\n(Invitrogen, Carlsbad, CA). Using a previously described method, complementary DNA\nwas synthesized and analyzed by real-time polymerase chain reaction (RT-PCR) using\nFast SYBR ( 37 ). Relative transcript levels\nwere quantified in comparison with the WT to WT vehicle group and normalized to\n Rpl7  using the model described by Pfaffl ( 38 ). Primer sequences ( Supplemental Table 1 ) purchased from\nSigma-Aldrich were selected using Primer Express (Applied Biosystems, Foster City,\nCA), Harvard Primer Bank (Harvard University, Cambridge, MA), or PrimerBot!\n(McDonnell Laboratory, Duke University, Durham, NC).\nPeritoneal cavity lavage fluid was used neat according to the manufacturer’s\nprotocol for multiplex analysis (BioRad, Hercules, CA). Bio-Plex\nPro ™  Cytokine 23-plex Assay (M60009RDPD) and Bio-Plex Custom\nAssays were used for the detection of vascular endothelial growth factor (VEGF),\nIL-6, granulocyte colony-stimulating factor (G-CSF), monocyte chemotactic protein 1\n(MCP1), granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-10, and\nIL-17.\nPeritoneal fluid cells were spun and resuspended in 450 µL of FACS buffer\n(0.5% BSA, 0.1% NaN 3 , 2 mM EDTA in PBS). Two antibody panels were run for\neach sample and 200 µL of cells (1–2 × 10 6  cells)\nwere placed into a round bottom 96-well plate. Cells were spun and resuspended and\nblocked for 30 minutes in nonspecific binding blocking reagent cocktail made in FACS\nbuffer with 5% normal mouse serum (#015-000-120; Jackson ImmunoResearch, West Grove,\nPA), 5% normal rat serum (#012-000-120; Jackson ImmunoResearch), and 5 µg/mL\nanti-CD16/32 (2.4G2 hybridoma). Antibody cocktails ( Table 1 ) in FACS buffer were added to the samples for an additional 30\nminutes. For staining, cells were incubated with fluorochrome Allophycocyanin (APC),\nAPC-Alexa Fluor-647, APC-Cy7, eFluor-450, eFluor-605, eFluor-780, phycoerythrin,\nPacific Blue, PerCP-Cy5.5, or biotin-conjugated antibodies against mouse B220/CD45r\n(RA3-6B2), CD4 (L3T4), NK1.1 (PK136), CD3e (145-2C11), EpCAM/CD326 (G8.8), Ly-6G\n(1A8), MHC class II/I-Ab (AF6-120.1), CD11b (M1/70), CD11c (N418), F4/80 (BM8), CD115\n(AFS98), and Ly-6C (AL-21) from BD Biosciences (San Jose, CA), Thermo Fisher\nScientific (formerly eBiosciences; Waltham, MA), and BioLegend (San Diego, CA).\nStained cells were analyzed on a FACS LSRII flow cytometer (BD Biosciences). Data\nfrom these studies were analyzed using FlowJo software (Treestar, Ashland, OR). Only\nsingle cells were analyzed.\nAntibodies Used\nAbbreviation: RRID, Research Resource Identifier.\nOne-way analysis of variance (ANOVA) with Tukey posttest, two-way ANOVA with\nBonferroni posttest, and one-way ANOVA with Bonferroni Multicomparison posttest\n P  < 0.05 were performed using GraphPad Prism version 7.01\n(GraphPad Software, San Diego, CA). Means not sharing a letter are significantly\ndifferent from each other ( P  < 0.05). Means sharing a same\nsingle letter or a letter in combination with other letters are not significantly\ndifferent from each other ( P  > 0.05).\n\nOur previous findings demonstrated the importance of ER α  and\nE 2  in chronic endometriosis ( 9 ).\nIn the current study, we evaluated the role of ER α  and\nE 2  in the early development of endometriosis with the hypothesis that\nER α -mediated signaling is critical for lesion development.\nA syngeneic mouse model of endometriosis was used to assess early endometriosis\nlesion formation ( Supplemental Fig. 1 ). We first chose to examine\nlesion development 72 hours post disease initiation to capture early lesion\ndevelopment. At necropsy, lesions and peritoneal fluid/peritoneal cells were\ncollected. Representative macroscopic and microscopic images of lesions collected\nfrom WT to WT, WT to  α ERKO,  α ERKO to\nWT 72 hours after disease initiation are shown ( Fig.