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Introduction

Endometriosis (EMs) is a painful disorder in which the uterine lining (endometrium) grows abnormally outside the uterus, and it frequently occurs in the lower abdomen or pelvic area near the ovaries and fallopian tubes (1). Patients with endometriosis often suffer from dysmenorrhea, pain during sexual intercourse, lower abdominal pain and infertility (1).

Endometriosis is generally considered to be a disease associated with chronic inflammation (1). The tissues associated with endometriosis, especially in abdominal wall and ovarian endometriosis, are infiltrated by a large number of inflammatory cells, including CD3+ T cells, CD4+ T helper (Th) cells, CD8+ cytotoxic T cells, CD450+ memory T cells, CD68+ macrophages, CD20+ B cells, Th17 cells and regulatory T cells (2). M2 macrophages exist in greater numbers in endometriotic tissues compared with the surrounding tissues (3). In addition, Th17 cells and macrophages are abundant in the peritoneal fluid of patients with endometriosis, and the Th17 cell number has been indicated to positively correlate with the disease severity (4-6). Although peritoneal macrophages in patients with endometriosis produce and secrete large amounts of inflammatory mediators, such as TNF-α, IL-1β, IL-6 and vascular endothelial growth factor (VEGF) (7), the phagocytic ability of the macrophages is reduced. Therefore, the macrophages fail to effectively remove the uterine endometrial debris in the context of an inflammatory environment, thereby promoting the implantation of the endometrial cells and subsequent endometriosis (8).

Currently, major therapeutic strategies for endometriosis include surgical and medical treatment (9). However, surgical treatment for the removal of endometrioma is associated with a high risk of recurrence of endometriosis (10), and medical treatment causes a decrease in estrogen levels, leading to premature menopause (11). Therefore, there is increasing interest in the biological treatment of endometriosis through which the production of inflammatory cytokines may be inhibited by regulating the differentiation of macrophages and T cells.

Protein arginine methyltransferases (PRMTs) serve a role in protein methylation. PRMT5 catalyzes the formation of monomethylarginine and symmetric dimethylarginine in proteins, regulates a variety of target genes such as CDKN2A and multiple signaling pathways, such as the SNAIL/cadherin-1 pathway and the leukemia inhibitory factor/signal transducer and activator of transcription 3 signaling pathway (12), and participates in the post-transcriptional splicing and processing of RNA, cell proliferation, differentiation, apoptosis and tumorigenesis (12). A previous study demonstrated that PRMT5 is involved in the inflammatory response in endothelial cells, contributing to the pathogenesis of various diseases such as atherosclerosis (13). In addition, overexpression of PRMT5 has been indicated to promote the proliferation of memory T cells, thereby enhancing IL-2 expression in multiple sclerosis (14). It has been demonstrated that inhibition of PRMT5 expression can reduce the risk of colitis by promoting the regulatory T cell differentiation of Th cells and subsequently suppressing the production of TNF-α, IL-6 and IL-13(15).

As the production of cytokines by activated macrophages serves an important role in the pathogenesis of endometriosis, it was hypothesized that PRMT5 contributes to macrophage activation, thereby promoting endometriosis development. The present study examined the effects of serum and extracts of eutopic endometrium from patients with endometriosis on PRMT5 expression in THP-1-derived macrophages, as well as the underlying signaling pathways. Furthermore, the role of PRMT5 in macrophage activation in vitro and in vivo was also investigated.

Materials and methods

Reagents

Phorbol 12-myristate 13-acetate (PMA; cat. no. P8139) and lipopolysaccharide (LPS; cat. no. L2630) were purchased from Sigma-Aldrich (Merck KGaA). The Luminex cytokine panel kit (cat. no. LXSAHM) was purchased from R&D Systems, Inc. Rabbit PRMT5 antibody (cat. no. 18436-1-AP) was purchased from ProteinTech Group, Inc. IL-6 (cat. no. BMS213-2) and IFN-γ-induced protein 10 (IP-10) ELISA kits (cat. no. KAC2361) were purchased from Thermo Fisher Scientific, Inc. SB431542 (cat. no. S1067), which is used as an inhibitor of Smad 2/3(16), was purchased from Selleck Chemicals. NF-κB inhibitor SN50 (cat. no. SML1471) and PRMT5 inhibitor EPZ015666 were obtained from MedChemExpress.

