C5aR1+ macrophages are involved in the progression of endometriosis

Objective: Immune dysfunction plays an important role in the pathogenesis of endometriosis. C5a, a by-product of complement activation, binds to its receptor (C5a receptor 1, C5aR1) in various cells, including macrophages. The combination of C5a and C5aR1 in macrophages induces M2 polarization. There is no consistent conclusion regarding macrophage polarization status and its participation in the pathogenesis of endometriosis.

We analyzed the expression of C5aR1 and the immunophenotype of macrophages in samples from patients with endometriosis using immunohistochemistry and flow cytometry. We tested the equivalent item in a mouse model and the THP-1 cell line in vivo and in vitro. Single-cell RNA sequencing (scRNA-seq) from the Gene Expression Omnibus (GEO, GSE213216) was used to investigate the phenotype and function of C5aR1+ macrophages.

The infiltration of C5aR1+ macrophages was associated with the severity of endometriosis. C5aR1+ macrophages polarized more toward the M2 phenotype and transformed into an immunosuppressive state. Inhibiting C5aR1 using PMX-53 decreased the expression of C5aR1+ macrophages and reduced their polarization to the M2 phenotype, consequently shrinking lesion size in a mouse model.

Our study emphasizes the importance of C5aR1 and the macrophage phenotype in the pathogenesis of endometriosis. We propose PMX-53 as a potential therapeutic agent for endometriosis.

Endometriosis is a common disease that severely affects the health and quality of life in women of childbearing age and causes pelvic pain and infertility[1]. Owing to the nature of asymptomatic and invasive diagnoses, the prevalence of endometriosis is unclear[2]. This disease has considerable physical, psychological, and economical effects[3] and has attracted increasing attention and research interest. However, its pathogenesis remains controversial. The theory proposed by Sampson[4] that menstrual shredding refluxes through the fallopian tubes into the abdominal cavity and is implanted is generally accepted. Endometriosis is a chronic inflammatory-related disease[5]. Compared with the endometrium inside the uterine cavity, the outside endometrium is described as ectopic. Survival of the ectopic endometrium is closely related to regional and systemic immune abnormalities[6]. Macrophages play a key role under both physiological and pathological conditions. Exposure to microenvironmental factors can induce macrophages to polarize into two distinct subsets: classically activated (M1) macrophages and alternatively activated (M2) macrophages[7]. Macrophages with the M1 phenotype exert strong cytotoxic and anti-proliferative effector activities by producing both reactive oxygen and nitric reactive species. M2 macrophages are associated with tissue repair, angiogenesis, neurogenesis, and pro-tumor activity. M1 macrophages express biomarkers, including CD40, CD80, CD86, and secrete cytokines such as IL-1β, IL-6, IL-12, and TNF-α. Conversely, M2 macrophages express biomarkers, including CD163, CD206, and secrete cytokines such as IL-10 and TGF-β[8]. Macrophages and secreted cytokines are strongly associated with endometriosis. Without macrophages, endometriotic lesions cannot organize and develop into the peritoneum; furthermore, blood vessels cannot penetrate the lesion and stop growing[9]. Many in vivo and in vitro studies have attempted to elucidate the activation status of peritoneal macrophages in endometriosis and their influence on the pathogenesis of endometriosis. However, the findings of these studies are inconsistent. Bacci et al.[9] showed that peritoneal macrophages differentiate into M2 macrophages in mouse endometriosis models and patients with endometriosis. In addition, transplantation of bone marrow-derived M2 macrophages promotes endometriosis in BALB/c mouse models[10]. In contrast, in another study, there was no significant difference in the proportion of CD163+ (M2) macrophages between patients with endometriosis and those without[10,11]. Anaphylatoxin C5a is a vital component of this complement system. C5a expression was significantly increased in endometriosis[12]. Its cognate receptor, C5aR1, is expressed in various cells, including neutrophils, macrophages, astrocytes, mast cells, and immature dendritic cells[13]. The C5a–C5aR1 axis has been found to be crucial in the inflammatory response and oncogenesis, even in relation to coronavirus disease[14,15]. Plasmin-generated C5a targets the infiltrating C5aR1+ myeloid cells and skews the tumor microenvironment toward an anti-inflammatory phenotype that dampens CD8+ T cell responses[16]. In a mouse model of squamous carcinogenesis, urokinase-positive macrophages stimulated C5a generation, which, in turn, regulated the pro-tumorigenic properties of C5aR1+ M2 macrophages. Specific antagonist PMX-53 of C5aR1 could reprogram macrophage into the M1 type and create an antitumor microenvironment[16]. Endometriosis is characterized by a tumor-like biological behavior. Aslan et al.[12] evaluated the expression changes of 84 immune-response genes in endometriotic tissues in the lesions of 20 patients with endometriosis and found that compared with the C5 expression level seen in the control group, the expression of C5 was most significantly upregulated among the overexpressed genes. Therefore, it is reasonable to assume a similar association between C5aR1 expression, macrophages, and endometriosis. The present study focused on the role of C5aR1 and macrophage polarization in endometriosis.

