1-Methyl-tryptophan attenuates regulatory T cells differentiation due to the inhibition of estrogen-IDO1-MRC2 axis in endometriosis

Foxp3+ regulatory T (Treg) cells contribute to the local dysfunctional immune environment in endometriosis, an estrogen-dependent gynecological disease, which affects the function of ectopic endometrial tissue clearance by the immune system. The reason for the high percentage of peritoneal Treg in endometriosis patients is unknown. Here, we show that the proportion of peritoneal Treg cells increases as endometriosis progresses. To determine the probable mechanism, we established a naive T cell-macrophage-endometrial stromal cell (ESC) co-culture system to mimic the peritoneal cavity microenvironment. After adding 1-methyl-tryptophan (1-MT), a specific inhibitor of indoleamine 2,3-dioxygenase-1 (IDO1), to the co-culture system, we found that the differentiation of Treg cells, mainly IL-10+ Treg cells, decreased. Therefore, 1-MT-pretreated ESCs-educated Treg cells performed impaired suppressive function. Moreover, estrogen promoted the differentiation of Treg cells by elevating IDO1 expression in the ectopic lesion. Subsequently, we examined mannose receptor C, type 2 (MRC2), which is an up-stream molecule of IL-10, by bioinformatics analysis and real-time PCR validation. MRC2 expression in ectopic ESCs was notably lower than that in normal ESCs, which further negatively regulated the expression of IDO1 and Ki-67 in ESCs. Furthermore, MRC2 is required for Treg differentiation in the ectopic lesion, especially that for CD4high Treg. Therefore, MRC2-silenced ESCs-educated Treg manifested a stronger suppressive function in vitro. Consistently, the percentage of Treg increased when MRC2-shRNA was administered in the peritoneal cavity of endometriosis-disease mice model. Besides, 1-MT improved the condition of endometriosis, in terms of reducing the number and weight of total ectopic lesions in vivo. These results indicate that the estrogen-IDO1-MRC2 axis participates in the differentiation and function of Treg and is involved in the development of endometriosis. Thus, blockage of IDO1 in the ectopic lesion, which does not influence physiological functions of estrogen, may be considered a potential therapy for endometriosis.

Under the influence of various factors, sloughed endometriallike tissue in retrograde menstruation reaches the peritoneal cavity and adheres to endoabdominal structures to form ectopic lesions, resulting in dysmenorrhea, chronic pelvic pain and infertility, referred to as endometriosis (EMS). 1,2 At first, endometriosis was considered a benign, estrogen-dependent gynecological disease. However, it has been subsequently recognized as not only an endocrine disorder, but also a chronic inflammatory condition.
To date, three main aspects have been noted in the pathogenesis of endometriosis. Firstly, with regard to endometrial tissue, a lot of genes are differentially expressed in the ectopic endometrium compared with that in the eutopic and normal endometrium, 3,4 which may play pivotal roles in the development of endometriosis. We previously showed that the expression of indoleamine 2,3-dioxygenase-1 (IDO1), a ratelimiting enzyme that catalyzes the synthesis of tryptophan, is higher in ectopic endometrial stromal cells (ESCs) than that in normal ESCs. Additionally, IDO1 suppresses T-cell responses, promotes immune tolerance, and influences the differentiation of regulatory T (T reg ) cells. 5 We found that IDO1 promotes survival, proliferation, and invasion of ESCs via the JNK signaling pathway, but inhibits apoptosis of ESCs. 6 The second aspect involved is abnormal endocrine function. High expression of mitochondrial cholesterol side-chain cleavage enzyme (CYP11A1) and hydroxysteroid (17 beta) dehydrogenase (HSD17B) in ectopic endometrial tissue increases local estrogen levels, which influences biological activities of ESCs, 7,8 leading to the development of endometriosis. Finally, the immunological aspect has been implicated, owing to evidence of immune tolerance in the endometriosis microenvironment, which is affected by changes in the proportion of T reg cells, 9 which in turn plays an important role in the maintenance of immune homeostasis to prevent potentially severe autoimmunity. 10,11 It has been reported that the percentage of T reg cells in the peritoneal fluid of patients with endometriosis is higher than that in healthy women, 12 and that Foxp3 expression by T reg cells and T reg function is increased in estrogen-treated mice. 13 However, the exact mechanisms are unknown.
