{"paper_id":"2c470af1-bc42-4f97-8d06-fb8bee71de41","body_text":"R E S E A R C H Open Access\nRecombinant human IL-37 inhibited\nendometriosis development in a mouse\nmodel through increasing Th1/Th2 ratio by\ninducing the maturation of dendritic cells\nLijie Li, Zhouzhou Liao, Mingzhu Ye and Jianfa Jiang *\nAbstract\nBackground: Endometriosis is a serious reproductive and general health consequences. Recombinant human IL-37\n(rhIL-37) is an inhibitor of inflammation.\nMethods: ELISA assay was performed to detect the concentration of cytokines. Flow cytometry was used to\nanalyze cell proportion. Besides, qRT-PCR and western blotting assay were used to detect the level of gene and\nprotein, respectively. Transwell co-culture system was used for the co-culture of dendritic cells (DCs) and CD4 +T\ncells.\nResults: Our data showed that rhIL-37 inhibited the development of ectopic lesions in the mice with\nendometriosis, increased Th1/Th2 ratio and induced DCs maturation. The co-culture system of DCs and CD4 +T cells\ndemonstrated that rhIL-37 increased Th1/Th2 cell ratio through promoting DCs maturation. Moreover, the\nexpression of IL-4 in the DCs derived from healthy mice was inhibited by rhIL-37 treatment. rhIL-37 increased Th1/\nTh2 cell ratio through inhibiting IL-4 in DCs. Subsequently, our results proved that rhIL-37 promoted the maturation\nof DCs via inhibiting phosphorylation of STAT3. Activation of STAT3 could reverse rhIL-37-induced maturation of\nDCs.\nConclusion: Overall, rhIL-37 could protect against endometriosis through increasing the ratio of Th1/Th2 cells via\ninducing DCs maturation and inhibiting IL-4 expression in the DCs. Furthermore, rhIL-37 induced DCs maturation\nby inhibiting STAT3 phosphorylation. Our data confirmed the protective effect of rhIL-37 in endometriosis. These\ndata may provide a novel idea for the treatment of the disease.\nKeywords: Endometriosis, Recombinant human IL-37, Dendritic cell, T cell differentiation\nBackground\nIn clinical, endometriosis (EMs) is a common\ngynecological disease characterized by activated endo-\nmetrial cells plant onto the outside of endometrium [ 1].\nThe incidence of endometriosis in women of childbear-\ning age is about 10% ~ 15%, but the incidence of it is up\nto 30% in the patients with infertility or chronic pelvic\npain [ 2]. Endometriosis is a serious reproductive and\ngeneral health consequences. Importantly, it was re-\nported that the patients with endometriosis have a\nhigher risk of developing ovarian cancer [ 3]. Currently,\nthe goals of endometriosis treatment are to reduce op-\nerative intervention, fertility preservation, prevent dis-\nease recurrence, improve the quality of life, and pain\ncontrol. Although some drugs, such as GnRH\n© The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License,\nwhich permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give\nappropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if\nchanges were made. The images or other third party material in this article are included in the article's Creative Commons\nlicence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons\nlicence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain\npermission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.\nThe Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the\ndata made available in this article, unless otherwise stated in a credit line to the data.\n* Correspondence: jjfxy3yy@163.com\nDepartment of Gynecology, The Third Xiangya Hospital of Central South\nUniversity, NO.138 tongzipo, Yuelu District, Hunan 410013 Changsha, China\nLi et al. Reproductive Biology and Endocrinology          (2021) 19:128 \nhttps://doi.org/10.1186/s12958-021-00811-3\n\nantagonists, aromatase inhibitors, and antiprogestins,\ncould effectively protect against endometriosis, the treat-\nment of the disease still is a challenge [ 4, 5]. It is very\nnecessary to explore the pathogenesis of endometriosis,\nand explore a novel idea for the treatment of the disease.\nIt was well known that endometriosis is a choric and\ninflammatory disease [ 6]. The concentration of pro-\ninflammatory cytokines like tumor necrosis factor- α\n(TNF-α) was highly expressed in the peritoneal fluid of\nthe patients with endometriosis [ 7]. Abnormal immune\nsystem is closely associated with the development of\nendometriosis. The number of immune cells was obvi-\nously increased in the serum and peritoneal fluid of the\npatients with endometriosis, and the proportions of T\nhelper (Th) cells (Th1 and Th2 cells) were imbalanced\nin the serum of patients [ 8, 9]. It was reported that the\nconcentration of Th1 cell-related cytokines like\ninterferon-γ (IFN-γ) was lowly expressed, while Th2\ncell-related