{"paper_id":"90249cde-9c37-4500-b60f-48f88b6d1235","body_text":"1\nVol.:(0123456789)Scientific Reports |          (2021) 11:853  | https://doi.org/10.1038/s41598-020-79578-3\nwww.nature.com/scientificreports\nCD206+ macrophage \nis an accelerator \nof endometriotic‑like lesion \nvia promoting angiogenesis \nin the endometriosis mouse model\nYosuke Ono1, Osamu Yoshino2*, Takehiro Hiraoka2, Erina Sato2, Akiko Furue2, Allah Nawaz3, \nHideki Hatta4, Yoshiyuki Fukushi1, Shinichiro Wada1, Kazuyuki Tobe5, Yasushi Hirota6, \nYutaka Osuga6, Nobuya Unno2 & Shigeru Saito7*\nIn endometriosis, M2 MΦs are dominant in endometriotic lesions, but the actual role of M2 MΦ is \nunclear. CD206 positive (+) MΦ is classified in one of M2 type MΦs and are known to produce cytokines \nand chemokines. In the present study, we used CD206 diphtheria toxin receptor mice, which enable \nto deplete CD206+ cells with diphtheria toxin (DT) in an endometriosis mouse model. The depletion \nof CD206+ MΦ decreased the total weight of endometriotic‑like lesions significantly (p < 0.05). In \nthe endometriotic‑like lesions in the DT group, a lower proliferation of endometriotic cells and the \ndecrease of angiogenesis were observed. In the lesions, the mRNA levels of VEGFA and TGFβ1, \nangiogenic factors, in the DT group significantly decreased to approximately 50% and 30% of control, \nrespectively. Immunohistochemical study revealed the expressions of VEGFA and an endothelial cell \nmarker CD31 in lesions of the DT group, were dim compared to those in control. Also, the number \nof TGFβ1 expressing MΦ was significantly reduced compared to control. These data suggest that \nCD206+ MΦ promotes the formation of endometriotic‑like lesions by inducing angiogenesis around \nthe lesions.\nEndometriosis is a gynecological disorder occurring in women of reproductive  age1–3. The primary symptoms \nare severe pelvic pain and  infertility4,5. Multiple lines of evidence suggest that inflammation plays a pivotal role \nfor the pathogenesis of this disease and MΦs play central role for this  process1,6–8.\nIt has been reported that the number of MΦs is elevated in the peritoneal fluid of women with \n endometriosis6,8,10,11, and a significant increase in MΦ numbers is also observed in the eutopic and ectopic \nendometrium in women with  endometriosis6,9,12. Recent pieces of evidence suggest that MΦs directly affect endo-\nmetriotic cells and promote the  disease6,13–15. Activated MΦs in the peritoneal fluid and endometriotic implants \nproduce numerous cytokines, such as including IL-1β, IL-6, IL-8, IL-12, and TNFα, which promote proliferation, \nangiogenesis, and inflammatory  responses1,16–22. Using a rat model for endometriosis, Haber et al. demonstrated \nthat peritoneal MΦ depletion with liposomal bisphosphonate resulted in a reduction in the development and \ngrowth of endometrium  implants23. This finding suggests that MΦs play essential roles in the establishment of \ndisease and its progression.\nMΦs are known to be polarized by the distinct signals depending on their biological  conditions24–26. MΦs \nhave the plasticity and can be polarized into classically activated (M1) and alternatively activated (M2) MΦs. M2 \nMΦs are divided into M2a, M2b, M2c, and M2d subcategories. These MΦs differ in their cell surface markers, \nsecreted cytokines and biological functions, suggesting that there are a variety of populations in MΦs 27–29. In \nOPEN\n1Department of Obstetrics and Gynecology, Teine Keijinkai Hospital, Sapporo, Japan. 2Department of Obstetrics \nand Gynecology, Kitasato University School of Medicine, Sagamihara, Japan. 3Department of Molecular and \nMedical Pharmacology, Faculty of Medicine, University of Toyama, Toyama, Japan. 4Department of Diagnostic \nPathology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, \nJapan. 5First Department of Internal Medicine, University of Toyama, Toyama, Japan. 6Department of Obstetrics \nand Gynecology, The University of Tokyo, Tokyo, Japan. 7Department of Obstetrics and Gynecology, University of \nToyama, Toyama, Japan. *email: oyoshino624@gmail.com; s30saito@med.u-toyama.ac.jp\n\n2\nVol:.