Sample collection

A total of 25 female patients with ovarian cysts or infertility were enrolled from the Department of Obstetrics and Gynecology of The Second Xiangya Hospital of Central South University (Changsha, China) between December 2018 and October 2019. These patients were diagnosed with endometriosis by laparoscopic and histopathological examination. According to the revised American Fertility Society endometriosis classification, the study population included 2 stage I-II cases, 7 stage III cases and 16 stage IV cases (17). Of these, 20 cases were in the proliferative phase and 5 cases were in the secretory phase. The control group consisted of 12 patients who were diagnosed with cervical neoplasia or uterus septum and underwent laparoscopy, during which no endometrial lesions were observed in the pelvic cavity. All patients (aged 20-45 years) had regular periods and did not receive hormone therapy within 3 months prior to surgery. There were no significant differences in age, pregnancy, gravidity or proliferative vs. secretory phase between the two groups (Table I).

Table I General characteristics of the females in the two groups.

Table I

General characteristics of the females in the two groups.

Characteristic	EMS (n=25)	Control (n=12)	t-, u- or χ2-value	P-value
Age, years	28.0±4.99	31.75±5.05	-1.84	0.07
Age range, years	20-37	22-45
Parity, n	1.44±1.16	2.00±1.28	-1.30	0.19
Gravidity, n	0.60±0.50	1.00±0.60	-1.90	0.06
Menstrual cycle, number (P/S)	20/5	8/4		0.43

[i] Data are presented as the mean ± SD, range or ratio. EMS, endometriosis; P, proliferative phase; S, secretory phase.

During surgery, endometrial biopsies were collected via excision under sterile conditions from the control and research groups, and they were promptly transported to the laboratory on ice in PBS. A portion of each tissue sample was fixed in 10% formalin solution for 12 h at room temperature. and then processed for histological examination to exclude pathological abnormalities. Hematoxylin-eosin staining was performed for histological examination, and the staining was completed according to the a previously described protocol (18).

The present study was approved by the Ethics Committee of The Second Xiangya Hospital of Central South University (Changsha, China). Written informed consent was obtained from all participants.

Isolation of peritoneal monocytes

During the laparoscopy, the peritoneal cavity was rinsed with 50 ml normal saline solution, and cell pellets were collected via centrifugation at 376 x g for 5 min at 4˚C. Monocytes were isolated following a standard protocol of Ficoll density gradient centrifugation (19-21). Briefly, Ficoll-loaded samples were centrifuged at 455 x g or 25 min at 20˚C. The buffy coat layer containing cells was then harvested and washed three times with PBS followed by centrifugation at 35 x g for 10 min at 20˚C to obtain monocytes. The monocytes were then stored at -80˚C until multiple samples were collected before performing subsequent reverse transcription-quantitative PCR (RT-qPCR) and western blot analyses.

Serum collection

Samples of 5 ml peripheral blood were collected from each patient into a coagulation tube. The serum was separated by centrifugation at 845 x g for 5 min at 4˚C and then stored at -80˚C until further use.

Preparation of endometrial homogenates

Endometrial homogenates were prepared as described previously (22). Briefly, endometrial tissues were excised during laparoscopy. A total of ~200 mg tissue was minced and homogenized in 5 ml RPMI-1640 (Gibco; Thermo Fisher Scientific, Inc.), followed by centrifugation at 9,391 x g for 10 min at 4˚C. The supernatant was collected, and protein concentration was determined using the BCA method. The samples were then stored at -80˚C until further use.