cytokines like interleukins (IL)-4, IL-10, and\nIL-13 were highly expressed in the serum of the patients\nwith endometriosis [ 10, 11]. Moreover, dendritic cells\n(DCs) also play a crucial role in the development of\nendometriosis. DCs are specialized immune cells, which\ninvolve in both innate and adaptive T cells-mediated im-\nmunological responses [ 12]. It was demonstrated that\nthe number of immature dendritic cells (iDCs) was not-\nably higher than mature dendritic cells (mDCs) in the\nendometriosis tissues from a non-human primate model\nof the disease [ 13]. In previous study, Fainaru et al. re-\nvealed that immature bone marrow-derived DCs, not\nmature bone marrow-derived DCs, contribute to the de-\nvelopment of endometriosis [ 14]. These studies sug-\ngested the important role of mDCs in the improvement\nof endometriosis.\nIL-37 is a unique member of the IL-1 family, and par-\nticipates in the development of multiple diseases, for\nexample, colitis, arthritis, sepsis, and endotoxin shock.\nIL-37 is a natural suppressor of inflammatory, and play a\nprotective role in above diseases [ 15, 16]. Recently, some\nstudies demonstrated that IL-37 could suppress the pro-\nduction of pro-inflammatory cytokines like IL-1 β, IL-6,\nand IL-10, and inhibit the occurrence and development\nof endometriosis through targeting multiple signaling\npathways, such as mitogen-activated protein kinase sig-\nnaling and Wnt/ β-catenin [ 17]. He et al. indicated that\nIL-37b splice variant could effectively suppress the\ngrowth of lesion in an endometriotic mouse model\nthrough regulating the invasion, angiogenesis, prolifera-\ntion and inflammation by affecting AKT and ERK1/2\nsignaling [ 18]. However, the effect of IL-37 on the ab-\nnormal immune cell remains unclear. Here, in our\npresent study, the data revealed that recombinant hu-\nman IL-37 (rhIL-37) could inhibit the development of\nendometriosis through increasing the ratio of Th1/Th2\ncells. Mechanismly, rhIL-37 increased Th1/Th2 ratio\nthrough inducing the maturation of DCs and inhibiting\nIL-4 production via suppressing the phosphorylation of\nsignal transducer and activator of transcription 3\n(STAT3). Our study proved again the therapeutic ability\nof rhIL-37 in endometriosis, and may provide a novel\nidea for the treatment of endometriosis.\nMaterials and methods\nReagents\nFemale C57BL/6 mice (6 – 8 weeks; 19 – 24 g) were ob-\ntained from the Jiangsu Ailingfei Biotechnology Co., Ltd.\n(Nanjing, China). Here, rhIL-37 was obtained from\nAbcam (ab151873, USA). Estradiol benzoate (E8875-\n250MG), lipopolysaccharide (LPS, SMB00704), and IL-4\n(SRP3093) were purchased from Sigma-Aldrich (Califor-\nnia, USA). Colivelin, an activator of STAT3, was bought\nfrom Santa Cruz Biotechnology, Inc. (CAS 867021 – 83-8,\nDallas, TX, USA). FITC-labeled anti-CD4 (11 – 0048-42),\nAPC-labeled anti-CD11c (11 – 0116-42), APC-labeled\nanti-interferon-γ (IFN-γ,1 7 – 7319-82), PE-labeled anti-\nIL-4 (Th2 cells, 12 – 7049-42), PE-labeled anti-CCR5\n(12– 1956-42), PE-labeled anti-CD83 (12 – 0839-42),\nBiotin-labeled anti-MHC II (MA1 – 12180), FITC-labeled\nanti-CD40 (11 – 0409-42), PE-labeled anti-CD80 (12 –\n0809-42), APC-labeled anti-CD86 (MHCD8605) were\npurchased from eBioscience (California, USA). RPMI-\n1640 medium (11875119), fetal bovine serum\n(10100147), and penicillin-streptomycin sulfate\n(15140148) were obtained from Gibco (USA). The\nTranswell-6 co-culture system with a 0.4 μm porous\nmembrane was bought from Corning (NY, USA). More-\nover, the ELISA kits, including mouse IL-37 ELISA kit\n(ml058377), mouse IFN- γ ELISA kit (ml058350-J),\nmouse TNF- α ELISA kit (ml002095), mouse IL-4 ELISA\nkit (ml063156-J), and mouse IL-13 ELISA kit\n(ml063123), were purchased from Shanghai Enzyme-\nlinked Biotechnology Co., Ltd. (Shanghai, China). TRIzol\nreagent was from Invitrogen (Carlsbad, CA, USA). Tran-\nscriptor First Strand cDNA Synthesis Kit was from\nRoche (04379012001, Basel, Switzerland). SYBR Premix\nEx Taq was from Takara (Dalian, China). RIPA lysis buf-\nfer (R0010), BCA Protein Assay Reagent Kit (PC0020),\nand enhanced chemiluminescence kit (PE0010) were ob-\ntained from Solarbio (Shanghai, China). The primary\nantibodies like anti-STAT3 (ab68153) and anti-p-STAT3\n(ab267373), and the secondary goat anti-rabbit (ab6721)\nwere purchased from Abcam (Cambridge, MA, USA).