\n1 ). Regardless of ER α  genotype, lesions are found\nthroughout the peritoneal cavity. At the injection site, these lesions are visually\noften hemorrhagic, vascularized, and not securely attached to the peritoneal wall\n( Fig. 1A–1C ), whereas lesions distal\nto the injection site are also often hemorrhagic, vascularized, and beginning to\nattach by 72 hours ( Fig. 1D–1F ). Lesions\nwere similar across groups ( Supplemental Fig. 1 ), which is in contrast to\nwhat is observed at 3 weeks ( 9 ). At 3 weeks,\n α ERKO to WT do not develop lesions, and WT to WT or WT to\n α ERKO lesions are not hemorrhagic, but cystic with clear\nfluid, which demonstrates the disease has progressed and established ( 9 ). At 72 hours postinjection, the transferred\nuterine tissue is localized to the same sites of attachment found 3 weeks post\ndisease initiation. Lesions are often attached to the peritoneal wall, intestinal\nmesentery, fat pads, behind the stomach, in the rectouterine cul-de-sac area, and to\nthe uterine blood supply. Lesions are not found attached to the spleen, liver, or\nkidneys. Histological evaluation of 72-hour lesion tissue ( Fig. 1G–1I ) shows lesion tissue is highly disorganized and\ninfiltrated with white and RBCs. The disorganization at 72 hours is in contrast to\nwhat was observed 3 weeks after disease initiation where lesions are organized with\ndistinct epithelial and stromal cell layers ( 9 ). These data show similar lesion number, weight, and histopathological\ncharacteristics between all experimental groups.\nOur laboratory’s previous work with ER α  chromatin\nimmunoprecipitation sequencing of whole uterine chromatin uncovered a host of\nER α  bound genes associated with angiogenesis and\ninflammatory factors ( 39 ). To investigate how\nthese ER α  bound genes were altered by early endometriosis (72\nhours), ER α  status, and E 2  treatment,\nendometriosis was initiated in WT to WT,  α ERKO to WT, and WT\nto  α ERKO with and without E 2  treatment. When\nexamining lesion gene expression of a multitude of these factors, with a focus on\nimmune and angiogenic factors 72 hours post disease initiation, no statistical\ndifferences were found between the groups or with E 2  treatment (data not\nshown). Therefore, to more closely examine the regulation surrounding the development\nof a blood supply and the seemingly E 2 -independent effect, we isolated\nlesions 24 and 48 hours after disease initiation in WT to WT lesions. Genes expressed\nin lesions were compared with noninjected minced uterine tissue by RT-PCR analysis.\nLesions removed 24 and 48 hours after disease initiation have increased gene\nexpression of inflammatory factors ( Fig. 2A :\n S100a8 ,  F4/80/Adgre1 ,\n G-CSFR/Csf3r ) and angiogenic factors known to be regulated in\nendometriosis ( Fig. 2B :  Timp1 ,\n Vegfa ) compared with minced uterine tissue (set to 1) at both\ntime points. Again, no further increase in gene expression was observed with\nE 2  treatment. These data suggest increased activity of angiogenic and\nimmune factors in early lesion development are independent of E 2 \ntreatment.\nEstrogen does not further increase lesion marker gene expression 24 or 48 hours\nafter endometriosis disease initiation. (A) Gene expression of immune cell\nmarkers for neutrophils ( S100A8 ), macrophages\n( F4/80 ), and granulocytes ( G-CSFR ) from WT\nto WT lesions at 24 and 48 hours after endometriosis-like disease initiation.\nLesions are compared with minced uterine tissue (set to 1). (B) Gene expression\nfrom angiogenic factors ( Timp1  and  Vegfa )\nfrom WT to WT lesions at 24 and 48 hours after endometriosis-like disease\ninitiation. Lesions were removed, RNA was isolated, and gene expression was\ndetermined by RT-PCR. Means not sharing a letter are significantly different\nfrom each other ( P  < 0.05). Means sharing a same single\nletter or a letter in combination with other letters are not significantly\ndifferent from each other ( P  > 0.05; one-way ANOVA).\nError bars represent standard error of the mean; n = 8 to 11.\nAs women with endometriosis often have an aberrant peritoneal fluid cytokine milieu\n( 40 ), we next evaluated peritoneal fluid\ncytokines from mice induced with endometriosis. At necropsy, peritoneal fluid was\ncollected from WT to WT at 24, 48, and 72 hours after disease initiation and compared\nwith sham peritoneal fluid (24, 48, and 72 hours) or 3-week peritoneal fluid to\ndetermine the role E 2  plays in early disease initiation. All animals\nreceived vehicle or E 2  and surgery, but sham animals were injected with\nPBS alone. MCP1/CCL2, G-CSF, IL-6, and VEGF increase in endometriosis peritoneal\nfluid independent of E 2  treatment at 24, 48, and 72 hours after disease\ninitiation and return to sham levels by 3 weeks after disease initiation ( Fig. 3A–3D ). Although other cytokines, such\nas GM-CSF, IL-10, and IL-17, remain chronically elevated at 3 weeks, these pro- and\nanti-inflammatory cytokines show no E 2 -mediated differences ( Fig. 3E–3G ). These data again suggest that\nduring early disease development the immune-mediated responses outweigh potential\nE 2 -mediated effects.