Cell culture and treatment

THP-1 cells were generously provided by Dr Joseph Huang (University of South Florida). The cells were grown in RPMI-1640 supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.), 1% penicillin and 1% streptomycin at 37˚C in a humidified atmosphere with 5% CO2. THP-1 cells were differentiated into macrophage-like cells through treatment with 25 ng/ml PMA for 3 days at 37˚C in a humidified incubator with 5% CO2. THP-1-derived macrophage-like cells were pretreated with 100 ng/ml LPS for 24 h at 37˚C in a humidified incubator with 5% CO2 and then cultured in RPMI-1640 containing 10% serum from controls and patients with endometriosis for 6 days or cultured in homogenized solution of endometrium from controls and eutopic endometrium from patients with endometriosis (100 µl homogenized solution/ml medium) for 3 days. The inhibitors SN50 (50 µg/ml; treated for 1 h), SB431542 (10 µM; treated for 24 h) and EPZ015666 (10 µM; treated for 24 h) were added to the medium before treatment with the homogenized solution.

Western blot assay

THP-1 cells and the isolated peritoneal monocytes were washed with cold PBS and lysed with RIPA lysis buffer (Beyotime Institute of Biotechnology; cat. no. P0013B) on ice. Cell lysates were collected by centrifugation at 13,523 x g for 10 min at 4˚C, after which the supernatant was obtained and the protein concentration was determined using a BCA assay kit (Beyotime Institute of Biotechnology; cat. no. P0010S). Samples were then heated at 95˚C for 5 min in loading buffer. Protein samples (10 µg) were separated via 10% SDS-PAGE, transferred to a 0.2-µm nitrocellulose membrane, blocked with 5% nonfat milk in TBS containing 0.1% Tween-20 (TBST) for 2 h at room temperature, and incubated overnight with primary antibodies against PRMT5 (1:1,000) and β-actin (ProteinTech Group, Inc.; cat. no. 20536-1-AP; 1:10,000; internal control) at 4˚C. The membranes were then washed with TBST three times, incubated with HRP-Goat Anti-Rabbit IgG (H+L) (ProteinTech Group, Inc.; cat.no. SA00001-2; 1:5,000) for 1 h at room temperature, and then washed again with TBST three times. The chemiluminescence signal, visualized using SuperSignal West Pico PLUS (Thermo Fisher Scientific, Inc.; cat. no. 34580) was detected using the ChemiScope 5300 chemiluminescence system (Clinx Science Instruments Co. Ltd.) and quantified using Quantity One software (version 4.6.6; Bio-Rad Laboratories, Inc.).

RT-qPCR

Total RNA was extracted from THP-1 cells and the isolated peritoneal monocytes using TRIzol® reagent (Thermo Fisher Scientific, Inc.; cat. no. 15596026). cDNA was synthesized using the PrimeScript RT reagent kit with gDNA Eraser (Perfect Real Time; Takara, cat. no. RR047A) from 1 µg total RNA in accordance with the manufacturer's protocol. Relative gene expression levels were determined using the SYBR green PCR Master Mix (Applied Biosystems; Thermo Fisher Scientific; cat. no. 4309155) using the 2-ΔΔCq method (23) with normalization to β-actin expression. The thermocycling conditions were as follows: Initial denaturation at 95°C for 30 sec, followed by 40 cycles of 95°C for 5 sec, 60°C for 20 sec, 95°C for 1 sec, 65°C for 15 sec and 95°C for 1 sec for the dissociation curve. The primer sequences were as follows: PRMT5 forward, 5'-GGTGAACGCTTCCCTG-3' and reverse, 5'-TGAGACTACGGTCACTTGG-3'; β-actin forward, 5'-GAGCGCGGCTACAGCTT-3' and reverse, 5'-TCCTTAATGTCACGCACGCACGATTT-3'.

ELISA

After being treated as aforementioned, THP-1 cells with eutopic endometrial tissue extract from patients with endometriosis and the PRMT5 inhibitor EPZ015666, IL-6 and IP-10 levels in these cells were measured using ELISA kits (Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions.