\nEstablishment of endometriosis mouse model\nAll mice were housed in a standard environment with\n60– 70% relative humidity, 22 – 24 °C of temperature, and\n12 h light/12 h dark cycle. At 1 week after adapt, 3 μgo f\nestradiol benzoate dissolved in 50 μl of soybean oil was\nLi et al. Reproductive Biology and Endocrinology          (2021) 19:128 Page 2 of 13\n\nsubcutaneously injected into each donor mouse. After\n1 week of estradiol benzoate injection, the uteri tissues\nof donor mice were dissected, and then the endometrial\ntissues were obtained. After that, all endometrial tissues\nwere cut into < 1 mm 3 pieces. These pieces were sus-\npended in normal saline. The pieces from one mouse\nwas suspended in 1 ml normal saline. Subsequently, each\nrecipient mouse were administrated with 500 μl of tis-\nsues suspension. Then, the recipient mice were ran-\ndomly assigned to different groups. All animal\nexperiments were approved by the ethical committee of\nthe Third Xiangya Hospital of Central South University\n(No. 2018-S146), and performed strictly in accordance\nwith animal experiment guidelines and regulations in\nthe Third Xiangya Hospital of Central South University.\nIsolation of CD4 +T cells and DCs, and cell culture and\ntreatment\nPeritoneal lavage fluid samples were obtained from\nhealthy mice and the mice with endometriosis at the\ntime of sacrifice through peritoneal lavage with 5 ml of\nice-cold PBS. Then, the peritoneal lavage fluid samples\nwere centrifuged at 1500 g for 5 min to obtain the cells\nof intraperitoneal lavage. Cells were subsequently resus-\npended in PBS, and were stained with FITC-labeled\nanti-CD4 (CD4 +T cells), or APC-labeled anti-CD11c\n(DCs) antibodies in the dark for 30 min at 4 °C. Subse-\nquently, a flow cytometer (FACSVerse, BD, New York,\nUSA) was utilized to separate CD4 +T cells from the in-\ntraperitoneal lavage of healthy mice, and isolate DCs\nfrom the intraperitoneal lavage of healthy mice (control-\nDCs) and endometriosis mouse model (EMs-DCs).\nCD4+T cells were cultured in the RPMI-1640 medium\nsupplemented with 10% fetal bovine serum, 100 U/mL\npenicillin and 100 μg/mL streptomycin sulfate. DCs were\ncultured in the RPMI-1640 medium supplemented with\n10% fetal bovine serum, 100 U/mL penicillin, 100 μg/mL\nstreptomycin sulfate, 10 ng/ml IL-4, and 20 ng/ml re-\ncombinant mouse GM-CSF. All cells were grown at\n37 °C in a humidified atmosphere with 5% CO 2. For the\ndifferent cellular experiments, 100 ng/mL rhIL-37 was\nused to stimulate DCs, 100 ng/mL LPS was utilized to\ninduce the maturation of iDCs, and 100 ng/mL IL-4 was\nused to stimulate DCs. Besides, 0.5 μM Colivelin was\nchosen as the STAT3 activator.\nAdministration of rhIL-37 to mouse\nFor animal experiments, the endometriosis mouse model\nwas randomly divided into three groups: EMs, EMs +\nNS, and EMs + rhIL-37. A total of six model mice of\nendometriosis were assigned to the EMs group. Besides,\n12 mice with endometriosis were balanced distributed to\nEMs + NS and EMs + rhIL-37 groups. The mice in\nEMs + NS group were intraperitoneally injected with\nnormal saline at 24 h before modeling. The mice in\nEMs + rhIL-37 group were intraperitoneally injected with\nrhIL-37 which was dissolved into normal saline, for\n1000 ng per mouse at 24 h before modeling. The admin-\nistration of normal saline and rhIL-37 was performed for\nonce every 2 days, and was performed for a total of 10\ntimes. Besides, the C57BL/6 mice in control group were\nsuffered from acupuncture, but were injected with noth-\ning. At 24 h after the last normal saline and rhIL-37 in-\njection, all mice were sacrificed through cervical\ndislocation. Next, the ectopic lesions were observed, and\nthe weight and volume of tissues were evaluated. Mean-\ntime, the peripheral blood samples of each mice were\nobtained for next experiments. The drug administration,\nsampling, and sacrifice process to the mice was shown\nin Supplementary Fig. 7.\nDetection of IL-37, IFN- γ, TNF- α, IL-4, and IL-13\nThe concentrations of IL-37, IFN- γ, TNF- α, IL-4, and\nIL-13 in serum, and the production of IL-4 in DCs were\nmeasured by ELISA assay. All experiment were carried\nout strictly in accordance with the manufacture ’ s intro-\nductions of the mouse IL-37 ELISA kit, mouse IFN- γ\nELISA kit, mouse TNF- α ELISA kit, mouse IL-4 ELISA\nkit, and mouse IL-13 ELISA kit. The OD values at 450\nnm of each well were examined utilizing a microplate\nreader (BioRad Model 680, USA), and the reading results\nwere saved in the instrument.