Sample collection The present study was approved by the institutional ethics board of the Obstetrics and Gynecology Hospital of Fudan University (Approval no.: 2019-89). Written consent was obtained from all enrolled patients. Tissues from 36 patients diagnosed with ovarian endometriosis and 10 controls who underwent surgery at our hospital between April 2016 and January 2018 were collected. The disease stage was determined based on the ASRM staging method, and basic information was collected from the medical history records. Immunohistochemistry For immunohistochemical staining, Put the paraffin sections into a 60° oven at constant temperature and bake them for about 6 hours. All paraffin sections were placed in two cylinders of dimethylbenzene solution, each of which was left for 15 minutes to make the solution completely submerge the tissue and ensure that the histone wax was removed. The paraffin sections were dewaxed and placed successively in ethanol at a gradient concentration for a certain time (100% for 5 minutes, 95% for 5 minutes, and 75% for 5 minutes). They were immersed in phosphate-buffered saline (PBS) and placed on a shaker for 15 minutes, three times. The slides were incubated with 3% hydrogen peroxide for 10 minutes to quench endogenous peroxidase activity. After antigen retrieval by EDTA, the slides were blocked with non-immune serum at room temperature (20°C–28°C) for 1 hour and then incubated with primary antibodies against rabbit anti-human C5aR1 and CD68 (dilution 1: 500; BioLegend, San Diego, CA, USA) at 4°C overnight. After rinsing with PBS, the slides were incubated with a biotinylated goat anti-rabbit secondary antibody (Proteintech) for 30 minutes. Replacement of the primary antibody with PBS was used as the negative control. The reaction was visualized using 3,3′-diaminobenzidine (DAB; Zhongshan Golden Bridge Biotech, China). The slides were then rinsed with water and counterstained with Mayer’s hematoxylin. The immunological reaction was quantified as the average integrated optical density (IOD) in five randomly selected fields (400×) using Image-Pro® Plus analysis software (Media Cybernetics, Rockville, MD, USA). Flow cytometry The remaining peritoneal lavages were divided equally into three parts for flow cytometry (FCM) analysis of the macrophage proportion and polarization. The cytokine profile of M1 macrophages includes increased TNF-α, IL-6, IL-1, IL-12, IL-23, and type 1 interferon (IFN), as well as expression of inducible NO synthase (NOS2), CCR7, and HLA-DR. The cytokine release profile of M2 macrophages includes increased IL-4, IL-10, IL-13, and IL-1ra production, as well as the expression of CD206, Ym1, CD163, CCL1, CCL18, FIZZ1, arginase 1, and chitotriosidase[17]. F4/80 and CD11b were used as pan-macrophage markers[18]. NOS2 and CD206 are markers of M1 and M2 macrophages, respectively[17,19]. To maintain the original polarization state and avoid adherence to the tube wall, macrophages were inactivated by incubation in 2% paraformaldehyde for 10 minutes on ice before antibody staining. After centrifugation (300 g), the FC receptor was blocked by incubation with anti-mouse CD16/CD32 (14-0161; eBioscience, USA) at a concentration of 1:100 for 10 minutes on ice. The cells were subsequently incubated with APC-conjugated anti-mouse F4/80 (17-4801, eBioscience) at a ratio of 1:25 and PerCP-Cyanine5.5-conjugated anti-mouse CD11b (45-0112, eBioscience) at a ratio of 1:100 for 25 minutes on ice in the dark. After centrifugation (300 g), the cells were incubated in fixation/permeabilization buffer (BD Biosciences, USA) for 45 minutes. Mice and models Twenty-six-week-old virgin female C57BL/6 mice used throughout this study were purchased from Beijing HFK Bioscience Company (Beijing, China). All mice were maintained under controlled conditions with a light/dark cycle of 12/12 hours, stable temperature of 25 ± 1°C, and a relative humidity of 55% (±10%). All the animals had free access to laboratory food and water. Several murine endometriosis models have been established in the former studies for different research objectives[9]. In this study, we focused on macrophages, which are sensitive to the surrounding microenvironment. Inflammatory changes can generate a molecular memory that primes the macrophage inflammatory response. Hence, operations during modeling, such as abdominal surgery and ovariectomy, may