HSD11B1 overexpression in dendritic cells and stromal cells relates to endometriosis by inhibiting dendritic cell proliferation and maturation

Aims This study aims to explore the alterations of dendritic cells (DCs) subpopulations in ectopic endometrial lesions and unveil the underlying mechanisms.

Patients with endometriosis (n = 81) and women without endometriosis (n = 19) were recruited in this study. Dendritic cells (DCs) in the endometrial samples were counted after immunohistochemistry staining. The proportion of myeloid DCs and plasmacytoid DCs was calculated by flow cytometry. Primary DCs were isolated from tissues, and the cell viability and apoptosis were examined by MTT assay and flow cytometry. Cytokines were detected by the enzyme-linked immunosorbent assay. Differentially expressed genes were filtered by analyzing two datasets that were downloaded from GEO database and detected by RT-qPCR in tissues and isolated DCs. The function of HSD11B1 was examined in an endometrial stromal cell-DCs co-culture system and in vitro cultured DCs.

Reduced myeloid DCs and increased CD11c-CD304-DCs were found in ectopic endometrium compared to control endometrium and eutopic endometrium from endometriosis patients. Myeloid DCs isolated from ectopic endometrium expressed less CD80, CD83, CD86 and had reduced proliferation, increased apoptosis, and reduced cytokine production. The expression of HSD11B1 was significantly increased in both ectopic endometrium and isolated myeloid DCs. Overexpression of HSD11B1 in immature DCs could repress DCs maturation and cytokine production. Endometrial stromal cells overexpressing HSD11B1 secreted increased cortisol, which repressed DCs maturation.

HSD11B1 is upregulated in ectopic endometrial lesions, which may contribute to endometriosis through repressing myeloid DCs maturation.

Endometriosis, defined as the presence of endometrial type mucosa outside the uterine cavity, is a multifactorial gynecological disorder that affects 10–15% of women of reproductive age and 20–50% of infertile women [Citation1]. It is identified that endometriosis is related to infertility and ovarian cancer [Citation2]. Dysmenorrhea, dyspareunia, persistent pelvic discomfort, and irregular uterine bleeding are among the main signs and symptoms of endometriosis, and surgery is the mostly used method to remove ectopic endometriosis lesions. However, nearly 40% patients experienced the recurrence [Citation3]. Abnormal proliferation of endometrial tissues and chronic inflammation have been found to be involved in the onset and progression of endometriosis, but the detailed mechanisms remain unclear [Citation4]. It is identified that women with endometriosis have altered immune systems in their peripheral and uterine tissues, which may contribute to aberrant tissue homeostasis, infertility, and early pregnancy failure [Citation5–8]. Dendritic cells (DCs) are antigen-presenting cells that play a key role in the immunological responses on mucosal surfaces, including the endometrium [Citation9]. DCs can be divided into two main subgroups depending on their surface molecules: plasmocytoid DCs (CD123+, HLA-DR+) and myeloid DCs (CD1a+, CD11c+, HLA-DR+) cells. Plasmocytoid DCs are involved in viral detection and produce interferon, whereas myeloid DCs are engaged in T cell activation, which are mostly relevant to endometriosis [Citation10]. Myeloid DCs are the DCs mostly relevant to endometrium and can be divided into immature DCs (iDCs) and mature DCs (mDCs) according to their maturation status [Citation9]. iDCs can detect and react to foreign antigens that also produce cytokines and chemokines, including IL-6, IL-10, IL-12, TNFα, RANTES, and MCP-1, that regulate other lymphocyte populations [Citation9]. Schulke et al. detected DCs subpopulations in endometrium using immunohistochemistry and found reduced mDCs and increased iDCs signals in the ectopic endometrium compared with normal endometrium [Citation11]. Maridas et al. observed that peripheral CD141+ myloid DCs were decreased between secretory and menstrual phases in women with endometriosis [Citation12]. Research using mice indicated that disturbed DCs populations may contribute to the escape of endometrial cells from the uterus and their subsequent development into endometriotic lesions [Citation13–15]. Recently, altered DC subgroups have also been confirmed by single-cell sequencing methods; however, the mechanism is unknown [Citation16, Citation17]. HSD11B1(11-hydroxysteroid dehydrogenase type-1) is a endoplasmic reticulum membrane enzyme broadly expressed in the human body [Citation18]. It works by transforming inactive cortisone into active cortisol [Citation19]. Cortisol levels were found higher in the serum and hair, but lower in the saliva samples from patients with endometriosis [Citation20–22]. Cortisol level is an indicator for stress, and previous study using rat endometriosis model indicates that stress increases the size and severity of the ectopic endometrial lesions as well as inflammation responses [Citation23, Citation24]. As the main enzyme catalyzing cortisol production, HSD11B1 is found to play a role in female reproductive system disorders. For example, three polymorphisms in the HSD11B1 coding region are found to be related to increased risk for developing pregnancy-induced hypertension and preeclampsia [Citation25]. Meanwhile, Monsivais et al. identified the upregulation of HSD11B1 by microarray analysis and immunoblotting in ectopic endometrium and their findings suggest that pro-inflammatory milieu of the endometriotic lesion stimulates cortisol synthesis [Citation26]. However, the roles of HSD11B1-cortisol in regulating DCs maturation in endometrium remains unclear. In this study, we investigated the impact of HSD11B1 overexpression on myeloid DCs. We identified that upregulation of HSD11B1 may contribute to the aberrant proliferation and maturation of DCs in patients with endometriosis.