\nAnalysis of Th1/Th2 cells ratio, iDCs and mDCs\npercentages, and MHC II-, CD40-, CD80-, and CD86-\npositive DCs cells\nFlow cytometry was carried out to analyze the pro-\nportions of Th1, Th2, iDCs, and mDCs, and the ex-\npression of MHC II, CD40, CD80, and CD86 in the\nsurface of DCs cells. In order to detect the differenti-\nation of Th1 and Th2 cell, the CD4 +T cells were\nstained with APC-labeled anti-IFN- γ (IFN-γ+T cells,\nTh1 cells) and PE-labeled anti-IL-4 (IL-4 +Tc e l l s ,T h 2\ncells). In order to analyze the maturation of DCs, the\nDCs were stained with APC-labeled anti-CD11c and\nPE-labeled anti-CCR5 (CD11c +CCR5+DCs, iDCs), or\nAPC-labeled anti-CD11c and PE-labeled anti-CD83\n(CD11c+CD83+DCs, mDCs). Moreover, DCs were\nstained with PE-labeled anti-MHC II, FITC-labeled\nanti-CD40, PE-labeled anti-CD80, and APC-labeled\nanti-CD86. During the process, the cells were incu-\nbated with above antibodies in the dark for 30 min at\n4 °C. Finally, the percentages of the Th1, Th2, iDCs\nand mDCs, and the percentages of MHC II-, CD40-,\nCD80-, and CD86-positive cells were analyzed utiliz-\ning a flow cytometer (FACS Aria; BD) with a Flow Jo\nv10.0.7 software.\nLi et al. Reproductive Biology and Endocrinology          (2021) 19:128 Page 3 of 13\n\nCo-Culture system of CD4 +T cells and DCs\nThe co-culture system of CD4 +T cells and DCs was con-\nducted using a Transwell system. CD4 +T cells were\nplanted into the upper chamber of Transwell chamber,\nand DCs were seeded into the bottom chamber. CD4 +T\ncells were co-treated with LPS, rhIL-37 and IL-4 for 24\nh. Subsequently, the maturation of DCs, ratio of Th1\nand Th2 cells, and expression of molecules were\nmeasured.\nMeasurement of genes expression\nThe expression levels of IFN- γ mRNA, TNF- α mRNA,\nIL-4 mRNA, and IL-13 mRNA were measured by qRT-\nPCR. Total RNA was isolated from CD4 +T cells using\nTRIzol reagent. Then, the total RNA served as the tem-\nplate in reverse transcription, which was carried out ac-\ncording to the manufacture ’ s protocol of the\nTranscriptor First Strand cDNA Synthesis Kit. Subse-\nquently, real-time PCR was performed on an ABI 7500\nReal-time PCR system (Applied Biosystems, Foster City,\nCA, USA) using the SYBR Premix Ex Taq. The relative\nexpression levels of IFN- γ mRNA, TNF- α mRNA, IL-4\nmRNA, and IL-13 mRNA were normalized to GAPDH,\nand were calculated in accordance with 2 -ΔΔ Ct method.\nThe gene sequence of primers were as follows: IFN- γ:\n5′-CTTCTTCAGCAACAGCAAGG-3′ (F) and 5 ′-\nTGAGCT CATTGAATGCTTGG-3 ′ (R); TNF- α:5 ′-\nGCTCTTCTGTCTACTGAACTTCGG-3′ (F) and 5 ′-\nATGATCTGAGTGTGAGGGTCTGG-3′ (R); IL-4: 5 ′-\nCACAACTGAGA AGGAAACCTTCTG-3 ′ (F) and 5 ′-\nCTCTCTCATGATCGTCTTTAGCCTTTC-3′ (R); IL-\n13: 5 ′-GCTCCTCAATCCTCTCCTGTT-3′ (F) and 5 ′-\nGCAACTTCAATAGTCAG GTCC-3 ′; GAPDH: 5 ′-\nTCCACCACCCTGTTGCTGTA-3′ (F) and 5 ′-ACCA-\nCAGTC CATGCCATCAC-3 ′ (R).\nDetection of the expression of STAT3 and its\nphosphorylation\nThe expression levels of STAT3 and p-STAT3 were\nmeasured using western blotting assay. Total protein\nwas separated from DCs using RIPA lysis buffer. Then,\nthe concentration of protein was determined using a\nBCA Protein Assay Reagent Kit. After that, 25 μg of pro-\ntein was separated on 12% SDS-PAGE gel, and were\ntransferred onto PVDF membranes. The membranes\nwere then maintained with 5% non-fat milk for 1 h at\nroom temperature followed by the anti-STAT3 and anti-\np-STAT3 antibodies incubation overnight at 4 °C. Next\nday, the membranes were incubated with secondary goat\nanti-rabbit for 1 h at room temperature. At last, an en-\nhanced chemiluminescence kit was utilized to determine\nthe protein bands, and the optical density of the western\nblot was analyzed using the Image-Pro Plus 6.0 (Media\nCybernetics, lnc., USA) software. The relative expression\nof STAT3 and p-STAT3 was normalized to β-actin.\nStatistical analysis\nSPSS 19.0 (SPSS Inc., USA) software was utilized for all\ndata analysis, which was displayed as mean ± standard\ndeviation (SD). The statistical difference among multiple\ngroups were determined using one-way analysis of vari-\nance (ANOVA) followed by Bonferroni ’ s test. The statis-\ntical difference between two independent groups were\ndetermined by Student ’ s t-test. The value of P lower\nthan 0.05 was recognized as statistically significant. All\nexperiments were independently repeated for three times\nat least.\nResults\nrhIL-37 inhibited lesion development, increased serum\nTh1/Th2 ratio, and induced DCs maturation in the mice\nwith endometriosis\nHere, compared with the mouse with endometriosis and\nnormal saline-treated endometriosis mouse model, de-\nclined weight of ectopic lesion and reduced volume of\nectopic lesions were found in the rhIL-37-treated endo-\nmetriosis mouse model, suggesting that rh-IL-37 treat-\nment effectively inhibited the development of ectopic\nlesions (Fig. 1A-C). ELISA assay displayed that rhIL-37\nwas highly existed in the serum of the mice with endo-\nmetriosis, and no rhIL-37 was found in the serum of\ncontrol mice, endometriosis mouse model, and normal\nsaline-treated endometriosis mouse model (Fig. 1D).