(1234567890)Scientific Reports |          (2021) 11:853  | https://doi.org/10.1038/s41598-020-79578-3\nwww.nature.com/scientificreports/\nendometriosis, it has been reported that M2 MΦs are dominant in endometriotic  lesions13,14. Bacci et al. showed \nthat intraperitoneal injection of M2 MΦ promoted the growth of endometriotic like lesions in a mouse model, \nsuggesting M2 MΦs are responsible for promoting endometriotic  lesions13. However, the actual role of MΦ in \nthe pathogenesis of endometriosis is unclear.\nTo elucidate the role of MΦ in vivo, the use of neutralization antibody has been utilized in eliminating \nMΦs but is less effective in removing MΦs within lesions owing to differential concentrations of  antibody30. \nTherefore, in the present study, we used CD206 diphtheria toxin receptor (DTR) mice, which enable to deplete \nCD206+ MΦs, one of a specific marker of M2 MΦ, with diphtheria toxin (DT)  injection31,32 to examine the role \nof CD206+ MΦ in progression of endometriosis.\nMaterial and methods\nReagents and materials. Roswell Park Memorial Institute (RPMI)-1640 medium and Diphtheria Toxin \n(DT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Estradiol valerate was from Asuka Pharmaceu-\ntical Co., Osaka, Japan. Fetal bovine serum (FBS) was from Life Technologies (Tokyo, Japan). Antibiotics (a mix-\nture of penicillin, streptomycin, and amphotericin B) were from Wako Pure Chemical Industries (Osaka, Japan).\nImmunohistochemistry. Paraffin-embedded tissues were cut 5-μm thick and mounted on slides. The sec-\ntions of endometriotic-like lesions formed in endometriosis model mice were deparaffinized in xylene, rehy-\ndrated through a graded series of ethanol, and washed in water. Antigen retrieval was performed in 10 mM \nsodium citrate buffer (pH 6.0) by microwaving for 10 min and then cooling to room temperature. Control IgG \nwas used as a negative control. Slide staining with antibodies was performed according to the manufacturer’s \ninstructions. The immunostaining was performed using specific antibodies to Ki-67 (Abcam, Tokyo, Japan, \nCat# 15580, 1:100 dilution), Vascular endothelial growth factor (VEGFA) (Abcam, Tokyo, Japan, Cat# 46154, \n1:100 dilution), CD206 (Abcam, Cat# 64693, 1:100 dilution), TGFβ1 (Abcam, Tokyo, Japan, Cat# 92486, 1:500 \ndilution), and CD31 (Abcam, Tokyo, Japan, Cat# 124432, 1:200 dilution). All images were taken with Keyence \nBZ-X800 (Keyence, Tokyo, Japan). Immunofluorescence analysis of endometriotic-like lesions was performed \nusing CD206 and TGFβ1. The primary antibodies were incubated overnight at 4 °C, and as a second antibody, \nthe gout anti-rabbit antibody (Alexa Fluor 488, ab150077, Abcam, Tokyo, Japan) was used. 4′,6-diamidino-\n2-phenylindole (DAPI; 1:500) was used to detect nuclei. Rabbit IgG was used instead of the primary antibody \nfor negative control.\nThe intensity of the staining of VEGFA, CD31, and TGFβ1 was analyzed by a semi-quantitative method, \nH-scoring33. H-score was calculated by the following equation: H-score = ∑Pi × i, where i is the intensity of stain-\ning with a value of 0, 1, 2, or 3 (negative, weak, moderate, or strong, respectively) and  Pi is the corresponding per-\ncentage of the cells. As for the Ki-67 evaluation, the percentage of Ki-67 positive cells per total cells was calculated.\nModel of endometriosis using CD206 DTR mouse. All animal experiments were approved by the \nethical committee of University of Toyama (G2015MED-38 and A2013MED-33), and performed in accord-\nance with animal experiment guidelines and regulations in University of Toyama. Female, CD206 DTR mice, \nfrom 12 to 20-weeks-old mice were used. We housed in a specific pathogen-free (SPF) animal facility with a \ncontrolled environment, 22–24 °C and 60–70% relative humidity, and on a light/dark cycle (12 h light/12 h dark) \nwith food and water ad libitum. CD206 DTR mice are genetically engineered transgenic (Tg) mice based on \nthe transgenic expression of the diphtheria toxin receptor (DTR) under the control of the CD206 promoter