\nPeritoneal lavage fluid from WT to WT endometriosis mice treated with and\nwithout E 2  have increased cytokine and chemokine production 24, 48,\nand 72 hours after disease initiation that is disease dependent using\nenzyme-linked immunosorbent assay (ELISA). (A–D) Transient increase is\nseen with MCP1, G-CSF, IL-6, and VEGF. (E–G) Chronic increase is seen\nwith GM-CSF, IL-17, and IL-10. Endometriosis (Endo) was induced with injection\nof minced uterine tissue and compared with sham operated animals. At necropsy,\n1 mL of saline was injected into the peritoneal cavity, mice were gently\nmassaged, and fluid was removed for cytokine/chemokine analysis by ELISA. Means\nnot sharing a letter are significantly different from one another\n( P  < 0.05). Means sharing a same single letter or a\nletter in combination with other letters are not significantly different from\none another ( P  > 0.05; two-way ANOVA). Error bars\nrepresent standard error of the mean. Three weeks: n = 5; 24 to 72 hours: n = 8\nto 12. ND, not detected.\nAs mentioned, menstruation is an inflammatory process with 40% of menstrual tissue\nbeing composed of neutrophils, macrophages, and uNK cells ( 22 ,  34 ,  35 ). Leukocytes secrete cytokines into the\nperitoneal cavity, and because our data display no differences between vehicle and\nE 2  treatment, we next examined the cell population(s) infiltrating into\nthe peritoneal cavity after the initiation of endometriosis in mice only treated with\nE 2 . A representative differential from each group is shown in\n Supplemental Fig. 2 . The cell differentials from\nthe sham groups are visually different from the endometriosis groups. Leukocytes are\npresent in the peritoneal cavity of endometriosis animals and sham animals\n( Supplemental Fig. 2 ). In the endometriosis\nsamples, increased numbers of recruited neutrophils and activated macrophages are\nseen. Total cell counts in the peritoneal fluid determined from WT to WT, WT to\n α ERKO, and  α ERKO to WT 24, 48, and\n72 hours after disease initiation were compared with WT and\n α ERKO sham animals. Total cell counts demonstrated immune\ncells are infiltrating into the peritoneal cavity and are increased transiently\nregardless of ER α  genotype 24 and 48 hours after disease\ninitiation ( Fig. 4A ). By 72 hours, cell numbers\ndecreased and were statistically unchanged from sham levels.\nInnate immune cells infiltrate into the peritoneal cavity after the initiation\nof endometriosis in a disease-dependent manner. (A) Total cell counts in sham\nWT (W), sham  α ERKO ( α ),\nendometriosis (endo) WT to WT (WW), endo WT to  α ERKO\n(W α ), and endo  α ERKO to WT\n( α W) 24, 48, and 72 hours after the initiation of\nendometriosis. A representative experiment is shown (n = 5). (B) Quantitation\nof peritoneal neutrophil counts. A representative experiment is shown (n = 5).\n(C) Neutrophils were gated for Ly6G + . (D) Macrophage gating\nstrategy. Regions (R1, R2, and R3) were gated. R1 was then gated for\nF4/80 +  and Ly6C +  to determine M1, M2, M-inflammatory\n(M-inflam) macrophages. (E) Quantitation of total macrophages\n(SSCA hi /CD115 + ,F4/80 + ). (F) Quantitation of\ninflammatory macrophages\n(SSCA hi /CD115 + ,F4/80 lo /Ly6C hi )\n(n = 8 to 12). (G) Quantitation of M1 macrophages\n(SSCA hi /CD115 + ,F4/80 med /Ly6C − )\n(n = 8 to 12). (H) Quantitation of M2 macrophages\n(SSCA hi /CD115 + ,F4/80 + /Ly6C − )\n(n = 8 to 12). (I) Quantitation of NK cells\n(NK1.1 + ,CD3e − ). A representative experiment is\nshown (n = 5). Means not sharing a letter are significantly different from each\nother ( P  < 0.05). Means sharing a same single letter or\na letter in combination with other letters are not significantly different from\neach other ( P  > 0.05; two-way ANOVA). Error bars\nrepresent standard error of the mean.