disturb and prime the original peritoneal cavity immunity. We established a murine endometriosis model by intraperitoneal injection of allogeneic endometrial segments, mimicking the natural spontaneous generation of endometriosis in women and avoiding tremendous disturbance to the peritoneal immune microenvironment caused by abdominal surgery[9]. Briefly, 7-week-old mice were initially treated s.c. with estradiol benzoate (3 μg/mouse; Aladdin, Shanghai, China). Seven days later, the mice treated with estradiol benzoate were sacrificed and their uteri were collected in a petri dish containing warm 0.01 mol/L PBS (pH 7.2–7.4). All uterine horns were processed identically, including a longitudinal split with a pair of scissors, subtle isolation of the endometrial tissue, and careful disruption into fragments that were consistent and smaller than 1 mm. These fragments were then injected intraperitoneally into recipient mice using a 1-mL syringe and 25-g needle. To eliminate any potential bias, we mixed endometrial fragments from every 2.5 donor mice, which were randomly distributed into five different experimental groups, and injected the mixture equally into five recipient mice. Mice that received intraperitoneal injections of PBS were randomly and equally distributed into five control groups. Hence, at each time point (0.25, 3, 14, 28, and 42 days), five endometriotic and five control mice were included. Peritoneal cells were harvested by injecting and shaking 8 mL (4 mL × 2) of ice-bath washing buffer containing Advanced Roswell Park Memorial Institute medium 1640 (Gibco, USA) and 2% heat-inactivated fetal bovine serum (Gibco, USA). After peritoneal lavage harvest, 1.5 mL of the cell suspension was seeded onto a culture plate. To purify the macrophages, the cell culture medium was changed twice, at 3 and 6 hours after seeding, in a humidified atmosphere containing 5% CO2. The residual adherent macrophages were randomly counted in three chosen fields under a microscope (×400). In addition, when rinsing the peritoneal cavity of the mice 6 hours after the endometrial injection, the hole through the abdominal wall produced by the peritoneal injection was carefully blocked using hemostatic forceps to avoid additional outflow. Cell culture Human monocytic cell line THP-1 was purchased from Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany) and maintained in RPMI 1640 GlutaMAX™ medium supplemented with 10% (v/v) heat-inactivated fetal calf serum (FCS) and a 1% (v/v) mixture of penicillin and streptomycin, all reagents from Thermo Fisher Scientific, in a humidified incubator containing 5% CO2 at 37°C. Periodically, the cells were controlled for mycoplasma contamination using the MycoAlert™ PLUS Mycoplasma Detection Kit (Lonza, Basel, Switzerland). To obtain THP-1 macrophages, THP-1 cells were seeded at a density of 0.5 × 106 cells per well in a sixwell culture plate and stimulated with 50 ng/mL PMA for 48 hours, followed by further incubation in a complete medium in the absence of PMA for 3 days. For M1 polarization, macrophages in fresh medium were co-stimulated with LPS (50 ng/mL) and IFNγ (20 mg/mL), whereas the M2a and M2c subsets were obtained by stimulation with either IL-4 (20 mg/mL) or IL-10 (20 mg/mL), respectively. Macrophages were incubated with the corresponding stimuli for 6, 24, or 48 hours without replacing the medium. Statistical analysis All independent experiments were repeated at least thrice. Unless otherwise indicated, the data are expressed as means and standard deviations. Significant differences were evaluated by one-way ANOVA, followed by Tukey’s multiple comparison test, unpaired Student’s t test, or the Kruskal-Wallis test with Dunn’s correction using GraphPad Prism v6 (GraphPad Software, San Diego, CA, USA). Single-cell RNA-seq analysis Single-cell RNA sequencing (scRNA-seq) profiles and clinical information from 10 independent eutopic endometrial specimens and 20 independent endometrial specimens were obtained from the Gene Expression Omnibus (GEO, GSE213216). After data normalization, principal component analysis, and uniform manifold approximation and projection (UMAP), nine clusters were identified and defined using canonical cell markers. The R package “CellChat” was used to infer intercellular communication networks using a database of interactions among ligands, receptors, and their cofactors.