Luminex cytokine assay

TNF-α, IL-6, IL-10 and C-C motif chemokine 20 (CCL20) levels in endometrial extracts from patients with endometriosis and control individuals were determined using the Luminex cytokine kit (R&D Systems, Inc.) according to the manufacturer's instructions. Briefly, frozen endometrial extracts were homogenized in RIPA lysis buffer [Beyotime Institute of Biotechnology; cat. no. P0013B; 50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1% sodium deoxycholate] with protease inhibitor cocktail (Sigma-Aldrich; Merck KGaA). Total protein levels were measured using the BCA method. TNF-α, IL-6, IL-10 and CCL20 levels were measured using a Luminex 200 analyzer (Luminex Corporation). The ratio of cytokine concentration to total protein concentration (pg/ml/mg protein) was used to determine differences between the two groups.

Statistical analysis

All quantitative data are presented as the mean ± SD. Statistical analysis was performed using SPSS software (version 17.0; SPSS, Inc.) and visualized using GraphPad Prism software (version 8.0; GraphPad Software, Inc.). In vitro cell experiments were repeated three times per sample, and the average values were used. Quantitative data were firstly examined for normal distribution using the Kolmogorov-Smirnov test, and data with equal variances were compared using one-way ANOVA followed by the Least Significant Difference test for comparisons among three groups, while unpaired t-test was used for comparisons between two groups. Alternatively, Mann-Whitney U tests were used for comparisons. Categorical variables were compared using the χ2 test. The Fisher's exact test was used to analyze the menstrual cycle variable in Table I. P<0.05 was considered to indicate a statistically significant difference.

Results

Cytokine levels in endometrial extracts from patients with endometriosis and control individuals

To investigate the differential expression of cytokines in patients with endometriosis and control individuals, the Luminex assay was used to compare the levels of TNF-α, IL-6, IL-10 and CCL20 in endometrial tissue extracts from these patients. The results demonstrated that the endometrial extracts of patients with endometriosis contained significantly higher levels of TNF-α, IL-6 and IL-10 and a decreased level of CCL20 compared with the endometrial extracts of control individuals (Fig. 1), suggesting that there are differences in the expression of TNF-α, IL-6, IL-10 and CCL20 that may contribute to the inflammatory response under pathological conditions.

Figure 1 Cytokine levels in endometrial extracts from patients with endometriosis and control individuals. Cytokine levels in endometrial extracts were detected via Luminex cytokine panel assay. The TNF-α (*P<0.05), IL-6 (*P<0.05) and IL-10 (**P<0.01) levels were increased, and the CCL20 level was decreased in endometrial tissue extracts from the endometriosis group (*P<0.05). n=12 for control and n=25 for EMs. CCL20, C-C motif chemokine 20; EMs, endometriosis.

Serum from patients with endometriosis inhibits PRMT5 expression in macrophages

To investigate the possible role of macrophage-derived PRMT5 in endometriosis development, the expression pattern of PRMT5 in THP-1 cells treated with serum from controls and patients with endometriosis in the presence of LPS was examined. As indicated in Fig. 2, both the mRNA and protein levels of PRMT5 were significantly decreased in THP-1 cells treated with serum from patients with endometriosis, compared with the levels in cells treated with serum from controls. These results revealed that PRMT5 expression in macrophages could be downregulated in the context of endometriosis, suggesting a potential role for macrophage-derived PRMT5 in the pathogenesis of endometriosis.

Figure 2 Effect of serum from patients with endometriosis on macrophage PRMT5 expression. THP-1 cells were pretreated with phorbol 12-myristate 13-acetate and then stimulated with 100 ng/ml LPS for 24 h, followed by incubation with RPMI-1640 containing 10% serum from patients with endometriosis and controls for 6 days. Reverse transcription-quantitative PCR and western blot analyses were performed to examine the mRNA and protein expression levels of PRMT5 in THP-1 cells. β-actin was used as the internal control. *P<0.05 (n=25). PRMT5, protein arginine methyltransferase 5; LPS, lipopolysaccharide.