to \nablate CD206+ MΦs 31,32,34 specifically. In CD206 DTR mice, CD206+ MΦs are removed about more than 80% \nin various organs systemically, such as lung, adipose tissue, blood, spleen, and  ovary31,32,34. Induction of endo-\nmetriosis was studied as described  previously7. Briefly, mice were injected s.c. with 100 μg/kg estradiol valerate \nin sesame oil once per week for three weeks. After three weeks, endometrium-rich fragments from donor mice \nwere finely chopped using a razor blade. Fragments suspended in 0.6 ml phosphate buffered salts (PBS) were \ninjected with an 18-gauge needle through the abdominal wall into the peritoneal cavity of recipient mice with \nthe ratio of one donor to two recipients (designated day 0 when endometrial fragments were injected). We \nused wild type mice as donors and CD206 DTR mice as recipients. One week after endometrial inoculation, we \nchecked the formation of endometriotic-like lesions in the peritoneal cavity by sacrificing a few mice. Then, the \nrecipient mice underwent intraperitoneal injection of DT (DT group) or PBS for control group, every two days. \nDT of 20 ng/gram body weight was diluted with sterile PBS and injected  intraperitoneally31. Two weeks after \nthe inoculation of endometrial fragments, the recipient mice were sacrificed through cervical dislocation under \nanesthesia. Then, PBS (1 ml) was injected into the peritoneal cavity. After vigorous shaking, peritoneal fluid (PF) \nand PF cells were collected. Laparotomy was performed, and the numbers of endometriotic foci were counted. \nEach focus and uterus was excised to exclude as much normal surrounding tissues as possible. Immediately, the \nweight of the excised tissues was measured. During all the inspection procedures, examiners were blinded to the \ntreatment given to each mouse.\nReverse transcription (RT) and quantitative real‑time polymerase chain reaction (PCR) analy‑\nsis. Total RNA was extracted from mouse endometriotic-like lesions using the ISOGEN-II (NIPPON GENE, \nTokyo, Japan) according to the manufacturer’s instructions. About 1 μg of total RNA was reverse-transcribed \nusing Rever Tra Ace qPCR RT Master Mix with gDNA Remover (TOYOBO, Tokyo, Japan). For the quantifica-\ntion of various mRNA levels, real-time PCR was performed using the Mx3000P Real-Time PCR System (Agilent \nTechnologies, CA, USA) according to the manufacturer’s  instructions31. The PCR primers were selected from \ndifferent exons of the corresponding genes to discriminate PCR products that might arise from possible chro-\nmosomal DNA contaminants. The SYBR Green thermal cycling conditions were one cycle of 95 °C for 30 s, \n\n3\nVol.:(0123456789)Scientific Reports |          (2021) 11:853  | https://doi.org/10.1038/s41598-020-79578-3\nwww.nature.com/scientificreports/\nand cycles of 95 °C for 10 s, 60 °C for 10 s and 72 °C 10 s). The primer sequences were as follows: Glyceralde-\nhyde 3-phosphate dehydrogenase (GAPDH, NM_001289726.1: 801–822 and 883–862). CD206 (NM_008625.2: \n3335–3355 and 3630–3611), TNFα (NM_013693.3: 435–455 and 556–533), IL-1β (NM_008361.4: 338–359 and \n442–420), VEGFA (NM_001025257.3: 1103–1127 and 1261–1241), and TGFβ1(NM 1829–1810 and 1702–\n1722).　The relative mRNA levels were calculated using the standard curve method and were normalized to the \nmRNA levels of  GAPDH31.\nHuman peritoneal fluids MΦ (PF MΦ) and culture of human endometriotic stromal cells \n(ESCs). The experimental procedures were approved by the institutional review board of Kitasato Univer -\nsity (approved number: B18-265), and University of Toyama (Approved Number: 25–44) and signed informed \nconsent for the use of samples was obtained from each patient. PFMΦs were purified as previously  described35. \nCulture of primary ESCs was described  elsewhere7. Human samples were taken and used under each patient’s \nconsent. All experimental methods were carried out in accordance with relevant guidelines and regulations of \nDeclaration of Helsinki.\nStatistical analysis. Data were evaluated by Mann Whitney using Jump version 10. P less than 0.05 was \naccepted as statistically significant.