\nInterestingly, for the mice in Control, EMs, EMs + NS,\nand EMs + rhIL-37 groups, there was no significant dif-\nference in the level of serum IFN- γ (Fig. 1E). Neverthe-\nless, the production of serum TNF- α, a pro-\ninflammatory cytokine, was notably upregulated in the\nmice with endometriosis, which was partly downregu-\nlated by rhIL-37 treatment (Fig. 1F). Besides, the levels\nof serum IL-4 and IL-13 were upregulated in the mice\nwith endometriosis, but rhIL-37 treatment could effect-\nively decline the levels of them (Fig. 1G and H). IFN- γ,\nTNF-α, IL-4, and IL-13 are the important cytokines for\nTh1 and Th2 cells, hence, above results indicated that\nrhIL-37 maybe improve endometriosis through regulat-\ning Th1 and Th2 differentiation.\nFurthermore, we detected the percentages of Th1 and\nTh2 cells, and percentages of surface maturation\nmarkers-positive DCs, including CD40-positive DCs,\nCD86-potitive DCs, MHC-II-positive DCs, and CD80-\npositive DCs, in the blood of mice. The results showed\nthat there was no significant difference in the proportion\nof Th1 cells. The proportion of Th2 cells was signifi-\ncantly upregulated, while the ratio of Th1/Th2 cells was\ndownregulated in the mice with endometriosis, which\nwere partly recused by rhIL-37 treatment (Fig. 2A-C).\nLi et al. Reproductive Biology and Endocrinology          (2021) 19:128 Page 4 of 13\n\nThe percentages of CD40-positive DCs and CD86-\npositive DCs were declined in the mice with endometri-\nosis, but rhIL-37 treatment increased the percentage of\nthem (Fig. 2D-E, and Supplementary Fig. 1). Besides,\nthere was no significant difference in the percentages of\nCD80-positive DCs and MCH II-positive DCs (Fig. 2F-\nG, and Supplementary Fig. 1). Overall, above data indi-\ncated that rhIL-37 induced the maturation of DCs, in-\ncreased Th1/Th2 percentages, and improved\nendometriosis.\nrhIL-37 upregulated Th1/Th2 ratio through inducing DCs\nto mature\nIn order to explore whether rhIL-37 improves endo-\nmetriosis through increasing Th1/Th2 ratio by indu-\ncing DCs maturation, we separated CD4 +Tc e l l s\nsuccessfully from the peripheral blood of healthy mice\n(Supplementary Fig. 2), and DCs from the peripheral\nblood of healthy mice (control-DCs) and mouse with\nendometriosis (EMs-DCs) (Supplementary Fig. 3).\nControl-DCs and EMs-DCs were treated with 100 ng/\nmL rhIL-37 for 24 h. Our results displayed that rhIL-\n37 treatment could decline the percentage of iDCs in\nboth control-DCs and EMs-DCs, and the percentage\nof iDCs was higher in EMs-DCs than that in control-\nDCs (Fig. 3A and B). Oppositely, the percentage of\nmDCs was lower in EMs-DCs than that in control-\nDCs, rhIL-37 treatment could promote the maturation\nof DCs (Fig. 3C and D). The images of iDCs and\nmDCs were shown as in Supplementary Fig. 4.S u b s e -\nquently, LPS was used to stimulate the rhIL-37-\ntreated control-DCs and EMs-DCs for another 2 days.\nThe percentages of CD40-, CD80, CD86-, and MCH\nII-positive DCs were significantly lower in EMs-DCs\nFig. 1 rhIL-37 inhibited the development of lesion in the endometriosis mouse model. At 24 h after the last rhIL-37 administration, ( A) the\nformation of ectopic lesions were observed; ( B-C) the weight and volume of ectopic lesions were measured; ( D-H) the serum IL-37, IFN- γ, TNF-α,\nIL-4, and IL-13 levels were measured using ELISA assay. N= 6. **P < 0.01 compared with Control, and ##P < 0.01 compared with EMs\nLi et al. Reproductive Biology and Endocrinology          (2021) 19:128 Page 5 of 13\n\nthan that in control-DCs. In both control-DCs and\nEMs-DCs, rhIL-37 treatment increased the percent-\nages of CD40-, CD80, CD86-, and MCH II-positive\nDCs (Fig. 3E-H, and Supplementary Fig. 5). Above re-\nsults suggested that rhIL-37 could contribute to the\nmaturation of DCs.\nNext, we constructed a co-culture system of DCs\nwith CD4 +T cells. CD4 +T were co-cultured with\ncontrol-DCs, EMs-DCs, rhIL -37-treated control DCs,\nrhIL-37-treated EMs-DCs, and LPS-treated DCs, re-\nspectively. Our data showe d that DCs promoted Th1\ndifferentiation, which was not related to whether the\nDCs was control-DCs or EMs-DCs, and whether the\nDCs accepted with rhIL-37 treatment. Compared\nwith control-DCs, EMs-DCs signifi cantly promoted\nTh2 differentiation and downregulated Th1/Th2 ra-\ntio, which were partly reversed by rhIL-37 treatment\n(Fig. 4A-C). Moreover, our results also demonstrated\nthat the expression of IFN- γ,T N F -α, IL-4, and IL-13\nmRNAs was facilitated in the CD4 +Tc e l l sc o -\ncultured with DCs. Comp ared with control-DCs,\nEMs-DCs boosted the expression of TNF- α,I L - 4a n d\nIL-13 mRNAs in CD4 +T cell. Importantly, rhIL-37\nand LPS treatment could notably inhibit the expres-\nsion of TNF- α, IL-4, and IL-13 mRNAs (Fig. 4D-G).\nIn conclusion, rhIL-37 inhibited Th2 differentiation\nand increased Th1/Th2 ratio through inducing DCs\nto mature.