Considering the higher peritoneal T reg cell percentage in EMS patients than that in healthy women, as well as findings that IDO1 controls T reg cell function in response to inflammatory stimuli, 14,15 and higher expression of IDO1 in ectopic lesion regulates biological activities of ESCs in endometriosis, 6 we reasoned that excessive estrogen may regulate IDO1 expression in the ectopic lesion to induce T reg cell differentiation. In this study, we explored the origin of excessive T reg cells in the peritoneal fluid of patients with endometriosis. To our knowledge, this is the first report showing that mannose receptor C, type 2 (MRC2), which is related to collagen turnover 16 and cancer prognosis, 17,18 plays a vital role in T reg cell differentiation and function in endometriosis. Clinically, our findings may provide evidence that 1-methyl-tryptophan (1-MT) has potential applications in the treatment of endometriosis, retaining the physiological functions of estrogen.

Results
Peritoneal T reg increases as endometriosis progresses. The percentage of T reg cells in peritoneal fluid is higher in patients with endometriosis than in healthy women. 12 As shown in Figure 1a and b, the proportion of peritoneal T reg cells in EMS stage III/IV patients was significantly higher than that in EMS stage I/II patients and healthy women. TGF-β 1 expression in peritoneal T reg cells also showed a similar trend (Figure 1c and d); however, IL-10 expression in T reg cells did not (data not shown). These data demonstrated that the percentage of peritoneal T reg cells increases with exacerbation of endometriosis.
IDO1 participates in the differentiation of T reg cells in endometriosis. To verify whether IDO1 influences the differentiation of T reg cells in the ectopic lesion, we established co-culture systems to mimic the microenvironment of the peritoneal cavity (Supplementary Figure 1) and detected T reg cell differentiation and expression of functional molecules in T reg cells when treated with 1-MT or not. The Values indicate mean ± S.D., n (Control) = 6, n (EMS stage I/II) = 6, n (EMS stage III/IV) = 6, ***Po0.001, ****Po0.0001, two-tailed, unpaired t-test. EMS I/II, stage I and II of endometriosis; EMS III/IV, stage III and IV of endometriosis highest percentage of T reg was found in the naive T cellmacrophage-ESC co-culture system, compared with that in two other co-culture systems (Figure 2a and b). Therefore, we used this co-culture system for further analysis.
After adding 1-MT, a specific inhibitor of IDO1, to the co-culture system, the differentiation of T reg cells decreased significantly, especially that of IL-10 + T reg cells (Figure 2c and d), whereas, TGF-β 1 + T reg cell remained unchanged (data not shown). Moreover, 1-MT-pretreated ESCs-educated T reg performed less suppressive function, as the divided percentage of CD4 + CD25 − (T eff ) cells was higher than that of E+T reg group, while ESCs-educated T reg cells owned a more suppressive function compared with non-pretreated-T reg (Figure 2e and f). These results indicate that IDO1 is involved in the differentiation and suppressive function of T reg cells in endometriosis. To investigate whether IDO1 participates in the differentiation of T reg cells in endometriosis in vivo, we established an endometriosis-disease mouse model (Supplementary Figure 2). The results showed that 1-MT distinctly inhibited the percentage of T reg cells in peritoneal fluid of mice (Figures 2g and h), especially IL-10 and CD 73 expression in peritoneal T reg cells (Figures 2i-k), which were consistent with the in vitro results.