Data collection, processing, and identification of differentially expressed genes (DEGs) Gene expression datasets were downloaded from the Gene Expression Omnibus (GEO; https://www.ncbi.nlm.nih.gov/geo) database and filtered by characteristics of data, experiment, and sample size. Two high-throughput sequencing datasets (GSE99949 and GSE135485) were for DEGs analysis. Transcriptome analysis was conducted using R (version 4.2.2) via RStudio (Desktop version, 2022.12.0 + 353). The two datasets selected for DEG identification (GSE99949 and GSE135485) were downloaded from the GEO database of the National Center for Biotechnology Information using the GEOquery R package. DEGs were identified by the limma software package (version 3.52.4), with adjusted p values and Log2|FoldChange| (Log|FC|) less and greater than .05 and 1, respectively. Collection of endometriotic tissues A total of 81 Chinese-Han women with endometriosis (mean age: 36.9 years, range: 28–44 years) and 19 women without endometriosis with histologically normal endometrium (mean age 31.1, range 27–41) were included in this study. All participants underwent laparoscopic surgical examination of the abdominal cavity and complete excision of endometriotic tissue. None of the patients had received pre-operative hormonal therapy. The presence of endometriosis was suspected by either clinical or ultrasonography examination and confirmed by surgical findings and post-operative pathological examination. Laparoscopic examination of the abdominal cavity excluded the presence of any other pelvic pathology that could potentially confound the data. The menstrual phase was identified according to the day of the reproductive cycle and histological analysis of the endometrium. Written informed consents were obtained from all participants prior to participating in this study. This study was approved by West China Second University Hospital ethics committee (approval no.HX-20220133). Isolate primary DCs The primary DCs were isolated from endometrium tissues (five participants from each group in each menstrual cycle phase, patients were in ARSM endometriosis stages II–IV). Briefly, tissues were minced in 1 mL digestion enzyme cocktail (450 U/mL collagenase IV, .01% [wt/vol] deoxyribonuclease I) and then incubated at 37 °C for 45 min. The cells were filtered using a 250-µm tissue strainer, and washed by phosphate buffered saline (PBS) thrice. The mixed cell suspension were layered over 3 mL of polysucrose solution and centrifuged at room temperature at 500 g for 30 min with no brake. The white band is collected from the top of the polysucrose solution and washed with PBS. The cells are resuspended by magnetic selection buffer (PBS, .5% heat-inactivated human AB serum, 2 mM EDTA) at a density of 1 × 107/mL. The myeloid DCs were isolated using Myeloid Dendritic Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) through negative selection. Part of the cells was subjected to RNA isolation and gene expression analysis. Another part of the isolated cells was subjected to functional study. Generation of iDCs and in vitro stimulation The iDCs were generated from adherent mononuclear cells in human blood. Blood was freshly drawn from healthy volunteers. Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll/Hypaque density gradient centrifugation. DCs were isolated by using MagCellect Human Blood Dendritic Cell Isolation Kit (R&D Systems, Minneapolis, MN, USA) following the manufacturer’s instructions. Cells were cultured for 7 days, with GM-CSF (800 U/ml) plus IL-4 (1000 U/ml) addition every second day, to obtain immature cells. Cells were stimulated with Lipopolysaccharides(LPS) (1.0 μg/ml) for 18 h and then subjected to flow cytometry analysis. Immunohistochemistry (IHC) Paraffin-embedded sections were first deparaffinized and then incubated with rabbit anti-CD1c monoclonal antibody (Cell Signaling Technology, Inc., Cat.no. #45581) at 4 °C overnight. After three times wash by TBST (20 mM Tris base, 150 mM NaCl, .1% Tween-20 detergent, pH7.4), the sections were incubated with HRP conjugated goat anti-rabbit secondary antibody (Cell Signaling Technology, Inc.,Danvers, MA, USA, Cat.no. #7074). The sections were