CD206+ macrophage is an accelerator of endometriotic-like lesion via promoting angiogenesis in the endometriosis mouse model

Reagents and materials. Roswell Park Memorial Institute (RPMI)-1640 medium and Diphtheria Toxin (DT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Estradiol valerate was from Asuka Pharmaceu- tical Co., Osaka, Japan. Fetal bovine serum (FBS) was from Life Technologies (Tokyo, Japan). Antibiotics (a mix- ture of penicillin, streptomycin, and amphotericin B) were from Wako Pure Chemical Industries (Osaka, Japan). Immunohistochemistry. Paraffin-embedded tissues were cut 5-μm thick and mounted on slides. The sec- tions of endometriotic-like lesions formed in endometriosis model mice were deparaffinized in xylene, rehy- drated through a graded series of ethanol, and washed in water. Antigen retrieval was performed in 10 mM sodium citrate buffer (pH 6.0) by microwaving for 10 min and then cooling to room temperature. Control IgG was used as a negative control. Slide staining with antibodies was performed according to the manufacturer’s instructions. The immunostaining was performed using specific antibodies to Ki-67 (Abcam, Tokyo, Japan, Cat# 15580, 1:100 dilution), Vascular endothelial growth factor (VEGFA) (Abcam, Tokyo, Japan, Cat# 46154, 1:100 dilution), CD206 (Abcam, Cat# 64693, 1:100 dilution), TGFβ1 (Abcam, Tokyo, Japan, Cat# 92486, 1:500 dilution), and CD31 (Abcam, Tokyo, Japan, Cat# 124432, 1:200 dilution). All images were taken with Keyence BZ-X800 (Keyence, Tokyo, Japan). Immunofluorescence analysis of endometriotic-like lesions was performed using CD206 and TGFβ1. The primary antibodies were incubated overnight at 4 °C, and as a second antibody, the gout anti-rabbit antibody (Alexa Fluor 488, ab150077, Abcam, Tokyo, Japan) was used. 4′,6-diamidino- 2-phenylindole (DAPI; 1:500) was used to detect nuclei. Rabbit IgG was used instead of the primary antibody for negative control. The intensity of the staining of VEGFA, CD31, and TGFβ1 was analyzed by a semi-quantitative method, H-scoring33. H-score was calculated by the following equation: H-score = ∑Pi × i, where i is the intensity of stain- ing with a value of 0, 1, 2, or 3 (negative, weak, moderate, or strong, respectively) and Pi is the corresponding per- centage of the cells. As for the Ki-67 evaluation, the percentage of Ki-67 positive cells per total cells was calculated. Model of endometriosis using CD206 DTR mouse. All animal experiments were approved by the ethical committee of University of Toyama (G2015MED-38 and A2013MED-33), and performed in accord- ance with animal experiment guidelines and regulations in University of Toyama. Female, CD206 DTR mice, from 12 to 20-weeks-old mice were used. We housed in a specific pathogen-free (SPF) animal facility with a controlled environment, 22–24 °C and 60–70% relative humidity, and on a light/dark cycle (12 h light/12 h dark) with food and water ad libitum. CD206 DTR mice are genetically engineered transgenic (Tg) mice based on the transgenic expression of the diphtheria toxin receptor (DTR) under the control of the CD206 promoter to ablate CD206+ MΦs 31,32,34 specifically. In CD206 DTR mice, CD206+ MΦs are removed about more than 80% in various organs systemically, such as lung, adipose tissue, blood, spleen, and ovary31,32,34. Induction of endo- metriosis was studied as described previously7. Briefly, mice were injected s.c. with 100 μg/kg estradiol valerate in sesame oil once per week for three weeks. After three weeks, endometrium-rich fragments from donor mice were finely chopped using a razor blade. Fragments suspended in 0.6 ml phosphate buffered salts (PBS) were injected with an 18-gauge needle through the abdominal wall into the peritoneal cavity of recipient mice with the ratio of one donor to two recipients (designated day 0 when endometrial fragments were injected). We used wild type mice as donors and CD206 DTR mice as recipients. One week after endometrial inoculation, we checked the formation of endometriotic-like lesions in the peritoneal cavity by sacrificing a few mice. Then, the recipient mice underwent intraperitoneal injection of DT (DT group) or PBS for control group, every two days. DT of 20 ng/gram body weight was diluted with sterile PBS and injected intraperitoneally31. Two weeks after the inoculation of endometrial fragments, the