\nResults\nEndometriosis model using CD206 DTR mice. As shown in Fig.  1, we induced endometriotic-like \nlesions using CD206 DTR mice. After one week of inoculation of endometrial fragments into the peritoneal \ncavity, we started to deplete CD206+ MΦs for a week and measured the total weight and the number of endo-\nmetriotic-like lesions per mouse. As shown in Fig.  2, in CD206 DTR, the depletion treatment of CD206+ MΦ \nsignificantly decreased the total weight of endometriotic-like lesions (p < 0.05), but the number of lesions per \nmouse was not changed compared to control (1.4 ± 0.5 vs 1.2 ± 1.1).\nWe checked the depletion of CD206+ MΦ in peritoneal micro-environment by quantitative-PCR and found \nthat in peritoneal fluid cells, about 80% of CD206+ MΦ were reduced in CD206DTR mice with DT adminis-\ntration (supplemental Fig. 1). The mRNA expression of CD11b, which is a pan MΦ marker, was not decreased \nsignificantly with DT stimuli, suggesting that the amount of total MΦs was not changed (Supplemental Fig. 1). As \nfor other types of MΦs, the ratio of iNOS, a classical M1 MΦ marker, to CD11b was increased significantly, but \nthe ratio of inflammatory MΦs producing TNFα to CD11b was not increased significantly (Supplemental Fig. 1).\nDepletion of CD206+ MΦ lead to a histological change of the endometriotic‑like lesion. Endo-\nmetriotic lesion is composed by glandular epithelial cells and stromal cells. In the DT group, the appearance of \nendometriotic glandular epithelial cells was thinner, (Fig. 3b,d) compared to the appearance of epithelial cells in \ncontrol (Fig. 3a,c). These surface cells covering lesions were confirmed to be epithelial cells by cytokeratin stain-\ning (Fig. 3e,f). Figure 3g,h show the negative control of Fig. 3e,f, respectively.\nFigure 1.  The depletion protocol of CD206+ macrophages (MΦ) in the CD206 DTR mouse-derived \nendometriosis model. After one week of inoculation of endometrial fragments derived from donor mice (①), \nthe formation of an endometriotic-like lesion in the peritoneal cavity was confirmed in some mice. (②). The \nrecipient mice underwent intraperitoneal injection of Diphtheria Toxin (DT) for DT group or phosphate \nbuffered salts (PBS) for control group every two days starting from day 7 to day14 after endometrial inoculation. \nAfter 2 weeks of inoculation of the endometrium, the total weights of endometriotic-like lesions per mouse were \ninvestigated. Estrogen was injected every week.\n\n4\nVol:.(1234567890)Scientific Reports |          (2021) 11:853  | https://doi.org/10.1038/s41598-020-79578-3\nwww.nature.com/scientificreports/\nThe positive ratio per total cells of proliferation marker, Ki-67 + cells, was significantly decreased in both the \nglandular epithelial cells and stromal cells of endometriotic-like lesions of DT group, receptively (1.6  ± 1.8 and \n2.3 ± 1.2%, mean ± SD) compared to control (9.7 ± 13.4% and 3.3 ± 1.8, p < 0.001) (Fig.  4a–d), suggesting that \nlower proliferation ratio of endometriotic-like cells resulted in a decrease of endometriotic-like lesions in the \nabsence of CD206+ MΦ.\nFigure 2.  The depletion effect of CD206+ macrophages (MΦ) in the endometriosis mouse model. \nRepresentative appearances of endometriotic-like lesions are shown (a), and data of the total weight of lesions \nare shown as the dots and mean (b). In CD206 DTR mice, depletion of CD206+ MΦs was induced by injection \nof Diphtheria Toxin (DT).\nFigure 3.  Representative microscopic picture of the endometriotic-like lesion of the DT and control group. \nIn CD206 DTR mice, depletion of CD206+ macrophages (MΦ) was induced by injection of Diphtheria Toxin \n(DT). Representative endometriotic-like lesions in control (a and c) and DT group (b and d) were shown. The \narrows indicate the endometriotic epithelial and stromal cells. Immunostaining of cytokeratin was performed in \ncontrol (a and c) and DT (b and d) to confirm the presence of endometriotic epithelial cells in the same lesion as \nin both groups (e, f). Rabbit IgG was used for negative control (g, h).