washed by TBST three times, and the signals were detected using DAB Substrate kit following the manufacturer’s instructions. Images were obtained using microscopy. Isolation and purification of endometriotic stromal cells Endometrial stromal cells (ESCs) were isolated from eutopic endometrium according to the reported methods [Citation27, Citation28]. Briefly, tissue samples were minced and then digested in DMEM/F12 with type I collagenase (2.5 mg/mL, Sigma-Aldrich, St. Louis, MO, USA) and DNase I (15 U/mL, Sigma-Aldrich) at 37 °C for 1 h. Debris were removed by filtration by using 40-μm nylon cell strainers. Cells were collected after centrifugation at 400 × g for 10 min, and then cultured in DMEM/F12 with 10% fetal bovine serum (FBS), penicillin (100 U/mL), and streptomycin (100 µg/mL). When the cells became confluent, they were dissociated with .25% trypsin, harvested by centrifugation, and replanted in six-well plates at 2 × 105 cells/well. RNA extraction Total RNA was extracted from tissue and cell samples using Trizol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instruction. RNA concentration and purity were determined using a model ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE, USA). Only samples with absorbance ratios of 260 nm/280 nm of ∼2.0, and 260 nm/230 nm of 1.9–2.2 were considered for inclusion in the study. Vector construction and electrotransfection The full length of HSD11B1 coding sequence was cloned into pcDNA3.1 vector to form a HSD11B1 overexpression vector pcDNA3.1-HSD11B1. HSD11B1 overexpression or empty control vector was transfected into ESCs and DCs through electrotransfection by using a 4D-Nucleofector X Unit (Lonza, Tampa, FL, USA) and a P3 Primary Cell 4D-Nucleofector® X Kit (Lonza, Tampa, FL, USA) following the manufacturer’s instructions. Quantitative RT-PCR The levels of candidate genes were quantified by qRT-PCR using SYBR Green Real-Time PCR Master Mix (Thermal Fisher Scientific, Waltham, MA, USA), with β-actin level as the loading control. Each sample in each group was measured in triplicate and the experiment was repeated at least thrice. The method of -ΔΔct was used to determine the relative level of target genes. Immunoblotting Proteins were extracted from cells and then quantified by using the Bradford protein assay kit (Bio-Rad, Hercules, CA, USA) following the manufacturer’s instructions. Proteins were denatured through boiling in sodium dodecyl sulfate/β-mercaptoethanol sample buffer, and 10 μg of samples was loaded into each lane of 12% polyacrylamide gels. The proteins were separated by electrophoresis and then blotted onto a polyvinylidene fluoride membrane (Amersham Pharmacia Biotech, St. Albans, Herts, UK) by electrophoretic transfer. The levels of candidate proteins were detected by specific antibodies. The GAPDH signal was used as a loading control. Antibody information: Rabbit anti-HSD11B1 polyclonal antibody (Abcam, Cat.no. ab39364), HRP Conjugated Rabbit anti-GAPDH monoclonal antibody (Cell Signaling Technology, Inc., Cat.no. #3683), HRP conjugated goat anti-rabbit secondary antibody (Cell Signaling Technology, Inc., Cat.no. #7074). Cell proliferation assay Cell proliferation was estimated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cells were seeded in 96-well plates at a density of 2 × 103 cells/well and allowed to attach overnight. After treatment, 20 μL of MTT (5 mg/mL) (Sigma-Aldrich) was added to each well, and the cells were incubated for 4 h. The absorbance was recorded at 570 nm with a 96-well plate reader after the addition of dimethyl sulfoxide (DMSO). Flow cytometry DCs subpopulations were analyzed following the protocol reported by Hey-Cunningham et al. [Citation29]. Briefly, endometrial cells were washed by PBS and stained with a panel of monoclonal antibodies conjugated to fluorochromes that included pan leucocyte markers, CD45, lineage markers (CD3, CD19, CD20, CD14 and CD56) as well as HLA-DR to allow identification of the CD45+, Lineage-, HLA-DR+ DC populations. Further subset was divided into CD304+ plasmacytoid DCs, and CD11c + myeloid DCs was followed by identification of three myeloid DC subsets. Cells were analyzed by flow cytometry by using a