\nrhIL-37 upregulated Th1/Th2 ratio by suppressing the\nproduction of IL-4 in DCs\nSubsequently, we explored the mechanism of Th1/\nTh2 ratio increasing induced by DCs. IL-4 is a main\nfactor that induces the differentiation of Th2 cells.\nHere, rhIL-37 stimulation significantly suppressed\nt h ep r o d u c t i o no fI L - 4i nc o n t r o l - D C s( F i g .5A).\nFig. 2 rhIL-37 increased the serum Th1/Th2 ratio and promoted DCs to mature in the endometriosis mouse model. At 24 h after the last rhIL-37\nadministration, (A-C) the percentages of Th1 and Th2 cells, and the ratio of Th1/Th2 cells in serum were analyzed using flow cytometry; ( D-G) the\npercentages of CD40-, CD80-, CD86-, and MHC II-positive DCs in serum were determined using flow cytometry. N= 6. ** P < 0.01 compared with\nControl, and ##P < 0.01 compared with EMs\nLi et al. Reproductive Biology and Endocrinology          (2021) 19:128 Page 6 of 13\n\nHere, to ensure whether rhIL-37-treated DCs in-\ncreases Th1/Th2 ratio via regulating IL-4, we used\nIL-4 combined with rhIL-37 to treat control-DCs\nand EMs-DCs. The flow cytometry results revealed\nthat Th1/Th2 ratio was significantly lower in the\nCD4+T cells co-cultured with EMs-DCs than that in\nthe CD4 +T cell co-cultured with control-DCs (Fig.\n5B and C). Besides, compared with CD4 +T cell co-\ncultured with control-DCs, the proportion of Th1\nand expression of TNF- α were decreased, while the\nproportion of Th2 and expression of IL-4 and IL-13\nwere increased in the CD4 +T cells co-cultured with\nEMs-DCs (Fig. 5D and E). Summary, rhIL-37 could\nincrease Th1/Th2 ratio via inhibiting the production\nof IL-4 in DCs.\nrhIL-37 promoted DCs to mature via inhibiting the\nphosphorylation of STAT3\nHowever, how rhIL-37 induces the maturation of DCs\nremains unclear. Our results indicated that the\nFig. 3 rhIL-37 promoted the maturation of DCs. The control-DCs and EMs-DCs were separated, and were then treated with rhIL-37. ( A-H) The\npercentages of iDCs, mDCs, and the percentages of CD40-, CD80-, CD86-, and MHC II-positive DCs were determined using flow cytometry. N= 3.\n**P < 0.01 compared with Control, and ##P < 0.01 compared with EMs\nLi et al. Reproductive Biology and Endocrinology          (2021) 19:128 Page 7 of 13\n\nFig. 4 rhIL-37 increased the ratio of Th1/Th2 via inducing EMs-DCs to mature. The control-DCs and EMs-DCs were co-cultured with CD4 +T cells.\nAt the same time, the DCs were treated with or without rhIL-37 and LPS. ( A-C) The percentages of Th1 and Th2 cells, and the ratio of Th1/Th2\ncells in CD4 +T cells was analyzed by flow cytometry; ( D-G) the expression levels of IFN- γ mRNA, TNF-α mRNA, IL-4 mRNA, and IL-13 mRNA in\nCD4+T cells were measured by qRT-PCR. N= 3. **P < 0.01 compared with CD4 +T, ##P < 0.01 CD4+T + Control-DC, and &&P < compared\nwith CD4+T + EMs-DCs\nLi et al. Reproductive Biology and Endocrinology          (2021) 19:128 Page 8 of 13\n\nphosphorylation level of STAT3 was notably down-\nregulated in the control-DCs by rhIL-37 treatment\n(Fig. 6A and B). Then, control-DCs were pre-treated\nwith Colivelin, an activator of STAT3, followed by\nrhIL-37 treatment, and these control-DCs were co-\ncultured with CD4 +T cells. Colivelin pre-treated DCs\nnotably upregulated the percentage of iDCs and\ndownregulated the percentage of mDCs. The rhIL-37-\ntreated control-DCs-induced downregulation in the\npercentage of iDCs and upregulation in the percent-\nage of mDCs was markedly recused by rhIL-37 treat-\nment (Fig. 6C-E). Furthermore, Colivelin treatment\neffectively downregulated CD40-, CD80-, CD86-, and\nMHC II-positive DCs percentage. Meantime, rhIL-37-\ninduced upregulation in the percentages of CD40-,\nCD80-, CD86-, and MHC II-positive DCs were re-\nversed by the phosphorylation of STAT3 (Fig. 7A-D,\nand Supplementary Fig. 6). In summary, rhIL-37 in-\nduced DCs to mature through inhibiting the phos-\nphorylation of STAT3.\nDiscussion\nAccording to the study of Cavalli et al., IL-37 is\nexpressed in several human tissues and cell lines. Among\nimmune cells, IL-37 is expressed in circulating\nmonocytes and tissues macrophages, DCs, tonsillar B\ncells, and plasma cells [ 16]. Different with other mem-\nbers of IL-1 family, IL-37 has been proved to be an anti-\ninflammation cytokine in numerous inflammatory disor-\nders, including endometriosis [ 19]. However, the action\nmechanism of rhIL-37 in endometriosis development\nstill is not clear. In this study, our data showed that\nrhIL-37 treatment could effectively inhibit the develop-\nment of ectopic lesions in the mice with endometriosis.