\nCells infiltrating into the peritoneal cavity were immunophenotyped using flow\ncytometric analysis. The antibody panels used to stain and immunophenotype\nneutrophils, macrophages, epithelial cells, B cells, T cells, NK cells, and dendritic\ncells (DCs) are listed in the materials and methods. Cells were first gated for\nsingle cell populations and then gated based on the specific cell type antibody\nmarker(s). Neutrophils were gated based on side scatter (SSC hi ), exclusion\nof B cells (B220 − )/T cells (CD3e − ), and\nLy6G +  expression ( Fig. 4B and 4C ).\nNeutrophil infiltration increases 24 hours after the initiation of disease relative\nto sham animals. The numbers of neutrophils are reduced at 48 hours, but return to\nsham levels by 72 hours. No differences were observed regardless of the presence of\nER α  in the donor or host peritoneal cavity.\nTotal macrophages were gated following the peritoneal fluid gating strategy of Xia\n et al.  ( 41 ). In brief,\ntotal macrophages were gated for SSC-A hi , CD115 + , and\nF4/80 +  ( Fig. 4D ). No differences\nin total macrophages are seen between endometriosis and sham groups at 24, 48, or 72\nhours after the initiation of endometriosis ( Fig.\n4E ). The total macrophage sample was further immunophenotyped ( Fig. 4F–4H ) by gating for inflammatory\nmacrophages (CD115 + , F4/80 low , Ly6C hi ), M1\nmacrophage/proinflammatory macrophages (CD115 + , F4/80 med ,\nLy6C − ), and M2 macrophage/resident anti-inflammatory macrophages\n(CD115 + , F4/80 hi , Ly6C − ). No\nER α  genotype–dependent changes in macrophages were\nobserved. Inflammatory macrophages increased 24 hours after disease initiation and by\n72 hours return to sham levels. M1 macrophages increased 48 and 72 hours after\ndisease initiation in endometriosis animals compared with sham animals. M2\nmacrophages show no striking differences between sham animals or in any of the\nexperimental combinations at 24, 48, or 72 hours. Neutrophil and macrophage\npopulations are the predominant cell types recruited to the peritoneal cavity after\ndisease initiation.\nBecause uNK cells are a known component of menstrual effluent and can assist in\nvascular remodeling ( 42 ), uNK cells were\nimmunophenotyped by excluding any CD3e +  cells (excludes NK T cells) and\nthen gated for NK cell marker NK1.1 ( Fig. 4I ).\nNK cells increased 48 hours after disease initiation in endometriosis animals when\ncompared with sham animals at 24, 48, and 72 hours. To ensure robust characterization\nof the peritoneal cavity immune cell population after the initiation of\nendometriosis, we also examined DCs, B cells, and T cells. Additionally, endometrial\nglandular cells secrete chemokines ( 43 );\ntherefore, recruitment of epithelial cells was examined. DCs were characterized\n(Ly6C + , IA-b + , CD11c hi ) and, as previously\npublished by Stanic  et al.  ( 44 ), we observed a twofold increase in DCs compared with sham animals\n( Supplemental Fig. 3 ). In contrast, B cells\n(B220 + ), T cells (CD3e + , CD4 +  vs\nCD4 − ), and epithelial cells (EpCAM + ) exhibit no\nstatistical changes relative to sham operated or ER α  status\n( Supplemental Fig. 3 ). Additionally, naïve\nmice were staged based on estrus cycle (proestrus, estrus, metestrus, and diestrus)\nand the peritoneal cavity cell populations remained static throughout the cycle\nstages with our immunophenotyping parameters (data not shown). These data demonstrate\ninnate immune cell populations are involved in the early initiation of endometriosis.\nMacrophages and neutrophils predominate in the early initiation of endometriosis with\nneutrophils peaking and inflammatory macrophages at 24 hours, and M1 macrophages at\n48 hours.\nExamining the presence of factors known to be expressed in human endometriosis, we\nexamined relative messenger RNA expression in the two main groups, WT to WT and\n α ERKO to WT with E 2  treatment compared with WT\nand  α ERKO minced uterine tissue. Our focus was on IL-6\nsignaling as IL-6 was one of the highest modulated cytokines in our analyses ( Fig. 5A–5F ). IL-6 message mirrors the\nsecretory IL-6 levels and is not elevated at 48 hours after the initiation of\nendometriosis in the  α ERKO to WT group ( Fig. 5A ). ER α  is known to modulate IL-6\nvia CEBP β  and nuclear factor  κ B\n(NF- κ B) pathways ( 45 – 48 ); therefore, gene\ntargets in these pathways were examined. Prostaglandin-endoperoxide synthase 2\n( Cox2 / Ptgs2 ),  Timp1 ,\n Cxcl2 , and  Cebpβ  are significantly\ndecreased in the  α ERKO to WT lesions at 48 hours and suggests\nthis pathway is involved in lesion development.  