The expression of C5aR1 increases in abdominal environment of endometriosis Since there are no studies involving C5aR1 in endometriosis, we compared the expression of C5aR1 under normal and endometriotic conditions using immunohistochemistry (Fig. 1A). We enrolled 36 ovarian endometriosis cases and 10 controls in the research, with a mean age at 32.58 ± 5.71. Endometriosis was divided into four stages based on the rASRM score: 8 in stage I, 3 in stage II, 12 in stage III, and 21 in stage IV (Supplementary Table 1, https://links.lww.com/RDM/A80). The expression of C5aR1 correlates with the severity of EMS. (A) Representative image of C5aR1 staining in NEMS and EMS. (B) Comparison of the infiltration of C5aR1+ cells in stage III + IV (n = 25) and stage I + II (n = 11). P <0.001. (C) The correlation of the infiltration of C5aR1+ cells and the largest diameter of the lesion in endometriosis. Linear regression, P <0.001, R2 = 0.359. EMS: endometriosis; NEMS: non-endometriosis. Stages I and II are classified as mild, whereas stages III and IV are classified as severe. The level of C5aR1 was higher in severe endometriotic lesions than in mild lesions (Fig. 1B, P <0.001), and the infiltration of C5aR1+ cells in endometriosis was highly associated with the largest diameter of the lesion (Fig. 1C, P <0.001, R2 = 0.359). Infiltration of C5aR1+ cells indicates the severity and progression of endometriosis. The infiltration of C5aR1+macrophages increases in abdominal environment of endometriosis C5aR1 is expressed by macrophages and plays an important role in the immune system. We found that C5aR1 expression correlated with CD68+ cells, indicating that C5aR1 was mainly expressed in macrophages (Fig. 2A, P <0.0001). Peritoneal washes from 10 patients with endometriosis and 10 with non-endometriotic benign disease were collected to analyze macrophage infiltration using FCM. C5aR1+ macrophage infiltration was increased in the endometriotic environment compared to that in the normal environment (Fig. 2B). We also tested peritoneal washes from endometriotic and normal mice to determine the C5aR1+ macrophage levels. Consistent with the human specimens, the endometriosis group displayed more C5aR1+ macrophage infiltration (Fig. 2C). The infiltration of C5aR1+ macrophages in EMS is higher than that seen in NEMS benign diseases. (A) The correlation of the infiltration of C5aR1+ and CD68+ cells in EMS. Linear regression, P <0.0001, R2 = 0.383. (B) The infiltration of C5aR1+ macrophages was higher in peritoneal washes from mice with EMS model (n = 10) and normal mice (n = 10), as observed by flow cytometry. P = 0.001. (C) The correlation of the infiltration of C5aR1+ and CD68+ cells in EMS. Linear regression, P <0.0001, R2 = 0.471. (D) GSEA plot showing the enrichment scores for the antigen processing and presentation, CAM pathways in macrophages of NEMS. CAM: cell-adhesion molecule; EMS: endometriosis; GSEA: gene set enrichment analysis; NEMS: non-endometriosis. To examine the cellular source of macrophages, we analyzed publicly available scRNA-seq data from 10 biopsies of eutopic endometrial specimens and 20 biopsies of endometriotic specimens[20]. To investigate the function of macrophages, Kyoto Encyclopedia of Genes and Genomes (KEGG) term enrichment analysis revealed the upregulation of antigen processing and presentation, cell-adhesion molecule (CAM) pathways in the eutopic endometrium (Fig. 2D), which indicates different phenotypes of macrophages. In conclusion, C5aR1+ macrophage infiltration was higher in endometriotic environments and resulted in different phenotypes. C5aR1+macrophages present M2 phenotype in endometriosis To explore the phenotype of C5aR1+ macrophages, we compare the expression levels of CD163, CD206, and IL-10 between C5aR1+ and C5aR1- macrophages in endometriosis through FCM. The proportions of CD163+, CD206+, and IL-10+ cells were higher among C5aR1+ macrophages in peritoneal washes from patients with endometriosis, suggesting an M2 macrophage signature. We further analyzed the subtypes of C5aR1+ macrophages in peritoneal washes acquired from mice with endometriosis. Simultaneously, CD206 and IL-10, which are highly expressed and secreted by