The Formation And Pathogenesis Of IL-10~+ Th17 Cell In Endometriotic Milieu

Endometriosis is a common, complex gynecologic disorder characterized by the presence of endometrial glands and stroma at extrauterine (ectopic) sites. In women who develop this disease, alterations in specific change involving pelvic anatomic abnormalities and peritoneal microenvironment have been observed, which may lead to clinical symptoms, including chronic pelvic pain, dysmenorrheal and infertility. Multiple hypotheses have been proposed regarding the etiology of this disease, especially retrograde menstruation (implantation) theory. However, retrograde menstruation is believed to occur in the majority of fertile women[1]; and only 10% to 15% of women suffering from the disease. Endometriosis is generally considered to be a steroid-dependent disease; however defects of the immune system as well as genetic and epigenetic predisposition probably play equally important role in determining whether an individual will develop this condition [2].It has been reported that the abnormal local immune microenvironment in abdominal from women with endometriosis cannot effectively remove endometrial stromal cells (ESC) of the ectopic lesion. In contrast, it can promote the growth and implantation of ESC. Recent studies suggest that IL-10+Th17 cells have regulatory roles. We found that the peritoneal fluid (PF) of patients with endometriosis had high levels of Th17 cells and IL-17A. Moreover, IL-10 level in patients PF of Ⅲ-Ⅳ phase was higher than that of Ⅰ-Ⅱ phase. The results above suggest that the local microenvironment of women with endometriosis present a coexistence state of pro-inflammatory and tolerance. The initial stage of this disease is pro-inflammatory. On the contrary, such advantage tends to tolerance in the pathogenesis of endometriosis. Further studies showed that ESC of ectopic foci highly expressed IL-27. After co-culture with macrophage, it could induce naive T differentiate to IL-10+Th17 cells. However, the molecular mechanisms and the roles in the development of endometriosis are unclear. Therefore, taking advantage of in vitro experiment imitating the abdominal microenvironment in endometriosis and in vivo animal model, our study intends to investigate the downstream signal pathways and transcription factors which are involved in ESC and macrophage-derived IL-27 on IL-10+Th17 cells differentiation, to further explore whether these Th17 cells promote the progress of endometriosis by secreting IL-17A and IL-10, and to study the pathological mechanisms of the disease.1. The pro-inflammatory and tolerant state coexisted in the endometriotic milieuPeritoneal fluid (PF) and endometrial tissue were collected from patients with or without endometriosis. Immune cells from peritoneal fluid, including macrophages, NK cells, γδ T cells, Thl7 cells and Treg cells, were analyzed by Flow Cytometry (FCM). Compared to non-endornetriosis group, Th17 cells, CD56bright CD16low and D56dimCD16bright natural killer (NK) cells were increased in Ⅰ-Ⅱ stage, with no further increase in III-IV stage. On the contrary, Treg and y5 T cells were raised during the progress of disease, especially in the Ⅲ-Ⅳ stage. Both CD56brightCD16low and CD56dim CD16bright natural killer (NK) cells increased in the Ⅰ-Ⅱstage of endometriosis. CD 14+ macrophages were decreased in Ⅲ-Ⅳ stage.Immune cytokines in peritoneal fluid from patients with or without endometriosis were detected by cytometric bead array (CBA). Multiple cytokines, including INFy, TNFα, IL-6, IL-21, IL-17A, IL-1β, IL-4, IL-6 and TGFβ1, could be detected. Compared with normal fertile women, the peritoneal fluid concentration of TNFa, IL-6, IL-21, IL-17A, IL-1β in patients with endometriosis were increased in the early stage. The peritoneal fluid of women with advanced endometriosis were found to contain abnormally high levels of immune tolerant cytokines, including IL-4 and IL-10.The local microenvironment of women with endometriosis presents a coexistence state of pro-inflammatory and tolerance. The initial stage of this disease, a dominant position is pro-inflammatory, and such advantage tends to tolerance in the advanced stage of endometriosis.2. Endometriotic milieu expressed high level of IL-27The co-culture models of endometriotic-associated cells such as ectopic ESC, monocyte, were used to explore the cytokine feature in vitro. We measured the secretion levels of several cytokines by Bio-Plex Assay. It was found that the ectopic ESCs secreted relative high level of GM-CSF, MCP-1,RANTES, IL-6 and IL-27 in comparison with the normal ESCs, while the eutopic ESCs produced more GM-CSF, MCP-1, RANTES, IL-6. In the co-culture, high level of IL-6, IL-27, TGFβ1, RANTES and GM-CSF were detected in co-culture of the ectopic ESC. LPS simulation increased the production of IL-10, IL-1β1, IL-6, IL-27, TGFβ1, TNFa and GM-CSF, while PGE2 promoted IL-1β1, IL-10, IL-6 secretion.Thereafter, we detected the expression of IL-27p28 by immunohistochemical assay. We demonstrated that IL-27 was highly expressed in normal endometrial tissue and ectopic endometrial tissues, while it was less in eutopic endometrial tissues. We also analyzed IL-27p28 expression on three types of ESCs, which showed the percentage of IL-27p28 expressed on normal ESCs, eutopic ESCs and ectopic ESCs was 11.13%±1.204%,2.379%±0.774% and 34.50%±1.026%, respectively. Different types of ESCs were co-cultured with peripheral monocytes of the same person for 48h. Afterwards, CD14+IL-27p28+ cell were analyzed by Flow Cytometry. IL-27p28 was significantly up-regulated in the ectopic ESC-monocyte co-culture. Meanwhile, monocyte/macrophage increased IL-27p28 expression after co-culture with the ectopic