\nA recent study reported that IL-37 plays an anti-tumor\nimmunity role in the progression of hepatocellular car-\ncinoma through promoting the recruitment of DCs and\ninducing the activation of DCs [ 20]. However, in another\nstudy, IL-37 was proved to suppress the maturation of\nDCs through targeting IL-1R8/ toll-like receptor 4/NF-\nκB signaling pathway [ 21]. CD40, CD80, CD86, and\nMHC II are the surface markers of mature DCs [ 22].\nThese studies suggested that DCs is a target of IL-37.\nHere, rhIL-37 treatment significantly increased the pro-\nportion of CD40-, CD80-, CD86-, and MHC II-positive\nDCs in the blood of the mice with endometriosis, sug-\ngesting that rhIL-37 promoted DCs maturation in endo-\nmetriosis. Moreover, a previous study revealed that IL-\n37 notably inhibits the differentiation of Th2 and Th17,\nand suppresses the expression of effector cytokines like\nFig. 5 IL-4 mediated the promotion of EMs-DCs to Th2 differentiation. ( A) Control-DCs were treated with rhIL-37, and then the production of IL-4\nwas examined using ELISA. N= 3. ** P < 0.01 compared with Control. ( B-D) The percentages of Th1 and Th2 cells, and the ratio of Th1/Th2 cells in\nCD4+T cells was analyzed by flow cytometry; ( E) the expression levels of IFN- γ mRNA, TNF-α mRNA, IL-4 mRNA, and IL-13 mRNA in CD4 +T cells\nwere measured by qRT-PCR. N= 3. ##P < 0.01 compared with CD4 +T + Control-DCs + rhIL-37 + IL-4\nLi et al. Reproductive Biology and Endocrinology          (2021) 19:128 Page 9 of 13\n\nFig. 6 rhIL-37-induced the maturation of DCs was reversed by activation of STAT3. (A and B) Control-DCs were treated with rhIL-37, and then the\nexpression of STAT3 and STAT3 phosphorylation were detected using western blotting assay.N= 3. **P < 0.01 compared with Control. (C-E) The percentages\no fi D C sa n dm D C sw e r em e a s u r e db yf l o wc y t o m e t r y .N= 3. *P <0 . 0 5a n d* *P < 0.01 compared with Control.##P < 0.01 compared with Control + rhIL-37\nLi et al. Reproductive Biology and Endocrinology          (2021) 19:128 Page 10 of 13\n\nIL-4, IL-5, and IL-6. However, IL-37 treatment has no\nsignificant effect on Th1 and Treg cells differentiation,\nand the expression of IFN- γ and IL-10 [ 23]. It is not\nclear that how about the effect of rhIL-37 on Th1 and\nTh2 differentiation in endometriosis. In our present\nstudy, we found that rhIL-37 has no effect on Th1 differ-\nentiation, but inhibited Th2 differentiation in endometri-\nosis mouse model. Importantly, our results showed that\nrhIL-37 increasing Th1/Th2 ratio through inducing the\nmaturation of DCs.\nEndometriosis is a chronic and pro-inflammatory disease.\nT cells, NK cells, and other immune cells play a crucial role\nin the development of the disease. Immune cells, pro-\ninflammatory cytokines and adhesion molecules provide\nsuitable conditions for the differentiation, adhesion, prolif-\neration and survival of ec topic endometrial cells [ 24– 26].\nSöhngen at al. indicated that the T cell-deficient mice can\nbe used to establish endometriosis mouse model without\nadditional clearance the B ce ll, suggesting the important\nrole of T cells in endometriosis development [ 27]. The\nnumber of T cells was markedly increased in the peritoneal\nfluid of the patients with endometriosis at early stage. Sub-\nsequently, with the development of endometriosis, apop-\ntotic rate of T cells was increasing [ 28]. The percentage of\nTh1 cells was lower in the endometriosis tissues than that\nin the endometrial tissues [29]. Besides, Chen et al. demon-\nstrated that the Th1 cell-related cytokine was lower, and\nTh2 cell-related cytokine was higher in the endometriosis\nt i s s u e si nc o m p a r i s o nw i t ht h ee n d o m e t r i a lt i s s u e s[30].\nDue to rhIL-37 has no effect on the differentiation of Th1,\nwe explored only the pathway for rhIL-37 inhibiting Th2\ndifferentiation. IL-4 is a cru cial inducer for the differenti-\nation of Th2 cells [ 31] .T h ei n h i b i t o r ye f f e c to fr h I L - 3 7o n\nIL-4 expression in DCs was found in our study. Further-\nmore, we proved that rhIL-37 increased Th1/Th2 ratio\nthrough inhibiting the production of IL-4 in DCs.\nSTAT3 is a transcriptional factor. It was proved that\nthe hyperactivation of STAT3 may resulted in the occur-\nrence of autoimmunity and immunodeficiency through\nregulating immune cells [ 32]. In tumor, the hyperactiva-\ntion of STAT3 has been proved to suppress the matur-\nation of bone marrow-derived DCs [ 33]. However, in\nFig. 7 rhIL-37-induced the increasing of CD40, CD80, CD86, and MHC II in DCs was rescued by activation of STAT3. (A-D) The percentages of\nCD40-, CD80-, CD86-, and MHC II-positive DCs were determined using flow cytometry. N= 3. **P < 0.01 compared with Control, and ##P < 0.01\ncompared with Control + rhIL-37\nLi et al. Reproductive Biology and Endocrinology          (2021) 19:128 Page 11 of 13\n\nendometriosis development, the effect of STAT3 on DCs\nmaturation remains unclear. Kim et al. demonstrated\nthat the phosphorylation level of STAT3 is significantly\nhigher in the endometriosis tissues than that in the nor-\nmal endometrial tissues [ 34]. In addition, it was reported\nthat extracellular IL-37 can regulate the downstream\nSTAT3 signaling [ 35]. These researches suggested that\nrhIL-37 may affect DCs maturation via regulating the\nphosphorylation of STAT3. In our present study, our\ndata revealed that activation of STAT3 could effectively\nreverse rhIL-37-induced the maturation of DCs.