IDO1 is up-regulated by estrogen in the ectopic lesion. Patients with endometriosis show high local estrogen levels. 7 Additionally, IDO1 expression in ectopic ESCs is higher than that in normal ESCs, 6 leading us to consider that estrogen may regulate the expression of IDO1 in the ectopic lesion. We found that IDO1 expression in estrogen-conditioned ESCs and estrogen-conditioned macrophages were obviously higher than that in the control groups (Figures 3c-f). Besides, the effect of ESCs on up-regulating the expression of IDO1 in macrophages was more significant than that with estrogen alone (Figures 3e and f), which indicates a crosstalk between ESCs and macrophages that related to IDO1 expression.
To identify which sub-unit estrogen receptor (ER) that allows estrogen to promote the expression of IDO1 in ESCs, we blocked ER α , ER β , and ER respectively. We found that IDO1 expression in ESCs was down-regulated regardless of whether a single or both sub-unit ERs were blocked. This suggests that both sub-units of ER are involved in this activity, especially ER β (Figures 3g and h). Although the percentage of T reg cells in ectopic lesions of the estrogen receptor inhibitor (ER i ) group showed little changes in vivo (data not shown), the percentage of TGF-β 1 + , IL-10 + , CD73 + , and CTLA-4 + T reg cells decreased (Supplementary Figure 3).
Estrogen induces the differentiation of T reg cells via IDO1. Considering that endometriosis is an estrogendependent disease, and that estrogen enhances Foxp3 expression and T reg cell function, 13 we explored whether estrogen regulates the differentiation of T reg cells in endometriosis. After adding estrogen to the co-culture system, the differentiation of T reg cells notably increased (Figures 4a  and b), which indicates that local high estrogen level participates in inducing the differentiation of T reg cells in the peritoneal fluid of patients with endometriosis.
As mentioned above, estrogen promotes the expression of IDO1 in the ectopic lesion, and IDO1 influences the differentiation of T reg cells, 5 raising the possibility that estrogen may promote the differentiation of T reg cells via up-regulation of IDO1 expression. Compared with that in the estrogen group, the differentiation of T reg cells in estrogen plus 1-MT group was lower (Figures 4a and b), which manifested that 1-MT inhibits T reg cell differentiation induced by estrogen.
MRC2 is a downstream molecule of IDO1 and negatively regulates IDO1. As outlined above, IDO1 influences the differentiation of T reg cells, especially IL-10 + T reg cells. To identify factors that possibly connect IDO1 and IL-10, and are involved in interactions of IL10, IDO1, Foxp3, and TGF-β, we performed a search of the KEGG database as part of the bioinformatics analysis. We found that molecules up-stream of IL-10 include C-type lectin domain family 4 member M (CLEC4M), mannose receptor C, type 1 (MRC1), mannose receptor C, type 2 (MRC2), STAT6, phospholipase A2 receptor1 (PLA2R1), platelet-activating factor receptor (PTAFR), and CD209 ( Figure 5a). After treating ESCs with estrogen, 1-MT, or estrogen plus 1-MT, only MRC2 expression increased in the estrogen-treated group compared with that in the control group (Figure 5b), which was similar to our previous result where estrogen up-regulated IDO1 expression in ESCs (Figures 3c and d). Thus, the results demonstrate that 1-MT also promotes the expression of MRC2 in ESCs to a greater extent than estrogen (Figures 5b and c), which means that MRC2 is downstream to IDO1 and estrogen.
MRC2 is a constitutively recycling endocytic receptor belonging to the mannose receptor family. 19 The expression of MRC2 in ectopic ESCs was significantly lower than that in normal ESCs (Figure 5d). Combined with evidence that the expression of IDO1 in ectopic ESCs is higher than that in normal ESCs, 6 and 1-MT up-regulates the expression of MRC2, we reasoned that high levels of IDO1 might lead to low expression of MRC2 in ectopic ESCs. Besides, after silencing MRC2 in ESCs, the expression of IDO1 increased (Figures 5e and g), which indicates a negative feedback between MRC2 and IDO1. Therefore, the level of Ki-67 in MRC2-silenced ESCs increased compared with that in the vector group (Figures 5e and f), which is consistent with the notion that ectopic ESCs have a stronger ability for proliferation. 6 Similarly, the expression of Ki-67 and IDO1 also increased in ectopic lesions after MRC2 shRNA was intraperitoneal injected to the peritoneal cavity of endometriosis mice model in vivo (Figures 5h and i).