\n\n5\nVol.:(0123456789)Scientific Reports |          (2021) 11:853  | https://doi.org/10.1038/s41598-020-79578-3\nwww.nature.com/scientificreports/\nThe possible mechanism of CD206+ MΦ in the progression of endometriotic lesions. To inves-\ntigate the reducing mechanism of endometriotic-like lesions by the depletion of CD206+ MΦ, we checked the \ncritical factors for the formation of endometriotic-like lesions at the mRNA level. As shown in Fig.  5a, the \ndepletion of CD206+ MΦ significantly decreased the expression of CD206 mRNA by more than 90% compared \nto control (p < 0.01) in the lesions. Although the levels of TNFα and IL-1β were comparable in control and DT \ngroup (,c), the mRNA expression of CD11b, a pan- MΦ marker, was not changed (Fig.  5d). The proportion of \nCD206/CD11b was significantly decreased in DT group, while that of TNFα/CD11b was not changed in both \ngroups (data not shown). The expression of VEGFA (Fig. 5e) and TGFβ1 (Fig. 5f) in DT group were significantly \ndecreased to the levels of approximately 50% and 30% of control, respectively (p < 0.05, p < 0.05).\nCD206+ MΦ contributes to the angiogenesis of endometriotic‑like lesions.. We investigated \nthe localization of VEGFA and TGFβ1 protein in the lesions. Immunohistochemical study revealed, in the DT \ngroup, the expression of VEGFA in especially epithelial cells, was significantly lower (p < 0.001) compared to \ncontrol (Fig. 6a,b,g H-score; 201.8 ± 26.7 vs 126.7 ± 56.0, p < 0.001). Also, the number of TGFβ1-expressing cells \nwas significantly reduced compared to control (Fig. 6c,d,h; H-score; 22.7 ± 12.9 vs 3.0 ± 5.6, p < 0.001). Immuno-\nfluorescence study showed that TGFβ1 staining cells are CD206 positive cells, indicating that CD206+ MΦs are \nthe main cells that produce TGFβ1 (Fig. 7). The expression of CD31, an endothelial cell marker, was significantly \ndecreased in the endometriotic-like lesions with the depletion of CD206+ MΦ compared to control (p < 0.05, \nFig. 6e,f,i, H-score; 12.1 ± 12.0 vs 2.2 ± 1.7, p < 0.05).\nDiscussion\nIn endometriosis, it has been reported that M2 MΦs are dominant in endometriotic  lesions13,14, but the roles \nof MΦ for the pathogenesis of endometriosis are remained to be clarified. To understand the dynamics and \nroles of MΦ in the pathogenesis of endometriosis, animal models might be a useful tool. Recently, Johan et al. \ninvestigated whether infiltrating MΦs acquire functionally different phenotypes, M1 or M2 MΦ, during lesion \ndevelopment in an endometriosis mice  model36. They showed that until day 7, after grafting endometrial tissues \nto mice, M1 MΦs are dominant, while M2 MΦs exhibit dominancy after day  736. Therefore, in the endometriosis \nmouse model, M1 to M2 MΦ shift occurs on day 7 after the inoculation of endometrial tissue. This is consistent \nwith a transition from classical M1 MΦ activity to an alternate M2 profile, which correlates to the findings of \ninitially acute inflammation followed by tissue remodeling in the process of development of  endometirosis36.\nGiven that some M2 MΦs have a role of suppressing  inflammation37, the contribution of M2 MΦ to the \nendometriosis-like lesions is uncertain, suppressive, or progressive. To address this question, we used CD206 \nDTR mice and depleted CD206+  MΦs exclusively in mice from day 7 after induction of endometriotic-like \nFigure 4.  Cell proliferation at the endometriotic-like lesion of DT and control group. Immuno-staining of \nKi-67, a proliferation marker, was examined in control (a) and DT (b) group, and the percentage of Ki-67 \npositive cells per total cells was calculated in glandular epithelial cells and stromal cells, respectively (c and d). \nThe arrow indicates the endometriotic epithelial cells. Data were evaluated by Mann Whitney U. P less than 0.05 \nwas accepted as statistically significant.\n\n6\nVol:.