recipient mice were sacrificed through cervical dislocation under anesthesia. Then, PBS (1 ml) was injected into the peritoneal cavity. After vigorous shaking, peritoneal fluid (PF) and PF cells were collected. Laparotomy was performed, and the numbers of endometriotic foci were counted. Each focus and uterus was excised to exclude as much normal surrounding tissues as possible. Immediately, the weight of the excised tissues was measured. During all the inspection procedures, examiners were blinded to the treatment given to each mouse. Reverse transcription (RT) and quantitative real‑time polymerase chain reaction (PCR) analy‑ sis. Total RNA was extracted from mouse endometriotic-like lesions using the ISOGEN-II (NIPPON GENE, Tokyo, Japan) according to the manufacturer’s instructions. About 1 μg of total RNA was reverse-transcribed using Rever Tra Ace qPCR RT Master Mix with gDNA Remover (TOYOBO, Tokyo, Japan). For the quantifica- tion of various mRNA levels, real-time PCR was performed using the Mx3000P Real-Time PCR System (Agilent Technologies, CA, USA) according to the manufacturer’s instructions31. The PCR primers were selected from different exons of the corresponding genes to discriminate PCR products that might arise from possible chro- mosomal DNA contaminants. The SYBR Green thermal cycling conditions were one cycle of 95 °C for 30 s, 3 Vol.:(0123456789)Scientific Reports | (2021) 11:853 | https://doi.org/10.1038/s41598-020-79578-3 www.nature.com/scientificreports/ and cycles of 95 °C for 10 s, 60 °C for 10 s and 72 °C 10 s). The primer sequences were as follows: Glyceralde- hyde 3-phosphate dehydrogenase (GAPDH, NM_001289726.1: 801–822 and 883–862). CD206 (NM_008625.2: 3335–3355 and 3630–3611), TNFα (NM_013693.3: 435–455 and 556–533), IL-1β (NM_008361.4: 338–359 and 442–420), VEGFA (NM_001025257.3: 1103–1127 and 1261–1241), and TGFβ1(NM 1829–1810 and 1702– 1722).　The relative mRNA levels were calculated using the standard curve method and were normalized to the mRNA levels of GAPDH31. Human peritoneal fluids MΦ (PF MΦ) and culture of human endometriotic stromal cells (ESCs). The experimental procedures were approved by the institutional review board of Kitasato Univer - sity (approved number: B18-265), and University of Toyama (Approved Number: 25–44) and signed informed consent for the use of samples was obtained from each patient. PFMΦs were purified as previously described35. Culture of primary ESCs was described elsewhere7. Human samples were taken and used under each patient’s consent. All experimental methods were carried out in accordance with relevant guidelines and regulations of Declaration of Helsinki. Statistical analysis. Data were evaluated by Mann Whitney using Jump version 10. P less than 0.05 was accepted as statistically significant.

Endometriosis model using CD206 DTR mice. As shown in Fig.  1, we induced endometriotic-like lesions using CD206 DTR mice. After one week of inoculation of endometrial fragments into the peritoneal cavity, we started to deplete CD206+ MΦs for a week and measured the total weight and the number of endo- metriotic-like lesions per mouse. As shown in Fig.  2, in CD206 DTR, the depletion treatment of CD206+ MΦ significantly decreased the total weight of endometriotic-like lesions (p < 0.05), but the number of lesions per mouse was not changed compared to control (1.4 ± 0.5 vs 1.2 ± 1.1). We checked the depletion of CD206+ MΦ in peritoneal micro-environment by quantitative-PCR and found that in peritoneal fluid cells, about 80% of CD206+ MΦ were reduced in CD206DTR mice with DT adminis- tration (supplemental Fig. 1). The mRNA expression of CD11b, which is a pan MΦ marker, was not decreased significantly with DT stimuli, suggesting that the amount of total MΦs was not changed (Supplemental Fig. 1). As for other types of MΦs, the ratio of iNOS, a classical M1 MΦ marker, to CD11b was increased significantly, but the ratio of inflammatory MΦs producing TNFα to CD11b was not increased significantly (Supplemental Fig. 1). Depletion of CD206+ MΦ lead to a histological change of the endometriotic‑like lesion. Endo- metriotic lesion is composed by glandular epithelial cells and stromal cells. In the DT group, the appearance of endometriotic glandular epithelial cells was thinner, (Fig. 3b,d) compared to the appearance of epithelial cells in control (Fig. 3a,c). These surface cells covering lesions were confirmed to be epithelial cells by cytokeratin stain- ing (Fig. 3e,f). Figure 3g,h show the negative control of Fig. 3e,f, respectively. Figure 1. The depletion protocol of CD206+ macrophages (MΦ) in the CD206 DTR mouse-derived endometriosis model. After one week of inoculation of endometrial fragments derived from donor mice (①), the formation of an endometriotic-like lesion in the peritoneal cavity was confirmed in some mice. (②). The recipient mice underwent intraperitoneal injection of Diphtheria Toxin (DT) for DT group or phosphate buffered salts (PBS) for control group every two days starting from day 7 to day14 after endometrial inoculation. After 2 weeks of inoculation of the endometrium, the total weights of endometriotic-like lesions per mouse were investigated. Estrogen was injected every week. 4 Vol:.(1234567890)Scientific Reports | (2021) 11:853 | https://doi.org/10.1038/s41598-020-79578-3 www.nature.com/scientificreports/ The positive ratio per total cells of proliferation marker, Ki-67 + cells, was significantly decreased in both the glandular epithelial cells and stromal cells of endometriotic-like lesions of DT group, receptively (1.6 ± 1.8 and 2.3 ± 1.2%, mean ± SD) compared to control (9.7 ± 13.4% and 3.3 ± 1.8, p < 0.001) (Fig.  4a–d), suggesting that lower proliferation ratio of endometriotic-like cells resulted in a decrease of endometriotic-like lesions in the absence of CD206+ MΦ. Figure 2. The depletion effect of CD206+ macrophages (MΦ) in the endometriosis mouse model. Representative appearances of endometriotic-like lesions are shown (a), and data of the total weight of lesions are shown as the dots and mean (b). In CD206 DTR mice, depletion of CD206+ MΦs was induced by injection of Diphtheria Toxin (DT). Figure 3. Representative microscopic picture of the endometriotic-like lesion of the DT and control group. In CD206 DTR mice, depletion of CD206+ macrophages (MΦ) was induced by injection of Diphtheria Toxin (DT). Representative endometriotic-like lesions in control (a and c) and DT group (b and d) were shown. The arrows indicate the endometriotic epithelial and stromal cells. Immunostaining of cytokeratin was performed in control (a and c) and DT (b and d) to confirm the presence of endometriotic epithelial cells in the same lesion as in both groups (e, f). Rabbit IgG was used for negative control (g, h). 5 Vol.:(0123456789)Scientific Reports | (2021) 11:853 | https://doi.org/10.1038/s41598-020-79578-3 www.nature.com/scientificreports/ The possible mechanism of CD206+ MΦ in the progression of endometriotic lesions. To inves- tigate the reducing mechanism of endometriotic-like lesions by the depletion of CD206+ MΦ, we checked the critical factors for the formation of endometriotic-like lesions at the mRNA level. As shown in Fig.  5a, the depletion of CD206+ MΦ significantly decreased the expression of CD206 mRNA by more than 90% compared to control (p < 0.01) in the lesions. Although the levels of TNFα and IL-1β were comparable in control and DT group (,c), the mRNA expression of CD11b, a pan- MΦ marker, was not changed (Fig.  5d). The proportion of CD206/CD11b was significantly decreased in DT group, while that of TNFα/CD11b was not changed in both groups (data not shown). The expression of VEGFA (Fig. 5e) and TGFβ1 (Fig. 5f) in DT group were significantly decreased to the levels of approximately 50% and 30% of control, respectively (p < 0.05, p < 0.05). CD206+ MΦ contributes to the angiogenesis of endometriotic‑like lesions.. We investigated the localization of VEGFA and TGFβ1 protein in the lesions. Immunohistochemical study revealed, in the DT group, the expression of VEGFA in especially epithelial cells, was significantly lower (p < 0.001) compared to control (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 was significantly reduced compared to control (Fig. 6c,d,h; H-score; 22.7 ± 12.9 vs 3.0 ± 5.6, p < 0.001). Immuno- fluorescence study showed that TGFβ1 staining cells are CD206 positive cells, indicating that CD206+ MΦs are the main cells that produce TGFβ1 (Fig. 7). The expression of CD31, an endothelial cell marker, was significantly decreased in the endometriotic-like lesions with the depletion of CD206+ MΦ compared to control (p < 0.05, Fig. 6e,f,i, H-score; 12.1 ± 12.0 vs 2.2 ± 1.7, p < 0.05).