FACSCanto II flow cytometer (BD Biosciences,Franklin Lakes, NJ, USA). The results were analyzed by using FlowJo software (v10.4.1) (Tree Star, Inc., Ashland, OR, USA). For cell sorting, the CD45- cells were subdivided to CD13+ EPCAM- stromal cells and CD13- EPCAM+ epithelial cells. A total of 5 × 106 each subtype of cells were sorted from each samples and subjected to immunoblotting and RT-qPCR. Antibody information: BD Horizon™ V450 Mouse Anti-Human CD45 (Cat.No.560367, BD Biosciences, USA), PE Mouse Anti-Human CD3 (Cat.No.552127, BD Biosciences, USA), PE Mouse Anti-Human CD19 (Cat.No.555413, BD Biosciences, USA), CD20-PE (Cat.No.555623, BD Biosciences, USA), PE Mouse Anti-Human CD14 (Cat.No.557154, BD Biosciences, USA), PE Mouse Anti-Human CD56 (Cat.No.555516, BD Biosciences, USA), PerCP-Cy™5.5 Mouse Anti-Human HLA-DR (Cat.No.560652, BD Biosciences, USA), CD304-PE-Cy7 (Cat.No.145212, BioLegend, Inc. USA), BD Horizon™ V500 Rat anti-CD11b (Cat.No.562127, BD Biosciences, USA), Alexa Fluor™ 647 Mouse Anti-Human CD80 (Cat.No.567340, BD Biosciences, USA), APC/Cyanine7 labeled mouse anti-human CD83 Antibody (BioLegend, Inc. Cat.no. 305330), FITC labeled Mouse Anti-Human CD86 antibody (Cat.No.555657, BD Biosciences, USA). For apoptosis analysis, DCs were isolated from control, eutopic or ectopic endometrium, DCs transfected with empty or HSD11B1 overexpression vector, and DCs co-cultured with wildtype or HSD11B1 overexpressed ESCs were collected and incubated with Annexin V-FITC and propidium iodide (PI). The apoptotic cells were determined by flow cytometry, and the results were analyzed by using FlowJo software (v10.4.1) (Tree Star, Inc., Ashland, OR, USA). Statistical analysis Data was analyzed using SPSS Statistical Package version 16 (SPSS Inc., Chicago, IL, USA). Two-tailed Student’s t-test was used to calculate statistical significance between the two comparator groups. The findings were considered significant when p value <.05.

The number of DCs is reduced in the ectopic endometrium To investigate the role of DCs in endometriosis, endometrium samples were collected from 81 patients with endometriosis and 19 women without endometriosis. Immunohistochemistry detecting CD1c in paraphing embedded tissue samples to determine the number of DCs. We observed that the number of DCs in ectopic endometrial lesions was significantly reduced compared with eutopic and healthy control endometrium samples during proliferative and menstrual phase (). Meanwhile, primary DCs were isolated from random five samples in each group at each menstrual stage. The number of myeloid and plasmacytoid DCs was counted by flow cytometry. The proportion of myeloid DC was found reduced in ectopic endometrium at all the three main menstrual cycle stages and the proportion of CD11c CD304 double-negative DCs was increased (). The proportion of plasmacytoid DCs was slightly increased in ectopic endometrium but the difference was not significant (). Furthermore, the myeloid DCs in the ectopic endometrium expressed less CD80, CD83 and CD86 (). Myeloid DCs in patients with endometriosis have reduced viability and increased apoptosis To further examine whether myeloid DCs function is altered in ectopic endometrium, primary myeloid DCs were isolated from endometrium by negative selection () and then subjected to flow cytometry analysis and MTT assay. Myeloid DCs that were isolated from ectopic endometrium pretend to apoptosis compared to that isolated from control endometrium (). The viability of myeloid DCs isolated from ectopic endometrium significantly reduced (). Meanwhile, myeloid DCs from ectopic endometrium secreted reduced IL6, IL-12 and TNF-α compared to control and eutopic endometrium (). These results indicated that the dysfunctional myeloid DCs existed in the ectopic endometrium and may contribute to the pathogenesis of endometriosis. HSD11B1 is upregulated in endometrium and DCs from patients with endometriosis To explore the factors inducing the dysfunction of DCs in endometriosis, two high-throughput sequencing datasets (GSE99949 and GSE135485) were downloaded from GEO datasets for analyzing differentially expressed genes. These two datasets contained the gene expression