Vegfa  and\n Socs3 , also associated with these pathways, were increased in\n α ERKO to WT lesions similarly to WT to WT lesions 48 hours\nafter the initiation of endometriosis. These data at 48 hours suggest an\nER α -IL-6 axis may contribute to early lesion development\nand a potential pathway for therapeutic targeting.\nEndometriosis lesions exhibit changes in the IL-6 pathway that are blunted in\nthe absence of ER α  in donor tissue. (A–F) Gene\nexpression in endometriosis (Endo) lesions when compared with WT and\n α ERKO minced uterine tissue. Lesions were removed,\nRNA was isolated, and gene expression was determined by RT-PCR (n = 5 from a\nrepresentative experiment). (G–I) Gene expression for peritoneal fluid\ncytokines. Peritoneal fluid from sham and endo were removed and fluid was\nanalyzed by enzyme-linked immunosorbent assay (n = 5 to 10 from a\nrepresentative experiment). For gene expression data, means not sharing a\nletter are significantly different from one another ( P \n< 0.05). Means sharing a same single letter or a letter in combination\nwith other letters are not significantly different from one another\n( P  > 0.05; one-way ANOVA). For cytokine analysis,\nmeans not sharing a letter are significantly different from one another\n( P  < 0.05). Means sharing a same single letter or a\nletter in combination with other letters are not significantly different from\none another ( P  > 0.05; two-way ANOVA).\nTo further examine an ER α -mediated effect on early disease,\nperitoneal fluid from WT to  α ERKO and\n α ERKO to WT at 24, 48, and 72 hours was examined and\ncompared with WT to WT fluid for a specific subset of cytokines ( Fig. 5G–5I ). The proangiogenic factor VEGF is strongly\nincreased in the peritoneal fluid of all endometriosis groups at 24, 48, and 72 hours\nafter disease initiation when compared with sham. G-CSF shows increased levels in all\nendometriosis groups and times after disease initiation. IL-6 secretion decreased 48\nhours after disease initiation in  α ERKO to WT compared with\nWT to WT. These findings further indicate a role for the immune system in early\ndisease, but also implicate a specific role for\nER α -IL-6-mediated cross-talk in lesion development. It is\nnotable to see an increase in both angiogenic and early inflammatory cytokines,\nregardless of ER α  genotype, in factors known to be important\nfor angiogenesis and disease progression. On the other hand, IL-6 and targets\ndownstream of IL-6 ( Cox2  and  Timp1 ) are decreased\nat 48 hours when ER α  is knocked out of the donor uterine\ntissue, suggesting ER α -IL-6 cross-talk is important in lesion\ndevelopment.\nWith the decreased response of IL-6 48 hours after the initiation of endometriosis,\nwe examined the ability of endometriosis lesions to attach and grow using IL-6KO mice\nor in the presence of anti-IL-6 treatments at 3 weeks. IL-6KO or anti-IL-6 treatment\ndid not alter the uterine weight increase with E 2  (data not shown). After\n3 weeks, as expected, no increased number of lesions is observed in WT to WT with\nE 2  treatment; however, using IL-6KO animals increased the number of\nlesions in IL-6KO to IL-6KO with E 2  treatment ( Fig. 6A ). Additionally, anti-IL-6 treatment + E 2 \nincreased the number of lesions ( Fig. 6A ). WT to\nIL-6KO or IL-6KO to WT with E 2  treatment tended toward, but did not reach\nsignificance and demonstrate that both host and donor IL-6 plays a role in lesion\ndevelopment. Lesion weight was not altered based on IL-6 genotype, but lesion weight\nincreased with anti-IL-6 + E 2  cotreatment relative to E 2 \ntreatment alone ( Fig. 6B ). These findings\nsuggest IL-6 plays a larger role in the number of lesions formed and E 2 \nplays a role in lesion growth. These data further support cross-talk between\nER α  and IL-6 in the development of endometriosis.