In endometriosis, it has been reported that M2 MΦs are dominant in endometriotic lesions13,14, but the roles of MΦ for the pathogenesis of endometriosis are remained to be clarified. To understand the dynamics and roles of MΦ in the pathogenesis of endometriosis, animal models might be a useful tool. Recently, Johan et al. investigated whether infiltrating MΦs acquire functionally different phenotypes, M1 or M2 MΦ, during lesion development in an endometriosis mice model36. They showed that until day 7, after grafting endometrial tissues to mice, M1 MΦs are dominant, while M2 MΦs exhibit dominancy after day 736. Therefore, in the endometriosis mouse model, M1 to M2 MΦ shift occurs on day 7 after the inoculation of endometrial tissue. This is consistent with a transition from classical M1 MΦ activity to an alternate M2 profile, which correlates to the findings of initially acute inflammation followed by tissue remodeling in the process of development of endometirosis36. Given that some M2 MΦs have a role of suppressing inflammation37, the contribution of M2 MΦ to the endometriosis-like lesions is uncertain, suppressive, or progressive. To address this question, we used CD206 DTR mice and depleted CD206+ MΦs exclusively in mice from day 7 after induction of endometriotic-like Figure 4. Cell proliferation at the endometriotic-like lesion of DT and control group. Immuno-staining of Ki-67, a proliferation marker, was examined in control (a) and DT (b) group, and the percentage of Ki-67 positive cells per total cells was calculated in glandular epithelial cells and stromal cells, respectively (c and d). The arrow indicates the endometriotic epithelial cells. Data were evaluated by Mann Whitney U. P less than 0.05 was accepted as statistically significant. 6 Vol:.(1234567890)Scientific Reports | (2021) 11:853 | https://doi.org/10.1038/s41598-020-79578-3 www.nature.com/scientificreports/ lesions, when the shift from M1 to M2 MΦ could occur in endometriotic lesions36. CD206 is known to be a type 1 mannose receptor and one of M2a subtype markers in M2 MΦs. These MΦs secrete cytokines such as TGFβ1 and IL-10 and chemokines such as CCLs37. In CD206 DTR mice model, CD206+ MΦs were depleted by more than 80% in endometriotic-like lesion and PF cells (Fig. 5a and Supplemental Fig. 1). At mRNA levels, although the ratio of iNOS/CD11b was increased in PF cells, the mRNA expression of CD11b was unchanged, and there was no increase of TNFα/CD11b, suggesting that inflammatory MΦs which exacerbate endometriotic lesions6,38 did not increase in both PF cells and lesions. We found that the depletion of CD206+ MΦ resulted in the decrease of the size of endometriotic-like lesions without affecting the number of lesions, implying that the role of CD206+ MΦ is an accelerator of endometriotic-like disease in mice model. Duan et al. used CD11b DTR mice in which pan MΦs could be depleted with DT treatment, and showed the reduction of lesion weight in an endometriosis mice model. Moreover, they also presented that the adoptive transfer of M2a, a subtype of M2 MΦ, systemically after the MΦ depletion significantly increased the weight of endometriotic lesions39, suggesting that M2a MΦ play a role in the progression of the endometriotic lesion. In consistent with the notion, as CD206 is one of M2a subtype markers in M2 MΦs 37, our present results also suggest that CD206+ MΦs are involved in the exacerbation of endometriosis. In the DT group, endometriotic lesions exhibited thinner appearance, and lower proliferation rate of epithelial and stromal cells compared to control (Fig. 4), suggesting that CD206+ MΦs influence the growth of endometriotic cells. Among endometriosis-related factors, in the DT group, the levels of TGFβ1 and VEGFA mRNA, known as angiogenic factors40,41, were decreased in endometriotic-like lesions. In accordance with the notion, CD31, an endothelial cell marker, was almost disappeared in the endometriotic- like lesions with the depletion of CD206+ MΦ. It has been reported that the administration of bevacizumab, a VEGF-A antibody, resulted in reduced lesion formation in mouse endometriosis model studies41, suggesting neovascularization of ectopic endometrial tissue is crucial in the development of the endometriotic-like lesion. In the present study, immunohistochemical analysis showed that CD206+ MΦs expressed TGFβ1 (Fig.  7) and produced high level of VEGFA in the endometriotic-like lesions (Fig.  6). Therefore, we performed an in vitro study to examine