data of 58 patients with endometriosis and 8 health controls. Nine genes (C7, CFH, NR3C1, HSD11B1, MYH11, FGF7, FABP4, ADH1B and COL8A1) were overexpressed in the ectopic endometrial lesions in these two datasets (). To confirm these findings, the expression of nine candidate genes was quantified by RT-qPCR. HSD11B1 was found overexpressed in ectopic endometrial lesions at all the three menstrual stages (proliferative, secretory and menstrual) (), which was also upregulated in myeloid DCs from ectopic endometrial lesions (). The levels of C7, NR3C1 and MYH11 were increased in ectopic endometrial lesions during proliferative and menstrual phases (Fig. S1). COL8A level was increased in ectopic endometrial lesions at proliferative phase, and FABP4 level was reduced in ectopic endometrial lesions at menstrual phase (Fig. S1). However, no consistent finding was observed in the primary myeloid DCs (Fig. S2). To further explore what kinds of cells expressed HSD11B1 in endometrium, the HSD11B1 level was detected by immunohistochemistry in control and ectopic endometrium. We observed that HSD11B1 was undetectable in the endometrium glands and epitheliums (). Weak HSD11B1 signals were found in endometrial stroma, and some strong HSD11B1 signals were found in some specific types of cells around the glands in the ectopic endometrium (). Furthermore, endometrial stromal and epithelial cells were obtained by flow sorting () and subjected to immunoblotting and RT-qPCR with myeloid DCs. HSD11B1 was found mainly expressed in the endometrial stromal cells and myeloid DCs (). Therefore, the functional study was mainly processed in myeloid DCs and endometrial stromal cells. The overexpression of HSD11B1 in DCs inhibits DC maturation To investigate the function of HSD11B1 in DCs, we isolated DCs from peripheral blood. DCs were cultured in medium containing GM-CSF and IL-4 to obtain iDCs. Empty or HSD11B1 overexpressing vector was electrotransfected into iDCs, and the iDC was then stimulated by LPS. We observed that HSD11B1 upregulation in DCs (), increased cortisol production (), repressed viability () and increased the number of apoptotic cells (). LPS stimulation increased the levels of mature DC surface markers including MHC II, CD80, CD83 and CD86 when compared with iDC (). The overexpression of HSD11B1 significantly inhibited the expression levels of MHC II, CD80 and CD86 on the cell surface (). Meanwhile, LPS stimulation promoted the production of cytokines (TNF-α, IL-1β, IL-6, IL-10 and IL-12) (). The overexpression of HSD11B1 inhibited the production of TNF-α, IL-1β, IL-6 and IL-12, but not significantly altered the level of IL-10 (). The overexpression of HSD11B1 in ESCs promotes cortisol production and inhibits DCs maturation The stromal compartment, which makes up the majority of the endometrium, tightly controls tissue proliferation, remodeling and modulating the immune microenvironment during the menstrual cycle [Citation30, Citation31]. To investigate the impact of HSD11B1 upregulation in endometrial lesion on dysfunctional DCs, endometrial stromal cells (ESCs) were isolated from eutopic endometrial lesion. The HSD11B1 overexpression vector was transferred into ESCs by electrotransfection with empty vector as control (). ESCs were co-cultured with DCs for 3 days, and the DCs were stimulated by LPS. The DCs were subjected to functional study (). The medium from HSD11B1 overexpressed ESCs contained 3.32-fold cortisol compared with that from control ESCs (). DCs co-cultured with HSD11B1 overexpressed ESCs had reduced viability () and increased apoptosis (). DCs that co-cultured with HSD11B1 overexpressed ESCs expressed reduced surface MHC-II, CD80, CD83 and CD86 () and produced reduced cytokines including TNF-α, IL-1β, IL-6 and IL-12 (). These results indicated that ESCs overexpressing HSD11B1 secreted increased cortisol production and repressed DCs maturation. Finally, we obtained the working model of HSD11B1 in endometrium that the overexpression of HSD11B1 transformed inactive cortisone into active cortisol, which further repressed the maturation of DCs and impaired immune responses and induced a failure of removing ectopic endometrium lesions ().