MRC2 is required for the differentiation of T reg cells in endometriosis. According to the findings above, MRC2 is downstream to IDO1, and IDO1 is involved in the differentiation of T reg in ectopic lesion, hinting the possibility that MRC2 may participate in the activity that IDO1 regulates the differentiation of T reg in endometriosis. When MRC2silenced ESCs were co-cultured with naive CD4 + T cells and monocytes-derived macrophages, the percentage of CD4 low T reg and CD4 high T reg cells were higher in the MRC2silenced group than that in the vector group, especially CD4 high T reg cells (Figures 6a and b). Moreover, CD4 high T reg cells from the MRC2-silenced group showed a more immunosuppressive phenotype, with higher expression of TGF-β1, IL-10, CD39 and CTLA-4, than that in CD4 high T reg cells in the vector group and CD4 low T reg cells in MRC2-silenced group (Figures 6c-e). Furthermore, MRC2-silenced ESCs-educated T reg performed stronger suppressive function, as divided percentage of T eff cells was lower than that of vector group, which indicated that

MRC2 is involved in T reg suppressive function (Figures 6f and g).
In vivo, at two weeks after administrating MRC2-shRNA via the peritoneal cavity of the endometriosis-disease mouse model, the percentage of peritoneal T reg cells and T reg cells in ectopic lesions were significantly higher than that in the vector group (Figures 6h, i, l and m), and mainly comprised TGF-β1 + and CTLA-4 + T reg cells in the peritoneal fluid (Figures 6j and k). These results demonstrate that MRC2 is not only responsible for the differentiation of T reg cells, mainly that of CD4 high T reg cells; but also promotes T reg immunosuppressive function in endometriosis, which may exacerbate the development of endometriosis.

1-MT reverses the development of endometriosis in vivo.
To determine whether 1-MT improves the condition of endometriosis in vivo, we measured the total number and weight of ectopic lesions and found that both parameters decreased notably in the 1-MT administered group compared with that in the control group, as similar as ERi administered group (Figures 7a, b and d). In the   MRC2-shRNA administered group, the total number of ectopic lesions did not significantly increased (Figure 7c), but total weight of ectopic lesions was obviously higher than that in the vector group (Figure 7e), which indicates that MRC2 is responsible for ectopic lesion growth while not for the spread of the implant. Moreover, 1-MT notably restrained the proliferation of ectopic lesions in vivo, in terms of lower expression of Ki-67 compared with that in the control group, as seen with the ER i group (Figures 7f and g). These results suggest that 1-MT reverses the condition of endometriosis in vivo.

Discussion
Although endometriosis is an estrogen-dependent disease, it is not enough to determine the pathogenesis of endometriosis from the endocrine perspective alone. Multiple factors participate in the development and maintenance of endometriosis, including immunological dysfunction, genetic susceptibility, psychological factors, and environment factors. Among these, the immunological aspect of endometriosis has recently been extensively studied. Up to 90% of women of reproductive age exhibit retrograde menstruation, whereas only 6-10% of these women develop endometriosis. 20 This indicates that a microenvironment of immune tolerance is formed within the ectopic lesion in patients with endometriosis, where the function of menstrual debris clearance by the local immune responses is ineffectively. 21 Several types of immune cells are involved in the formation of the local immune tolerance environment in endometriosis, such as T reg cells, macrophages, and natural-killer (NK) cells. Disturbance of T reg cells, which are responsible for selftolerance, maintenance of immune homeostasis, and immunosuppressive functions, 22 aggravates the condition of endometriosis. In this study, we have demonstrated not only that the percentage of T reg cells in the peritoneal fluid of endometriosis patients higher than that in healthy women, which is consistent with results of previous published studies, 23 but also that the augmentation of T reg cell percentage, especially that of TGF-β1 + T reg cells, occurs in parallel with endometriosis exacerbation. This suggests that the detection of peritoneal T reg cells may be used as an indicator to assess the severity of endometriosis. However, the origin of excessive T reg cells in the peritoneal fluid of patients with endometriosis is yet unclear. Three assumptions may elucidate this clinic phenomenon. One is homing of peripheral T reg cells to the peritoneal fluid as the percentage of T reg cells decreases in the peripheral blood of EMS patients. 23 The second is that local naive T cells differentiate into T reg cells under the influence of various factors involved; and the third is self-proliferation of local T reg cells. In fact, all of these assumptions may be involved in the increase of peritoneal T reg cell percentage in endometriosis.