these relationships. It is well known that IL-33, an alarmin, deviates macrophage to classical M2 type42–45. We also have reported that IL-33 induced CD206+ MΦs in human peritoneal MΦs 35. In the pre- sent study, we found that induced CD206+ MΦs with recombinant IL-33 stimuli (100 ng/ml, 8hrs) increased the expression of TGFβ1 mRNA (Supplemental Fig. 2). In addition, to investigate the relationship between TGFβ1 derived from CD206+ MΦ and VEGFA of the endometriotic lesion, we checked the mRNA expression of VEGFA in human endometriotic stromal cells (ESCs) with TGFβ1 stimulation and found the significant increase of VEGFA mRNA expression (Supplemental Fig. 2) suggesting that increased expression of TGFβ1 in Figure 5. The expressions of mRNA in endometriotic-like lesions in control and DT injected mice. In CD206 DTR mice, depletion of CD206+ macrophages (MΦ) were induced by injection of Diphtheria Toxin (DT). The expression of CD206 (a), TNFα (b), IL-1β (c), CD11b (d), VEGFA (e), and TGFβ1 (f) mRNA expressions were measured with quantitative PCR in control and DT group. Data were normalized by GAPDH mRNA levels to show the relative abundance. Representative data from three different experiments were shown as the mean ± SEM. *p < 0.05. 7 Vol.:(0123456789)Scientific Reports | (2021) 11:853 | https://doi.org/10.1038/s41598-020-79578-3 www.nature.com/scientificreports/ CD206-skewed-MΦ may induce angiogenesis via increasing VEGFA expression in ESCs. TGFβ1 is related to angiogenesis40 and is also reported to induce VEGFA in other cells46. Therefore, the reduction in TGFβ1 expres- sion by the depletion of CD206+ MΦ might have led to the decrease in angiogenesis, resulting in the reduction of endometriotic-like lesion. In endometriosis patients, it is reported that Natural Killer (NK) cells impair their function in the intra- peritoneal environment, which makes endometriotic cells more likely to survive and proliferate in the abdominal cavity. TGFβ1 is known to be increased in the peritoneal fluids of patients with endometriosis47 and to exacerbate endometriosis by decreasing NK cell activity48. Therefore, decreasing of TGFβ1 by the depletion of CD206+ MΦ may contribute to the recovery of NK activity and result in the reduction of endometriosis lesions. Further studies are necessary to clary the role of CD206+ MΦ in the pathogenesis of endometriosis. In the present study, we proved that CD206+ MΦ played a vital role in the promotion of endometriosis via inducing angiogenesis using CD206 DTR mice. Therefore, CD206+ MΦ might be a target for a new therapy for Figure 6. The expressions of VEGF , TGF-β and CD31 protein in endometriotic-like lesions. In CD206 DTR mice, depletion of CD206+ macrophages (MΦ) were induced by injection of Diphtheria Toxin (DT). The immunohistochemistry of VEGF (a and b), TGFβ1 (c and d), and a marker of the endothelium, CD31 (e and f), were examined in the endometriotic like lesions of control mice (control) and CD206+ MΦ depletion mice (DT). H-scores of VEGFA (g), TGFβ1 (h), and CD31 (i) immunostaining in endometriotic-like lesions were calculated and compared in control and DT mice. Data were evaluated by Mann Whitney U. P less than 0.05 was accepted as statistically significant. 8 Vol:.(1234567890)Scientific Reports | (2021) 11:853 | https://doi.org/10.1038/s41598-020-79578-3 www.nature.com/scientificreports/ endometriosis. One can speculate that the depletion of CD206+ MΦ using a specific antibody could be a strategy. Further studies are needed to prove these notions. Received: 29 July 2020; Accepted: 23 November 2020

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This work was supported by Health and Labor Sciences Research Grants from the Ministry of Health, Labor and Welfare of Japan, Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology, and by The Smoking Research Foundation. Author contributions O.Y . and S.S. wrote the main manuscript text.O.Y . and K.T. set an experimental protocol.K.T. prepared for animal experimentsY .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. a critical discussion.All authors reviewed the manuscript. Competing interests The authors declare no competing interests. Additional information Supplementary Information The online version contains supplementary material available at https ://doi. org/10.1038/s4159 8-020-79578 -3. Correspondence and requests for materials should be addressed to O.Y . or S.S. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. © The Author(s) 2021