\nKO of IL-6 or anti-IL-6 treatment increases endometriosis lesion numbers.\nLesion number and lesion weight were examined in the absence of IL-6 using\nIL-6KO or anti-IL-6 treatment with or without E 2  treatment (n = 6).\nMeans not sharing a letter are significantly different from one another\n( P  < 0.05). Means sharing a same single letter or a\nletter in combination with other letters are not significantly different from\none another ( P  > 0.05; two-way ANOVA).\n\nUsing a mouse model of endometriosis to examine the early initiation of endometriosis\ndisease, we find that two phases contribute to the development and maintenance of the\ndisease—an immune predominant phase and a\nhormone/ER α /E 2  predominant phase ( Fig. 7 ). Based on our own and other’s findings,\nendometriosis develops in interconnected stages shown in  Fig. 7 . Herein, we find the early initiation phase of endometriosis\n(<72 hours) is largely modulated by the innate immune system. Many changes in\ngene expression, the infiltration of immune cells, and altered cytokine secretions are\ndisease-mediated and irrespective of E 2  or ER α \nstatus; however, a role for ER α -IL-6 cross-talk has emerged.\nFurther, IL-6KO to IL-6KO and anti-IL-6 treatment revealed increased lesion numbers in\nthe absence of IL-6 in both the host and the donor with E 2  treatment.\nTreatment with anti-IL-6 and E 2  additionally demonstrated an increase in\nlesion weight, suggesting even further a role for an\nER α –IL-6 axis in the development and subsequent\nproliferation of lesions. Our findings consistently align with the hormonal changes that\noccur cyclically each month in women at menstruation, hormone levels are low\n( i.e.,  immune-predominant phase) then during the follicular phase,\nhormone levels rise ( i.e.,  hormone/ER/E 2 -predominant phase).\nEach menstrual cycle has the potential to establish new endometriotic lesions, as\ngreater than 90% of women have retrograde menstruation ( 4 ); whereas, concomitantly, already established endometriotic lesions\ncontinue to respond to hormonal and paracrine signals during the menstrual cycle. Our\nfindings support the clinical observations about endometriosis; hormone alterations do\nnot cure disease, but render the disease in a suspended state.\nSchematic representation of proposed development of an endometriosis lesion. The\ninitiation phase of disease that is immune predominant includes the stages of\nattachment, angiogenesis, patterning, and immune modulation (<72 hours\nafter disease initiation—dotted gray line). The progression phase of\ndisease is hormone predominant and includes the proliferation and paracrine\nsignaling stages of disease (dotted pink line). From our studies, these two\ndifferent phases have emerged to increase understanding of the development of\nendometriosis lesions, with ER α  and IL-6 both having roles\nin disease development. Correlated with menses, during the initiation of\nendometriosis, hormone levels are low, but when lesions are established and\nproliferating, they respond to hormonal regulation.\nEndometriosis naturally occurs in humans and nonhuman primates because these species\nhave an open reproductive system ( 49 ). Mice, on\nthe other hand, have a closed reproductive system and thus, do not develop endometriosis\nnaturally. To circumvent this issue, we use a mouse model of disease that recapitulates\nendometriosis by injecting syngeneic minced uterine tissue into the peritoneal cavity of\na host mouse ( 9 ). In our model, endometriotic\nlesion development mimics human disease by forming lesions attached to the uterine blood\nsupply, cul-de-sac region, fat pads, peritoneal wall, bladder, and bowel. Also similar\nto human disease, we rarely find lesions that are attached to the liver, kidney, or\nspleen. As seen in human disease and presented in this study, mice display increased\nperitoneal cavity neutrophils, macrophages, NK cells, and increased levels of IL-6,\nVEGF, G-CSF, MCP1, and other chemokines/cytokines. The mouse lesions respond to hormonal\nstimulation and have altered gene expression similar to what is observed with human\nlesions ( 40 ). A limitation to the mouse model is\nthat in humans, the tissue shed from the eutopic uterus gives rise to the ectopic\nlesions; therefore, inherent