Extracts of eutopic endometrium suppress PRMT5 expression in macrophages

To further confirm the possible involvement of macrophage-expressed PRMT5 in endometriosis, PRMT5 expression was examined in THP-1 cells treated with extracts of normal and eutopic endometrium in the presence of LPS. As presented in Fig. 3, the extracts of normal endometrium induced a substantial increase in PRMT5 expression in THP-1 cells. However, treatment with extracts of eutopic endometrium significantly inhibited PRMT5 mRNA and protein expression in macrophages compared with extracts of normal endometrium, suggesting that macrophage-derived PRMT5 may serve a role in the interaction between macrophages and the eutopic endometrial microenvironment, which likely contributes to the inflammatory response under pathological conditions.

Figure 3 Effect of extracts of eutopic endometrium from patients with endometriosis on macrophage PRMT5 expression. THP-1 cells were pretreated with phorbol 12-myristate 13-acetate and then stimulated with 100 ng/ml LPS for 24 h, followed by incubation with RPMI-1640 containing homogenized solution of normal and eutopic endometrium (100 µl/ml) for 3 days. Reverse transcription-quantitative PCR and western blot analyses were performed to examine the mRNA and protein expression levels of PRMT5 in THP-1 cells. β-actin was used as internal control. *P<0.05; **P<0.01 (n=25). PRMT5, protein arginine methyltransferase 5; LPS, lipopolysaccharide.

Macrophage expression of PRMT5 is regulated in a NF-κB signaling-dependent manner

To investigate the mechanisms underlying macrophage PRMT5 expression in endometriosis, the NF-κB inhibitor SN50 or the Smad2/3 inhibitor SB431542 was added to cells together with homogenized endometrial tissue to examine PRMT5 expression in THP-1 cells in the presence of LPS. The results revealed that SB431542 significantly inhibited the mRNA expression of PRMT5 in cells treated with extracts of normal endometrium. However, SB431542 treatment demonstrated no inhibition of PRMT5 protein expression in cells treated with extracts of normal endometrium. Additionally, no inhibition was demonstrated in cells treated with extracts of eutopic endometrium at both the mRNA and protein level. The results demonstrated that SN50, but not SB431542, markedly inhibited PRMT5 mRNA and protein expression in THP-1 cells treated with extracts of both normal and eutopic endometrium (Fig. 4). The results indicated that the endometrial microenvironment regulated macrophage PRMT5 expression through NF-κB signaling, but not Smad2/3 signaling.

Figure 4 Effects of NF-κB inhibitor and Smad2/3 inhibitor on the macrophage expression of PRMT5. THP-1 cells were pretreated with phorbol 12-myristate 13-acetate and stimulated with 100 ng/ml LPS for 24 h before treatment with NF-κB inhibitor SN50 (50 µg/ml) for 1 h or Smad2/3 inhibitor SB431542 (10 µM) for 24 h, followed by incubation in RPMI-1640 containing homogenized solution of normal and eutopic endometrium (100 µl/ml) for an additional 3 days. Reverse transcription-quantitative PCR and western blot analyses were performed to examine the mRNA and protein expression levels of PRMT5 in THP-1 cells. β-actin was used as internal control. *P<0.05; **P<0.01 (n=15). PRMT5, protein arginine methyltransferase 5; LPS, lipopolysaccharide.

PRMT5 mediates endometrial tissue extract-induced macrophage activation

To examine the function of PRMT5 in the interaction between macrophages and the endometrial microenvironment, THP-1 cells were treated with eutopic endometrial tissue extract from patients with endometriosis containing the PRMT5 inhibitor EPZ015666. As presented in Fig. 5, EPZ015666 significantly inhibited IL-6 secretion and promoted IP-10 secretion from THP-1 cells activated by eutopic endometrial tissue extract. These data indicated that PRMT5 is essential for endometrial tissue extract-induced macrophage activation, suggesting an important role for PRMT5 in mediating the interaction between macrophages and the endometrial microenvironment.

Figure 5 Effect of PRMT5 inhibitor on macrophage activation. THP-1 cells were pretreated with phorbol 12-myristate 13-acetate and then treated with PRMT5 inhibitor EPZ015666 (10 µM) for 24 h, followed by incubation with RPMI-1640 containing homogenized solution of normal and eutopic endometrium (100 µl/ml) for additional 3 days. ELISAs were performed to determine the concentrations of IL-6 and IP-10 in the medium. **P<0.01 (n=15). PRMT5, protein arginine methyltransferase 5; IP-10, IFN-γ-induced protein 10.