IDO1 is essential for the generation and function of T reg cells. 14,24,25 Initially, IDO1 was thought as the first line of host defense against infectious pathogens, as it causes tryptophan shortage, which in turn restricts mammalian cell growth, 26 especially that of T cells. 27 Subsequent studies revealed that IDO1 has multiple immunological functions, such as suppressing T-cell responses, regulating functions of T reg cells, 14 and promoting immune tolerance. 15 Evidence of higher IDO1 expression in ectopic ESCs than in normal ESCs 6 and high percentage of peritoneal T reg cells in endometriosis 23 demonstrates that IDO1 may play an important role in inducing the differentiation of T reg cells in the ectopic lesion, which is a probable reason for excessive T reg cells in the peritoneal fluid of patients with endometriosis. In this study, we established a naive T cell-macrophage-ESC co-culture system to mimic the local microenvironment of the peritoneal cavity. The results of our study show that IDO1 participates in the differentiation of T reg cells in the ectopic lesion, evidenced by inhibition of T reg cell differentiation, especially that of IL-10 + T reg cells, by 1-MT. In addition, IDO1 regulates T reg differentiation in EMS C-Y Wei et al 1-MT-pretreated ESCs-educated T reg cells suppressed the proliferation of T eff cells less effectively compared with nontreated ESCs-educated T reg cells, which indicates that 1-MT is involved in T reg cell suppressive function. Moreover, estrogen promoted the expression of IDO1 in both ESCs and macrophages, which, on a side-note, explains that ectopic ESCs have high expression of IDO1 in endometriosis. Therein, the expression of IDO1 in ESCs-educated macrophages was notably higher than that in both estrogen-treated and untreated monocytes-derived macrophages. This demonstrates a crosstalk between ESCs and macrophages, involving IDO1. Besides, estrogen was found to induce T reg cell differentiation in the ectopic lesion and IDO1 was involved in this process, wherein, 1-MT down-regulated T reg cell differentiation induced by estrogen. These results indicate that estrogen regulates the expression of IDO1 in the ectopic lesion to induce the T reg cell differentiation. Based on the finding that 1-MT mainly influences the differentiation of IL-10 + T reg cells; we performed a search of the KEGG database to identify possible molecules that connect IDO1 and IL-10. The results show that MRC2, which is an upstream molecule of IL-10, is essential for the differentiation and function of T reg cells. Most researches on MRC2 to date have focused on its role in the development of cancer, as it can promote breast tumor growth, 17 co-operate with the matrix metalloproteinase to remodel of extracellular matrix that attenuate renal fibrosis, 28 and predict prognosis of hepatocellular carcinoma 18 and prostate cancer. 29 Besides, MRC2 is also closely related to collagen turnover. 30 In our study, we found that both estrogen and 1-MT promoted the expression of MRC2 in ESCs, which indicates that MRC2 is downstream to estrogen and IDO1. Therein, the regulatory ability of 1-MT was stronger than that of estrogen. Combined with the low expression of MRC2 by ectopic ESCs and MRC2 expression promoted by estrogen, we reasoned that other factors might be responsible for decreased MRC2 expression by ESCs. As shown in Figures 5b and c, we observed that 1-MT promoted the expression of MRC2 by ESCs to a greater extent than estrogen, whereas, decreased IDO1 levels caused an increase in MRC2 expression. Therefore, it is a possibility that increased IDO1 levels down-regulate the expression of MRC2 by ESCs. In addition, negative feedback was noted between IDO1 and MRC2, evidenced by increase in IDO1 expression when MRC2 in ESCs was silenced. Meanwhile, MRC2 negatively regulated proliferation of ESCs as the expression of Ki-67 increased in si-MRC2 ESCs compared with vector group. To explore the possibility that MRC2 influences the differentiation of T reg cells, we co-cultured MRC2-silenced ESC with macrophages and naive CD4 + T cells. Compared with that in the vector group, the percentage of T reg cells increased in the MRC2-silenced group, especially CD4 high T reg cells, which demonstrate that MRC2 plays a key role in the differentiation of T reg cells in endometriosis. Therefore, MRC2-silenced ESCs-educated T reg cells acquired stronger suppressive function than vector-pretreated ESCseducated T reg cells, which indicates that MRC2 negatively regulates T reg cell suppressive function. To our knowledge, this is the first report demonstrating the immunological functions of MRC2 as an important regulator of T reg cell differentiation and function, and showing that estrogen and IDO1 are up-stream to MRC2. These results illustrate that estrogen-IDO1-MRC2 axis is involved in the differentiation of T reg cells in endometriosis.