(1234567890)Scientific Reports |          (2021) 11:853  | https://doi.org/10.1038/s41598-020-79578-3\nwww.nature.com/scientificreports/\nlesions, when the shift from M1 to M2 MΦ could occur in endometriotic  lesions36. CD206 is known to be a \ntype 1 mannose receptor and one of M2a subtype markers in M2 MΦs. These MΦs secrete cytokines such as \nTGFβ1 and IL-10 and chemokines such as  CCLs37. In CD206 DTR mice model, CD206+ MΦs were depleted \nby more than 80% in endometriotic-like lesion and PF cells (Fig. 5a and Supplemental Fig. 1). At mRNA levels, \nalthough the ratio of iNOS/CD11b was increased in PF cells, the mRNA expression of CD11b was unchanged, \nand there was no increase of TNFα/CD11b, suggesting that inflammatory MΦs which exacerbate endometriotic \n lesions6,38 did not increase in both PF cells and lesions. We found that the depletion of CD206+ MΦ resulted in \nthe decrease of the size of endometriotic-like lesions without affecting the number of lesions, implying that the \nrole of CD206+ MΦ is an accelerator of endometriotic-like disease in mice model. Duan et al. used CD11b DTR \nmice in which pan MΦs could be depleted with DT treatment, and showed the reduction of lesion weight in an \nendometriosis mice model. Moreover, they also presented that the adoptive transfer of M2a, a subtype of M2 \nMΦ, systemically after the MΦ depletion significantly increased the weight of endometriotic  lesions39, suggesting \nthat M2a MΦ play a role in the progression of the endometriotic lesion. In consistent with the notion, as CD206 \nis one of M2a subtype markers in M2 MΦs 37, our present results also suggest that CD206+ MΦs are involved in \nthe exacerbation of endometriosis. In the DT group, endometriotic lesions exhibited thinner appearance, and \nlower proliferation rate of epithelial and stromal cells compared to control (Fig. 4), suggesting that CD206+ MΦs \ninfluence the growth of endometriotic cells. Among endometriosis-related factors, in the DT group, the levels \nof TGFβ1 and VEGFA mRNA, known as angiogenic  factors40,41, were decreased in endometriotic-like lesions. \nIn accordance with the notion, CD31, an endothelial cell marker, was almost disappeared in the endometriotic-\nlike lesions with the depletion of CD206+  MΦ. It has been reported that the administration of bevacizumab, a \nVEGF-A antibody, resulted in reduced lesion formation in mouse endometriosis model  studies41, suggesting \nneovascularization of ectopic endometrial tissue is crucial in the development of the endometriotic-like lesion.\nIn the present study, immunohistochemical analysis showed that CD206+ MΦs expressed TGFβ1 (Fig.  7) and \nproduced high level of VEGFA in the endometriotic-like lesions (Fig.  6). Therefore, we performed an in vitro \nstudy to examine these relationships. It is well known that IL-33, an alarmin, deviates macrophage to classical \nM2  type42–45. We also have reported that IL-33 induced CD206+  MΦs in human peritoneal MΦs 35. In the pre-\nsent study, we found that induced CD206+ MΦs with recombinant IL-33 stimuli (100 ng/ml, 8hrs) increased \nthe expression of TGFβ1 mRNA (Supplemental Fig. 2). In addition, to investigate the relationship between \nTGFβ1 derived from CD206+  MΦ and VEGFA of the endometriotic lesion, we checked the mRNA expression \nof VEGFA in human endometriotic stromal cells (ESCs) with TGFβ1 stimulation and found the significant \nincrease of VEGFA mRNA expression (Supplemental Fig. 2) suggesting that increased expression of TGFβ1 in \nFigure 5.  The expressions of mRNA in endometriotic-like lesions in control and DT injected mice. In CD206 \nDTR mice, depletion of CD206+ macrophages (MΦ) were induced by injection of Diphtheria Toxin (DT). \nThe expression of CD206 (a), TNFα (b), IL-1β (c), CD11b (d), VEGFA (e), and TGFβ1 (f) mRNA expressions \nwere measured with quantitative PCR in control and DT group. Data were normalized by GAPDH mRNA \nlevels to show the relative abundance. Representative data from three different experiments were shown as the \nmean ± SEM. *p < 0.05.\n\n7\nVol.:(0123456789)Scientific Reports |          (2021) 11:853  | https://doi.org/10.1038/s41598-020-79578-3\nwww.nature.com/scientificreports/\nCD206-skewed-MΦ may induce angiogenesis via increasing VEGFA expression in ESCs. TGFβ1 is related to \n angiogenesis40 and is also reported to induce VEGFA in other  cells46. Therefore, the reduction in TGFβ1 expres-\nsion by the depletion of CD206+ MΦ might have led to the decrease in angiogenesis, resulting in the reduction \nof endometriotic-like lesion.