PRMT5 expression is upregulated in the peritoneal fluid monocytes of patients with endometriosis

To further investigate the possible role of PRMT5 expression in macrophages in endometriosis, peritoneal monocytes were collected from controls and patients with endometriosis for measuring PRMT5 expression. Previous studies have indicated that among the isolated CD68+ monocytes in peritoneal fluid, 85-95% are macrophages (20,21). As demonstrated in Fig. 6, the protein levels of PRMT5 were significantly increased in the peritoneal monocytes from patients with endometriosis, compared with those in the peritoneal monocytes from controls. However, while the mRNA levels of PRMT5 appeared to also be increased, the difference was not statistically significant.

Figure 6 PRMT5 expression in peritoneal monocytes from patients with endometriosis. Peritoneal monocytes were collected from patients with endometriosis and controls. Reverse transcription-quantitative PCR and western blot analyses were performed to examine the mRNA and protein expression levels of PRMT5 in the cells. β-actin was used as internal control. *P<0.05. n=12 for control and n=25 for EMs. PRMT5, protein arginine methyltransferase 5; EMs, endometriosis.

Discussion

Previous studies have demonstrated that PRMT5 serves a critical role in inflammatory and autoimmune diseases, such as rheumatoid arthritis (14,15,22,24). However, the function of PRMT5 in macrophage activation has not yet been investigated. The present study demonstrated that the levels of cytokines in the endometrial extracts of patients with endometriosis were different from those in the endometrial extracts of control individuals. NF-κB-dependent PRMT5 may contribute to macrophage activation, resulting in the production of the pro-inflammatory mediators IL-6 and IP-10, which likely contributes to the pathogenesis of endometriosis (25).

Currently, the pathogenesis of endometriosis is incompletely understood. A generally accepted concept is the theory of retrograde menstruation, in which regurgitated endometrial cells during menstruation recruit blood monocytes into the peritoneal fluid and secrete large amounts of inflammatory mediators, such as TNF-α, IL-1β, IL-6, IL-8 and VEGF, thereby promoting invasion and angiogenesis of ectopic endometrial implants and subsequent lesion formation (26). It has been indicated that establishment and development of ectopic endometrial lesions are significantly suppressed if peritoneal macrophages are depleted in a mouse model of endometriosis (27), suggesting that interactions between macrophages and the ectopic endometrium may serve a key role in the development of endometriosis.

In the present study, the expression levels of cytokines TNF-α, IL-6 and IL-10 were increased and that of CCL20 was decreased in the endometrial extracts of patients with endometriosis compared with the endometrial extracts of control patients. The mechanisms via which cytokines regulate macrophage activation are relatively complex, as it has been reported that macrophages can be activated and differentiated into distinct subtypes under different cytokine-mediated microenvironments (28). A recent study demonstrated that in response to TNF-α, human endometrial stromal cell-derived IL-6 and monocyte chemoattractant protein-1 stimulated peritoneal macrophages toward M2-polarization, which could modulate endometriosis (29). It was also reported that osteoblast-derived CCL20 stimulates the recruitment of macrophages and T cells (30). In the current study, PRMT5 expression in THP-1-derived macrophages was markedly downregulated following treatment with either serum or extracts of eutopic endometrium from patients with endometriosis compared with the serum or extracts of control endometrium samples, indicating that PRMT5 may be a regulator of macrophage activation in the development of endometriosis. Consistently, overexpression of PRMT5 has been indicated to enhance the expression of major histocompatibility complex class II (MHC II) in macrophages (31). MHC II-dependent antigen presentation to CD4+ T cells orchestrates the interplay among a variety of immune cell types and regulates the humoral and cell-mediated immune responses (32). Therefore, MHC II expression may be inhibited via the downregulation of macrophage PRMT5 in the context of the microenvironment of eutopic endometrial lesions, resulting in a suppressed macrophage-dependent antigen-presenting capacity and an accelerated development of ectopic endometrial lesions.