Consistent with the in vitro results above, 1-MT dramatically reversed the condition of endometriosis in vivo, irrespective of total number or weight of ectopic lesions, expression of Ki-67 in total ectopic lesions, or the percentage of peritoneal T reg cells. These data suggest that locally applied 1-MT may clinically relieve sufferings of patients with endometriosis. Besides, MRC2 significantly induced the differentiation of T reg cells, increased weight of total ectopic lesions, and promoted the expression of Ki-67 in ectopic lesions in vivo, which is consistent with the in vitro results, suggesting that MRC2 is involved in the growth of ectopic lesions and T reg differentiation in endometriosis (Supplementary Figure 4).
Although endometriosis is a benign gynecological disease, its biological activities are similar to that of cancer, including metastasis, plantation, angiogenesis, immune tolerance, and recurrence. 31,32 In the present study, we focused on IDO1, MRC2, and T reg cells, which have been reported in previous cancer studies, 17,[33][34][35] to identify the endocrine-immune mechanism for the high percentage of peritoneal T reg cells in patients with endometriosis. The results revealed that the percentage of T reg cells in the peritoneal fluid increases as endometriosis progresses. Therein, we found that the lower expression of MRC2 in ectopic ESCs significantly promoted the differentiation and function of T reg cells. This is a novel function of MRC2, identified in this study. Future work would involve identifying probable mechanisms by which MRC2 influences the differentiation of T reg cells, including the transcription factors involved in this process and interactions of MRC2 and T reg functional molecules. Moreover, the possibility that MRC2 influences the biological activities of ESCs in endometriosis remains to be explored. Common treatment options for endometriosis, including progestogens, ovulation induction, GnRH analog and surgery, however, typically do not provide long-term relief. 36 Pharmacotherapy for endometriosis affects the physiological functions of estrogen and this limits its long-term use. In addition, high recurrence rate renders endometriosis clinically intractable. Therefore, to treat endometriosis, it is essential to identify possible targets that do not influence the physiological functions of estrogen. The in vivo results from this study showed that 1-MT, a specific inhibitor of IDO1, can notably improve the condition of endometriosis, as similar as ER inhibitor, which provide evidences that blocking IDO1 in ectopic lesions may be a novel treatment option for endometriosis, not only by virtue of its effects on biological activities of ESCs, but also on local immune-tolerance environment effected by inhibition of the differentiation and functions of T reg cells. To date, 1-MT is undergoing clinical phase II and I trials for application in cancer immunotherapy. 15 Intrauterine devices containing 1-MT can be considered for the treatment of endometriosis. These data indicate that endometriosis is more than a disease involving endocrine disorders, and also involves immunological factors. Moreover, it should be taken into consideration that assessment of the local immunological status of patients with endometriosis is essential for the treatment choice to be made. ESCs isolation. Endometrial stromal cells (ESCs) were purified as previously described. 37 Endometriotic tissues were cut intoo1mm-thick sections and digested with Dulbecco's Modified Eagle Medium (DMEM)/F12 (Hyclone, Logan, UT, USA) containing collagenase type IV (0.1%; Sigma, San Francisco, CA, USA) with constant agitation for 40 min at 37°C. The resulting suspension was then filtered through 100-to 70-μm nylon strainers (Becton Dickinson, Franklin Lakes, NJ, USA). After the filtrate was centrifuged at 1600 r.p.m. for 9 min at 4°C, the supernatant was discarded. Finally, ESCs were resuspended in DMEM/F-12 containing 10% fetal bovine serum (FBS; Hyclone) in the presence of 100 U/ml penicillin and 100 mg/ml streptomycin, and placed in culture flasks at 37°C under 5% CO 2 . The culture medium was replaced with fresh medium every 3 days.