defect in the eutopic uterus will not be reflected in the\nmouse model. However, suspected human eutopic uterine defects can be examined in the\nmouse model through the utilization of genetically modified mice to study lesion\ndevelopment. As we are unsure of how endometriosis develops in humans, our model, where\ntissue attaches naturally to sites within the peritoneal cavity, gives us the unique\nperspective into the early initiation of disease. Studying the early initiation of\ndisease in humans would require extensive efforts to follow a susceptible population of\nadolescents, perform surgery for endometriosis diagnosis, and acquire peritoneal fluid,\nmenstrual tissue, and serum at the time of menses to begin analyses; consequently, the\nuse of a mouse model with controlled variables sheds invaluable light into the\norchestration of the initiation of endometriosis.\nNeutrophils, macrophages, and uNK cells aid in orchestrating the simultaneous breakdown\nand repair of the eutopic endometrium during menstruation ( 22 ,  35 ,  50 ,  51 ). These leukocytes\nsecrete chemokines and cytokines, which then amplify inflammation and further leukocyte\nrecruitment. Women with endometriosis have increased numbers of immune cells in their\nperitoneal cavity ( 52 ). Although our\nendometriosis model is a mouse model of disease and can be viewed as a limitation, we\nfind similar increases in total cell recruitment into the peritoneal cavity of WT to WT,\nWT to  α ERKO, and  α ERKO to WT groups\nsupporting the findings that ER α  activity is not required in\neither the donor or the recipient in early endometriosis. In patients with\nendometriosis, peritoneal cavity neutrophil counts are approximately threefold to\nfivefold higher than healthy women ( 52 ). In our\nexperimental model, neutrophils are dominant in the initial leukocyte influx into the\nperitoneal cavity. Similar recruitment of neutrophils, dependent on disease state and\nnot ER α  status, is observed in WT to WT, WT to\n α ERKO, and  α ERKO to WT. Neutrophils,\nwhen activated, can release IL-6 that allows endothelial cells to express adhesion\nmolecules ( 53 ,  54 ). Although it seems most likely this orchestration is from neutrophils,\nuterine epithelial cells can also secrete IL-6 ( 55 ). Additionally, IL-17A ( 52 ,  56 ) and IL-6 ( 57 ,  58 ) are among the known\nchemokines/cytokines increased in the peritoneal fluid of women with endometriosis, and\nthese are also elevated in the peritoneal fluid of our mouse model. Neutrophils are\ncapable of a vast array of specialized functions, which will be the focus of future\nstudies, ranging from neutrophil activation to promotion of adhesion of endometrium\n( 59 ,  60 ).\nFollowing neutrophil activation monocytes are recruited and differentiate into\nmacrophages ( 54 ,  61 ). Women with endometriosis have a fourfold to sixfold increase in\nperitoneal fluid macrophages ( 12 ,  52 ,  62 ), have\nhigher peritoneal fluid volumes, higher protein concentrations, and, determined\nvisually, increased activated macrophages compared with healthy women ( 63 ). Macrophage activation is reflected, often, in a\ncontinuous spectrum of phenotypes that rapidly change in response to the local\nenvironment ( 64 ). Similarly to women with\nendometriosis, in our model, we observe different macrophage types have abundantly\ninfiltrated into the peritoneal cavity. This phenotype demonstrates complex macrophage\nplasticity that is dependent on the initiation of endometriosis and occurs irrespective\nof ER α  status in the host or donor mouse.\nEndometriosis is called a sterile inflammatory environment and a disease of the\nmacrophage ( 63 ,  65 ,  66 ). Although macrophage type was\nnot determined, peritoneal fluid of women with endometriosis, analyzed using a consensus\ncytokine signature enrichment analysis, found a macrophage-directed inflammatory\nphenotype ( 67 ). In our study, the total\nmacrophage population did not alter, but M1 and inflammatory macrophages play a\npredominant role in the initiation of endometriosis 48 and 72 hours after disease\ninitiation. Importantly, as described in women with endometriosis ( 63 ), we visualize activated macrophages by cell differential.