NF-κB, a major transcriptional factor of the inflammatory response in immunity, has been revealed to be involved in the pathophysiology of endometriosis (33). PRMT5 is responsible for the methylation of Arg30 on the NF-κB subunit p65, and may thereby regulate the expression of NF-κB target genes. In addition, PRMT5 activates NF-κB by binding to TNF-related apoptosis-inducing ligand (34). On the other hand, NF-κB may promote PRMT5 expression in Th cells as an upstream regulator of PRMT5(14), which is consistent with the present finding that the NF-κB inhibitor SN50 suppressed PRMT5 expression in THP-1-derived macrophages treated with extracts of either normal or eutopic endometrium. Further investigation is required to elucidate the molecular mechanisms underlying the interplay between PRMT5 and NF-κB.

EPZ015666 is a selective inhibitor of PRMT5 that can specifically block PRMT5 activity (35). A previous study has revealed that EPZ015666 inhibited IL-6 and IL-8 production by fibroblast-like synoviocytes (24), which supports the present finding that EPZ015666 reduced the secretion of IL-6 while increasing the secretion of IP-10 by THP-1 cells treated with extracts of eutopic endometrium. Both studies verified that PRMT5 possesses proinflammatory properties. Interestingly, the effect of PRMT5 on IP-10 production appears to depend on the cell type involved in the inflammatory response. For example, PRMT5 has been indicated to promote TNF-α-induced IP-10 production by endothelial cells (36).

Previous studies confirmed that 85-95% of the isolated monocytes in peritoneal fluid are macrophages (20,21). To further investigate the possible role of PRMT5 expression in peritoneal macrophages in endometriosis, peritoneal monocytes were obtained from patients and it was indicated that PRMT5 protein expression in peritoneal monocytes was significantly upregulated in patients with endometriosis compared with controls. Possible explanations for this discrepancy are as follows: i) T cells and natural killer (NK) cells are present in peritoneal fluid, although peritoneal macrophages are highly abundant; ii) the cytokines released by ectopic endometrium influence PRMT5 expression in peritoneal monocytes; and iii) the majority of samples were collected from patients with stage III-IV endometriosis. Due to the different immune profiles of eutopic endometrium between endometriosis stages I-II and III-IV, more samples are required from patients with stage I-II endometriosis to further examine the association between PRMT5 expression and the development of endometriosis (37).

Taken together, the results of the present study indicated that eutopic endometrium in patients with endometriosis induced a reduction in PRMT5 expression in THP-1-derived macrophages, resulting in the inhibition of IL-6 production and the increased production of IP-10 (Fig. 7). The mechanism of PRMT5 in endometriosis remains unknown. IP-10 is a key chemokine of Th1 and NK cells and the increase in IP-10 production reduces the number of Th1 and NK cells recruited to the peritoneal fluid (38). The inability of these cells to effectively clear peritoneal endometrial cells results in the successful establishment of endometrial implants (26). In this context, PRMT5 appears to be a novel regulator of macrophage activation.

Figure 7 Schematic diagram illustrating how the eutopic endometrial microenvironment regulates macrophage PRMT5 expression via NF-κB signaling. PRMT5, protein arginine methyltransferase 5; IP-10, IFN-γ-induced protein 10.

Acknowledgements

Not applicable.

Funding

Funding: No funding was received.

Availability of data and materials

The datasets generated and analyzed during the current study are not publicly available due to restricting patient privacy regulations by the different countries but are available from the corresponding author on reasonable request.

Authors' contributions

XW and XC collected and interpreted the patient samples and data. XC and LH performed the experiments of the study. XC and HD analyzed the data. HD designed the study was a major contributor in writing of the manuscript. All authors read and approved the final manuscript. HD and XC confirm the authenticity of all the raw data.

Ethics approval and consent to participate

The present study was approved by the Ethics Committee of The Second Xiangya Hospital of Central South University (Changsha, China). Written informed consent was obtained from all participants.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References