Generation of human naive CD4 + T cells and monocytes. Peripheral blood mononuclear cells (PBMCs) were isolated by Lymphoprep (Stemcell Technologies Inc., Vancouver, BC, Canada) density gradient centrifugation. CD14 + cells were obtained by positively selection using CD14 + cells micro-magnetic beads according to the manufacturer instructions (Miltenyi Biotec, Bergisch Gladbach, Germany). Naïve CD4 + T cells were obtained by negatively selection from the remaining immune cells according to the manufacturer instructions (Miltenyi Biotec). The purity of CD14 + cells was confirmed by flow cytometry, using PE/Cy7 anti-human CD14 monoclonal antibody (mAb) (Biolegend, San Diego, USA, clone: HCD14), was found to be495%.
Cell co-culture systems. Ectopic ESCs were cultured in 24-well plates (Corning, Steuben County, NY, USA) at a density of 1 × 10 5 cells/well. The co-culture systems were established by incubating 2 × 10 5 monocytes with ESCs or alone, adding of estrogen (   Western blot. Protein expression level of MRC2 and β-actin were measured by western blot as previously described. 38 The primary antibodies used were anti-MRC2 mAb (1:1000; Abcam Cambridge, MA, USA), and β-actin rabbit mAb Mice. We used seven-week-old female C57B/L6 mice (Slaccas Animal Laboratory, Shanghai, China) to establish an endometriosis-disease model as previously described. 39,40 Each donor mouse was administered 200 μl estrogen (10 − 8 M; Sigma) via the peritoneal cavity. One week later, donor mice were sacrificed and the uterine horn were collected and minced. Every two recipient mice were then intraperitoneally injected with minced uterine horn tissue from one donor mouse equally. All procedures were performed under aseptic conditions. One week after uterine tissue injection, mice were randomly divided into five groups and each received an intraperitoneal injection of 200 μl PBS (Ji-Nuo), estrogen receptors blockers (10 − 6 M; Sigma), 1-MT (0.05 mM; Sigma), vector (GenePharma, Shanghai, China), or mouse MRC2 shRNA (GenePharma, Shanghai, China) every week. Vector and mouse MRC2 shRNA were mixed with Lipofectamine 3000 (Invitrogen) and OPTI-MEM (Gibco BRL), according to the manufacturer protocol. Two weeks later, we measured the total number and weight of ectopic lesions, the expression of Ki-67 and IDO1 within ectopic lesions, the percentage and the expression of functional molecules of T reg in total ectopic lesions and peritoneal fluid of mice from groups administered PBS, estrogen receptor blockers, 1-MT, vector and MRC2 shRNA (Supplementary Figure 2).
Statistics. All studies were set up to include three wells per condition, and each experiment was independently repeated more than three times. Data collected from each independent experiment were analyzed using the Graphpad Prism (Graphpad software Inc., La Jolla, CA, USA) statistical package. Paired t-test of variance was performed when appropriate. Differences were considered statistically significant at ± S.D.o0.05.