\nAdditionally, in support of our findings that macrophages are important to lesion\nestablishment, 12 days after the initiation of endometriosis, Tie2 + \nmacrophages contribute to lesion tissue organization and are required for blood vessels\nto reach lesion inner layers ( 68 ,  69 ). Macrophage chemokines and cytokines, such as\nMCP-1, GM-CSF, and G-CSF, are secreted regardless of host or donor\nER α  status, which further suggests the initial phase of\nendometriosis is not dependent on ER α . Supported by other\nendometriosis studies, Cao  et al.  ( 70 ), without examining lesions or lesion development, find the presence of\nendometrial cells in the peritoneal cavity initiate recruitment of monocytes, and Zhao\n et al.  ( 10 ) demonstrated that\nchemicals suppressing both estrogenic and inflammatory activities are potential\ntherapeutic options for endometriosis. In contrast to our studies, a suture model of\nendometriosis that placed peritoneal cavity cells in  ex vivo  culture\nsuggests neutrophils and macrophages are important in early endometriosis; the results\nshowed changes at 4 days ( 71 ). In our studies, by\n4 days immune cell infiltration and dynamic signaling had already allowed uterine tissue\nto develop a blood supply to form endometriotic lesions, which, to us, demonstrates the\ncritical nature of using a dispersal mouse model to more accurately study the early\ninitiation of endometriosis, as it parallels more closely human disease.\nIL-6 has both pro- and anti-inflammatory properties ( 72 ). Invading neutrophils drive IL-6  trans -signaling that is\nimportant to recruit monocytes, stimulate the induction of integrins, cell adhesion,\nactin polymerization, chemotaxis, transmigration, and proliferation ( 72 ,  73 ). Once\nrecruited, monocytes can differentiate into macrophages that express\nER α , where migration and adherence have been associated with\nE 2  because ER α /E 2  can regulate the IL-6\npromoter through NF- κ B and CEBP β  ( 45 – 48 ,\n 74 – 77 ). Interestingly, these same responses do not occur via progesterone- or\nER β -mediated signaling ( 45 – 48 ,  74 – 77 ). Our data and\nothers suggest IL-6 has feed forward and feedback regulation with itself ( 78 ,  79 ),\nwhich can then signal via STAT3 ( 80 ,  81 ) to NF- κ B targets\n( Cox2 ,  Cebpβ ,  IL-6 ). In 60%\nof peritoneal endometriosis cases, NF- κ B is constitutively\nactive ( 82 ). Additionally, COX2 in combination\nwith SRC1/SRC1-isoform, known to play a role in endometriosis ( 83 ,  84 ), can signal to\nincrease mediators responsible for vascular permeability and cell sprouting ( 85 ,  86 ),\nsuggesting this dynamic pathway is important for endometriosis lesion vascularization.\nOur data fully support the current paradigm for endometriosis, but uniquely suggest the\nearly initiation of endometriosis is immune predominated to initiate chemotaxis and\nimmune cell infiltration into the peritoneal cavity, which then signals via an\nER α /IL-6-mediated axis when ectopic uterine cells are\ndeveloping a blood supply. E 2  through ER α  is required\nfor the repressive activity of IL-6, which leads to increased lesion number.\nAdditionally, anti-IL-6 treatment increased lesion size. We find with IL-6, as we found\npreviously for ER α  ( 9 ),\nthat both host and donor contribute to lesion properties because only when IL-6 is\nknocked out in the host and the donor, lesion number is affected.\nIn conclusion, we have uncovered a dynamic role for the innate immune system in the\nearly initiation of endometriosis that uniquely parallels human disease. Our findings\nsupport that the early initiation of endometriosis is predominated by the innate immune\nsystem less than 3 days after the initiation of disease. Further, our studies\ndemonstrate that an ER α /IL-6-mediated cross-talk is important\nfor disease development. Our findings strongly support the need for detailed studies\nthat focus on the innate immune system to further identify and characterize underlying\ncauses of endometriosis and the potential for appropriate therapeutic development. This\narea of research is paramount, not only to treat disease, but to prevent endometriosis\nin the millions of women around the world afflicted with this disease.","source_license":"CC0","license_restricted":false}