M2 macrophages, were elevated in C5aR1+ macrophages (Fig. 3D and E, P = 0.012, P = 0.005, P = 0.006, and P = 0.017, respectively). CD86 and TNF-β, highly expressed and secreted by M1 macrophages, were observed to be lower in C5aR1+ macrophages (Fig. 2F and G, P = 0.006; P = 0.017). These results indicate that C5aR1+ macrophages exhibit M2 features. Phenotype of macrophages in an abdominal endometriosis environment. (A–C) The proportion of CD163+, CD206+, and IL-10+ cells of C5aR1+ macrophages and C5aR1− macrophages in peritoneal washes from patients with endometriosis (n = 10) by flow cytometry. (A: P = 0.027; B: P <0.0001; C: P = 0.029). (D–G) The proportion of CD163+, CD206+, CD86+ cells, and TNF-α+ cells of C5aR1+ macrophages and C5aR1- macrophages in peritoneal washes of endometriosis mice model (n = 10) by flow cytometry. P = 0.012; P = 0.005; P = 0.006; P = 0.017. (H) Volcano plot showing differentially expressed genes (absolute log2FC >1.3, P <0.05) upregulated (right) or downregulated (left) in C5aR1+ macrophages vs. C5aR1- macrophages. (I) Bubble plot showing the results of GSEA based on differentially expressed genes. (J) GSEA plot showing the enrichment scores for chemokine signaling, cytokine–cytokine receptor interaction, the antigen processing and presentation, complement and coagulation pathways in C5aR1+ macrophages. (K) Bubble plot showing inferred ligand–receptor pairs between C5aR1+ macrophages or C5aR1− macrophages and other types of cells from scRNA-seq using CellChat. The depth of color from blue to red represents the low to high communication probability. The sizes of the bubbles represent the corresponding P values. Commun. Prob: Communication probability; GSEA: gene set enrichment analysis. Analysis of differentially expressed genes indicated that IL-10, CD163, and VEGFA were upregulated in the C5aR1+ macrophage-based scRNA-seq data (Fig. 3H). KEGG term enrichment analysis of differential genes revealed that in addition to the regulation of cytokine, chemokine, and complement pathways influences the immune microenvironment, C5aR1+ macrophages also regulated HIF1-α and mTOR signaling pathway which can promote the polarization of macrophages (Fig. 3I)[21,22]. In addition, antigen processing and presentation were decreased in C5aR1+ macrophages (Fig. 3J). In addition, C5aR1+ macrophages were predicted to interact with T/NK cells via the THBS1 − CD47, LAMB3 − CD44, and ADGRE5 − CD55 axes, suggesting that C5aR1+ macrophages regulate the functions of T and NK cells[23,24] (Fig. 3K). C5aR1+ macrophages were inferred to interact with mesenchymal and epithelial cells via the THBS1 − CD47 axis, which is reported to promote the epithelial-mesenchymal transition[25] (Fig. 3K). These data suggest that C5aR1+ macrophages exhibit more M2 than M1 features in endometriosis and play an important role in endometriosis promotion. C5aR1 inhibition reverses macrophage polarization and relieves progression of endometriosis PMX-53, a non-competitive inhibitor of C5aR1, is widely used in mouse models[26]. We introduced PMX-53 during the development of endometriosis mouse models and explored its influence on lesion size and macrophage polarization status. The volume of endometriotic lesions in PMX-53 treated mice shrank significantly compared to that in untreated mice (Fig. 4A). The infiltration of C5aR1+ macrophages decreases in PMX-53-treated group (Fig. 4B). We observed a decrease in CD206 and an increase in CD86, indicating that PMX-53 could reverse the alteration of CD206 and CD86 caused by the endometriotic environment (Fig. 4C and D). Although no significant alternation of IL-10 expression is detected, TNF-α revealed an increased tend after PMX-53 treatment (Fig. 4E and F). Inhibition of C5aR1 could retard endometriosis growth. (A) The lesion volume of lesions in endometriosis mice model with or without C5aR1 peptide antagonist (PMX-53; n = 10 per group; P = 0.030). The length, width, and height of endometriosis lesions in the two groups were measured by electronic vernimetric caliper and their volume (V) was calculated as 0.52 × L × W × H. (B) The proportion of C5aR1+ macrophages in peritoneal washes of endometriosis mice model with or without