Accumulating evidence suggests that defective immunosurveillance at the uterine may contribute to the failure of retrograde menstrual debris clearance and the onset of endometriosis [Citation32, Citation33]. Endometriosis patients have altered immune cell subpopulations in the peripheral blood and uterine tissues, including DCs [Citation29]. In this study, we gated the total DCs (HLA-DR+ Lin-) in the uterine by using CD1c and CD304. We observed increased CD1c-CD304- DCs in the ectopic endometrium which is in line with the findings of Hey-Cunningham et al. [Citation29]. What’s the difference is that we found reduced CD1c + myeloid DCs in the endometriosis uterine but in their research, the proportion of myeloid DCs was not significantly altered. Meanwhile, Jau-Ling Suen et al. observed an increased CD123+ plasmacytoid DCs in the endometrioma tissues that may secrete IL-10 and promote endometriosis development through activating angiogenesis [Citation15]. We found slightly increased CD304+ plasmacytoid DCs in the ectopic endometrium in this study, but the difference is not significant. These inconsistencies may be the result of different gating strategies, the complexity of endometriosis or the different race of the participants. Schulke et al. examined iDCs and mDCs in the eutopic and ectopic endometrium by detecting CD1a and CD83 using immunohistochemistry. They found decreased mDCs(CD83+) signal in women with endometriosis compared with controls in the endometrium across all phases of the menstrual cycle [Citation11]. In this study, we detected CD80, CD83 and CD86 in the subpopulation of myeloid DCs, and found myeloid DCs in ectopic endometrium expressed less CD80, CD83 and CD86 indicating an immature status. Meanwhile, we isolated primary myeloid DCs and found the myeloid DCs from ectopic endometrium pretended to be apoptosis and secreted less cytokines. These findings indicate that increased iDCs and impaired DCs function are involved in the development of endometriosis. Cortisol levels have been assessed in women with endometriosis in a number of studies, all focusing on stress reactivity at single points in time and chronic high cortisol levels always relate to advanced stages and contribute to the pathogenesis of the disease [Citation20–22, Citation34, Citation35]. It is identified that cortisol has a significant suppressive effect on myeloid DCs maturation, inhibiting CD86 and CD83 expression and cytokine production [Citation36–38]. HSD11B1 can transform cortisone to cortisol. Monsivais et al. screened differentially expressed genes in ovarian endometriosis samples by microarray and reported the upregulation of HSD11B1 for the first time [Citation26]. They concluded that overproduced TNF-α in endometriosis induced HSD11B1 expression and stimulated cortisol production. In this study, we analyzed two high-throughput sequencing datasets and found that nine genes were differentially expressed in ectopic endometrium. We examined these genes expressions in endometrium samples and confirmed the overexpression of HSD11B1 in patients with endometriosis. Furthermore, we identified the overexpression of HSD11B1 in the primary myeloid DCs from patients with endometriosis for the first time. We also confirmed that overexpression in either myeloid DCs or ESCs could upregulate cortisol production in the cell culture medium and inhibit DCs maturation that explained why immature DCs increased in ectopic endometrium. There are limits of this study: (1) we identified an increased proportion of CD1c-CD304- DCs, but these cells’ function is unclear; (2) we only examined the HSD11B1 function in myeloid DCs but do not understand its function in CD1c CD304 double-negative DCs and myeloid DCs; (3) the factors inducing HSD11B1 upregulation in ectopic endometrial lesions remain unclear. In conclusion, we identified that HSD11B1 is upregulated in ectopic endometrial lesions that may contribute to endometriosis through repressing myeloid DCs maturation. Author contributions YX and JL designed the research study; XY, JL and XY performed the research; XY and JL obtained the data; XY and JL processed and analyzed the data; XY and JL wrote the main manuscript text. All authors read and approved the final manuscript. Ethics approval and consent to participate Informed consent was obtained from all patients for being included in the study. This study was performed in accordance with the Declaration of Helsinki and approved by the Ethics Committee of West China Second University Hospital. All methods were carried out in accordance with relevant guidelines and regulations. Competing interest The authors declare that they have no competing interests. Supplemental material s2.tif Download TIFF Image (7.2 MB)s2.tifs1.tif Download TIFF Image (7.7 MB)s1.tifAcknowledgments The authors acknowledge all the volunteers for participation in this study. Disclosure statement No potential conflict of interest was reported by the authors. Data availability statement All data generated or analyzed during this study are included in this published article. Additional information Funding

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