\nConclusions\nOverall, our data demonstrated that rhIL-37 markedly\ninhibited the development of endometriosis via increas-\ning the ratio of Th1/Th2 cells by inhibiting the produc-\ntion of IL-4 in DCs and promoting the maturation of\nDCs. Mechanismly, rhIL-37 promoted DCs maturation\nthrough suppressing the phosphorylation of STAT3.\nOur research may provide a novel therapeutic idea for\nendometriosis.\nSupplementary Information\nThe online version contains supplementary material available at https://doi.\norg/10.1186/s12958-021-00811-3.\nAdditional file 1: Supplementary figure 1 . Detection of the mature\nDCs percentage. At 24 hours after the last rhIL-37 administration, the per-\ncentages of CD40-, CD80-, CD86-, and MHC II-positive DCs in serum were\ndetermined using flow cytometry. N =3 .\nAdditional file 2: Supplementary figure 2 . Analysis of the CD4 +T\ncells. Flow cytometry was used to isolate CD4 +T cells from the peripheral\nblood of healthy mice. N= 3.\nAdditional file 3: Supplementary figure 3 . Analysis of the DCs cells.\nFlow cytometry was used to isolate DCs from the peripheral blood of\nhealthy mice and endometriosis mouse model. N= 3.\nAdditional file 4: Supplementary figure 4 . Detection of the iDCs and\nmDCs percentages. The control-DCs and EMs-DCs were separated, and\nwere then treated with rhIL-37. The percentages of iDCs, mDCs were de-\ntermined using flow cytometry. N= 3.\nAdditional file 5: Supplementary figure 5 . Analysis of the maturation\nof DCs. DCs and EMs-DCs were separated, and were then treated with\nrhIL-37, and then the percentages of CD40-, CD80-, CD86-, and MHC II-\npositive DCs were determined using flow cytometry. N= 3.\nAdditional file 6: Supplementary figure 6 . Analysis of the maturation\nof DCs. The percentages of CD40-, CD80-, CD86-, and MHC II-positive DCs\nwere determined using flow cytometry. N= 3.\nAdditional file 7: Supplementary Fig. 7 . The drug administration,\nsampling, and sacrifice process to the mice.\nAcknowledgements\nNot applicable.\nAuthors’ contributions\nLL: substantial contributions to conception and design, acquisition of data, or\nanalysis and interpretation of data; draft the article or revising it; and final\napproval of the version to be published. ZL, MY, JJ: acquisition of data, or\nanalysis and interpretation of data; and final approval of the version to be\npublished. JJ: revise the article and final approval of the version to be\npublished. All authors read and approved the final manuscript.\nFunding\nThis study was supported by the grants from National Natural Science\nFoundation of China (No. 81801422).\nAvailability of data and materials\nThe datasets used and/or analysed during the current study are available\nfrom the corresponding author on reasonable request.\nDeclarations\nEthics approval and consent to participate\nThis study was approved by the ethical committee of the Third Xiangya\nHospital of Central South University (No. 2018-S146).\nConsent for publication\nNot applicable.\nCompeting interests\nThe authors declare that they have no competing interests.\nReceived: 31 May 2021 Accepted: 27 July 2021\nReferences\n1. Czyzyk A, Podfigurna A, Szeliga A, Meczekalski B. Update on endometriosis\npathogenesis. Minerva Ginecol. 2017;69(5):447 –61.\n2. Mehedintu C, Plotogea MN, Ionescu S, Antonovici M. Endometriosis still a\nchallenge. J Med Life. 2014;7(3):349 –57.\n3. Králí čková M, Laganà AS, Ghezzi F, Vetvicka V. 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Kim BG, Yoo JY, Kim TH, Shin JH, Langenheim JF, Ferguson SD, et al.\nAberrant activation of signal transducer and activator of transcription-3\n(STAT3) signaling in endometriosis. Hum Reprod (Oxford, England). 2015;\n30(5):1069–78.\n35. Mei Y, Liu H. IL-37: An anti-inflammatory cytokine with antitumor functions.\nCancer Rep (Hoboken). 2019;2(2):e1151.\nPublisher’sN o t e\nSpringer Nature remains neutral with regard to jurisdictional claims in\npublished maps and institutional affiliations.\nLi et al. Reproductive Biology and Endocrinology          (2021) 19:128 Page 13 of 13","source_license":"CC0","license_restricted":false}