PMX-53 through flow cytometry. P = 0.032. (C–F) The proportion of CD206+, IL-10+, CD86+, TNF-α+ macrophages in peritoneal washes of endometriosis mice model with or without PMX-53 through flow cytometry. P = 0.049; P = 0.516; P = 0.007; P = 0.138. THP-1 cells resemble primary macrophages in some functional properties and differentiation markers; therefore, they are widely used to study macrophage biology. Supernatants of endometriotic lesion and non-endometriotic ovarian cyst cell culture solutions were collected and co-cultured with induced THP-1 cells. The expression of C5aR1 was significantly higher in THP-1 cells co-cultured in endometriotic environments (Fig. 5A). The induced THP-1 cells were co-cultured with the supernatants of non-endometriotic ovarian cyst cells and endometriosis lesion culture solutions with or without PMX-53. PMX-53 recovered Ca5R1+ macrophage infiltration caused by endometriosis (Fig. 5B). Induced and inhibited C5aR1+ macrophages in vitro. (A) The proportion of C5aR1+ cells in THP-1-induced macrophages cultured under EMS (n = 10) and NEMS (n = 10) conditions through flow cytometry. P = 0.042. (B) The proportion of C5aR1+ macrophages in THP-1 co-cultured with supernatants of EMS lesion culture solutions with or without PMX-53, as observed via flow cytometry. P = 0.047. CM: conditional medium; EMS: endometriosis; NEMS: non-endometriosis. These results showed that PMX-53, a C5aR1 inhibitor, reversed the immunosuppressive microenvironment by reducing the proportion of C5aR1+ macrophage infiltration, thereby alleviating disease progression in a mouse model of endometriosis.

Endometriosis is closely associated with the immune system and inflammation. Macrophages play a vital role in the immune process, and the polarization switch between M1 and M2 phenotypes regulates the microenvironment. In the normal endometrium, macrophage polarization tends toward M2[27]. However, in the eutopic endometrium of patients with endometriosis, the balance shifts toward M1[28]. There is no consensus on the role of macrophages in the peritoneum and endometriotic lesions. The complement system is a component of the immune system that cooperates with macrophages. The complement system can be activated through three pathways, in which the C3 and C5 states are core locations. Previous studies have focused on the alteration and function of C3 in endometriosis. C3 increases in endometriosis and is associated with endometriosis-associated infertility[29,30]. Research on C5 and endometriosis is rare, but has been found to be the most significantly increased among the upregulated gene in endometriosis[15]. C5a is the degradation product of C5 and cross-talks with macrophages through C5aR1. Several studies have shown that the C5a-C5aR axis affects the immune function of macrophages. In squamous carcinoma models, C5a regulates the properties of C5aR1+-expressing mast cells and macrophages, thereby promoting tumor progression[16]. In addition, it has been reported that C5a is involved in M2-like TAM infiltration and protease secretion, promoting colon cancer cell metastasis. C5a mediated macrophage polarization and this process relied substantially on activation of the nuclear factor-kappa B (NF-κB) pathway[31]. C5a signaling increased the expression of chemokine monocyte chemoattractant protein-1, anti-inflammatory molecule arginase-1, interleukin-10, and transforming growth factor β, but was negatively correlated with the expression of pro-inflammatory molecules[32]. We found that C5aR1+ macrophages were increased in endometriosis according to the database and clinical samples. We confirmed that macrophages in endometriosis are polarized to the M2 phenotype based on clinical samples and mouse models. THP-1 cells co-cultured in an endometriotic environment tend to polarize into CD206+ M2 macrophages instead of the CD86+ M1 phenotype. M2 macrophages can regulate the peritoneal environment and may alter the functions of relevant immune cells. Future research should examine the infiltration of other immune cells, including plasma cells, T cells, neutrophils, and dendritic cells. Moreover, the expression of immune molecules is worth exploring to verify the