\nIn endometriosis patients, it is reported that Natural Killer (NK) cells impair their function in the intra-\nperitoneal environment, which makes endometriotic cells more likely to survive and proliferate in the abdominal \ncavity. TGFβ1 is known to be increased in the peritoneal fluids of patients with  endometriosis47 and to exacerbate \nendometriosis by decreasing NK cell  activity48. Therefore, decreasing of TGFβ1 by the depletion of CD206+ MΦ \nmay contribute to the recovery of NK activity and result in the reduction of endometriosis lesions. Further studies \nare necessary to clary the role of CD206+ MΦ in the pathogenesis of endometriosis.\nIn the present study, we proved that CD206+ MΦ played a vital role in the promotion of endometriosis via \ninducing angiogenesis using CD206 DTR mice. Therefore, CD206+ MΦ might be a target for a new therapy for \nFigure 6.  The expressions of VEGF , TGF-β and CD31 protein in endometriotic-like lesions. In CD206 DTR \nmice, depletion of CD206+ macrophages (MΦ) were induced by injection of Diphtheria Toxin (DT). The \nimmunohistochemistry of VEGF (a and b), TGFβ1 (c and d), and a marker of the endothelium, CD31 (e and \nf), were examined in the endometriotic like lesions of control mice (control) and CD206+ MΦ depletion mice \n(DT). H-scores of VEGFA (g), TGFβ1 (h), and CD31 (i) immunostaining in endometriotic-like lesions were \ncalculated and compared in control and DT mice. Data were evaluated by Mann Whitney U. P less than 0.05 was \naccepted as statistically significant.\n\n8\nVol:.(1234567890)Scientific Reports |          (2021) 11:853  | https://doi.org/10.1038/s41598-020-79578-3\nwww.nature.com/scientificreports/\nendometriosis. One can speculate that the depletion of CD206+ MΦ using a specific antibody could be a strategy. \nFurther studies are needed to prove these notions.\nReceived: 29 July 2020; Accepted: 23 November 2020\nReferences\n 1. Zondervan, K. T. et al. Endometriosis. Nat. Rev. Dis. Prim. 4, 9 (2018).\n 2. Eskenazi, B. & Warner, M. L. Epidemiology of endometriosis. Obstet. Gynecol. Clin. North Am. 24, 235–258 (1997).\n 3. Shafrir, A. L. et al. Risk for and consequences of endometriosis: a critical epidemiologic review. Best Pract. Res. Clin. Obstet. \nGynaecol. 51, 1–15 (2018).\n 4. 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Int. 2016, 1–6 (2016).\nAcknowledgements\nThis work was supported by Health and Labor Sciences Research Grants from the Ministry of Health, Labor and \nWelfare of Japan, Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, \nand Technology, and by The Smoking Research Foundation.\nAuthor contributions\nO.Y . and S.S. wrote the main manuscript text.O.Y . and K.T. set an experimental protocol.K.T. prepared for animal \nexperimentsY .O., O.Y ., T.H., E.S., A.N., H.H., Y .F ., and A.F . executed the experiments.N.U., S.W ., Y .H. and Y .O. \na critical discussion.All authors reviewed the manuscript.\nCompeting interests \nThe authors declare no competing interests.\nAdditional information\nSupplementary Information The online version contains supplementary material  available at https ://doi.\norg/10.1038/s4159 8-020-79578 -3.\nCorrespondence and requests for materials should be addressed to O.Y . or S.S.\nReprints and permissions information is available at www.nature.com/reprints.\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 \nCreative Commons licence, and indicate if changes were made. The images or other third party material in this \narticle are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the \nmaterial. If material is not included in the article’s Creative Commons licence and your intended use is not \npermitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from \nthe copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/.\n© The Author(s) 2021","source_license":"CC0","license_restricted":false}