downstream effect of macrophage M2 polarization and the possible mechanisms in the pathogenesis of endometriosis. Endometriosis is a chronic disease requiring long-term management, including medicine and surgery. Surgery can be adopted for some patients, whereas medication has a larger population. Current medications are divided into hormonal and non-hormonal medications. The former includes combined oral contraceptives, progestins, and gonadotropin-releasing hormone analogs. The latter are primarily painkillers. The tolerability of drugs, adverse reactions, and affordability vary among patients. However, the curative effect of medications is individualized. The cause and mechanism of endometriosis have not yet been determined; thus, there are no targeted drugs. Based on basic experimental results, drugs that interfere with inflammatory conditions, hormonal responses, cell survival, proliferation, angiogenesis, and invasion have been tested in preclinical models[31]. In this study, we found that PMX-53 restrained the polarization of macrophages into the M2 phenotype, thereby decreasing the expression of C5aR1+ macrophages and lesion size in a mouse model. This study suggests that the regulation of immune conditions may block the progression of endometriosis. Infertility has become an increasingly important issue in the management of endometriosis. It adversely affects fertility through different mechanisms that act on the pelvis, ovaries, and uterus. Possible pathological mechanisms include chronic inflammation of the peritoneal fluid leading to changes in fertilization, disruption of ovarian function, abnormalities of the normal endometrium, pelvic adhesions, and pain during intercourse, leading to reduced frequency of intercourse[32,33]. The immune system has been proposed to play a role in the pathophysiology of endometriosis-related infertility[34]. Endometrial receptivity is an essential index of fertility and decreases in endometriosis[35]. Inflammation altered endometrial receptivity[36]. Our study showed that the immune microenvironment in endometriosis is transformed. It is reasonable to hypothesize that there may be an association between the cytokines produced by polarized macrophages and reduced endometrial receptivity. Future studies should explore these hypotheses.

In summary, this study found that C5aR1+ macrophages infiltrate endometriotic tissues and exhibit an M2 phenotype. PMX-53 can reverse the polarization of macrophages by inhibiting C5aR1 and slowing the progression of endometriosis, which may provide new possibilities for the treatment of endometriosis. To verify the current conclusions, a larger sample size should be included. However, further research should be conducted to explore the mechanism of C5aR1-induced macrophage polarization in the endometriotic environment as well as the therapeutic value of PMX-53. Supplemental materials Supplementary information is linked to the online version of the paper on the Reproductive and Developmental Medicine website. Acknowledgments None. Author contributions C.X. and G.Z. performed the study concept, design, and acquisition; X.C. and W.Z. performed acquisition, analysis, and interpretation of data, and statistical analysis; and all authors read and approved the final manuscript. Funding(s) This work was supported by the Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases, the Obstetrics and Gynecology Hospital, Fudan University. Conflicts of interest All authors declare no conflicts of interest. Cong-Jian Xu is Editorial Board member of Reproductive and Developmental Medicine. The article was subject to the journal’s standard procedures, with peer review handled independently of these Editorial Board member and their research groups. Ethics approval The study was approved by the Institutional Review Board of the Obstetrics and Gynecology Hospital of Fudan University (IRB approval no. 2019-89). Informed consent statement Each patient provided informed written consent, and the hospital’s ethics committee approved their enrollment in the study. Data availability statement The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

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