ESCs. On the contrary, IL-27p28 production was lower in monocyte/macrophage after co-cultured with eutopic ESC. Interactions between differentiated macrophages and ectopic ESCs may play an important role in the development of endometriotic tolerant milieu.Moreover, we analyzed the effect of environmental factors on IL-27p28 expression. We found that ovarian steroid hormones and atmospheric pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)promoted IL-27p28 expression in ESCs or monocytes/macrophages, indicating microenvironment factors also made contributions to the formation of tolerant milieu.3. Ectopic ESCs or rhIL-27 treated monocytes/macrophages inhibited Th17 cell differentiation, but specially promoted IL-10+Th17 cells development.We next built the "M-E" and "M-T" co-culture model. Flow cytometry was used to analyze the percentage of total IL-17A+Th17 cells or IL-10+Thl7 in each co-culture system.In direct co-culture, the monocytes cocultured with eutopic ESCs induced more CD4+IL-17A+Thl7 cells compared with that of the normal ESCs, while the monocytes co-cultured with the ectopic ESCs induced less CD4+IL-17A+Th17 cells. In the following, we analyzed the effect of IL-27 in Th17 cell differentiation and found IL-27 inhibited the differentiation of IL-17A+Th17 cells, but specifically increased IL-10+IL-17A+Th17 cells.When rhIL-17or IL-27 antibody was added to the "M-E" co-culture, we found the rhIL-27-treated monocytes/macrophages inhibited the differentiation of Th17 cells, but increased more IL-10+Th17 cells. In the same way, we treated monocyte with PBS, LPS, PGE2 and rhIL-27, then analyzed the development of Thl7 cell induced by these treated monocytes. Data showed the rhIL-27-treated monocytes/macrophages inhibited the differentiation of total Th17 cells, but specially increased the differentiation of IL-10+Thl7 cells. While LPS and PGE2 had no such effect.4. Monocytes/macrophages increased IL-2 expression via IL-27 and tend to M2 phenotypeIL-27-IRES2-EGFP recombinant plasmid were successfully established and efficiently transfected into ESCs. Subsequently, we built "M-E" and "M-T co-culture model. Monocytes/macrophages and T cells from co-culture models were analyzed by microarray. IL-27 was highly up-regulated in the monocytes/macrophages cocultured with the IL-27 over-expressed ESCs. Also, some pro-inflammatory molecules and MMPs were down-regulated in these monocytes/macrophages, which tend to M2 phenotype. Most important of all, monocyte/macrophage expressed high level of IL-2 and IL-27RA in coculture with the over-expressed ESCs. For monocytes/macrophages differentiation, neutralizing antibody were added to antagonize EL-27 or IL-2. It was also demonstrated that IL-2 secretion was regulated by IL-27 during monocyte/macrophage differentiation.5. The down-regulation of IKZF3 in T cells mediated by IL-2 impaired Thl7 cell differentiationT cells from "M-T" coculture model were analyzed by microarray. We found that Th17 cell related genes, including IL-21, RORA, IL-17A, IL17C, IL-17F, IL-23R, CCR6 and IKZF3, were inhibited in the IL-27 over-expressed ESCs. On the contrary, some immune tolerant molecules and chemokines, such as CCL2, CCL8 and CCL24 were increased. In addition, we built signal network demonstrating the interaction regulatory network between monocytes/macrophages and T cells. We found that IL-2 from monocyte/macrophage up-regulated IKZF3 in T cell.In order to verify the relationship between IL-2 and IKZF3 in T cells, we treated monocyte/macrophages with IL-27 or IL-2 neutralizing antibody, and then built "M-T" co-culture. Flow Cytometry or ELISA were used to analyzed the IL-2 expression and Real time PCR were used to detect the mRNA level of ikzf3 which participate in the differentiation of helper T cells into the TH17 subset. We found neutralization of IL-27 in "M-E" co-culture could decrease IL-2 secretion. T cell induced by monocytes/macrophages from IL-27 or IL-2 neutralization expressed high level of ikzf3. Based on the IntAct database, we proposed that there was a negative regulation between IKZF3 (Aiolos) and Foxp3.IL-2 appeared to affect the imbalance of Th17/Treg differentiation via regulating IKZF3 (Aiolos) expression.Taken together, the transcription factor IKZF3 (Aiolos) was down-regulated in T lymphocytes in a manner dependent on the interleukin 2 from endometriotic milieu and lead to depression of Th17 cell differentiation. This acted as a cell-cell interactive safeguard mechanism for the differentiation of helper T cells into the Th17 subset.6. rhIL-27 directly inhibited Th17 cell differentiation but specially promoted IL-10+Th17 cells development.Naive T cells were stimulated directly with human recombinant IL-6, TGF-β1,IL-27 or different cytokines combinations for a period of 5 days in the presence of rhIL-2. Flow Cytometry were used to analyzed the differentiation of IL-10+T cells and Th17 cells. It was been demonstrated that TGFβ1, IL-27 or IL-27 added IL-6 significantly promoted CD4+IL-10+T cells in vitro; IL-27 effectively restrained Thl7 cells development induced by IL-6 plus low level of TGFβ1, but promoted IL-10+IL-17A+T cell differentiation. IL-27 also depressed the differentiation of Th17 cells induced by GM-CSF. In addition, IL-27 improved PD-L1 expression on T cells. These data suggest IL-27 might mediate immune tolerance deviation by inducing more IL-10+Th17 cell or anergic T cells.We built mice model of i.p. endometriosis and analyzed Th17 cell from endometriotic lesion by flow cytometry. The results showed antagonism of IL-27 by intraperitoneal injection with IL-27 neutralizing antibody could significantly decrease the 1L-10+IL-17A+T cell and IL-10+IL-17F+T cell differentiation.7. IL-27 mediated IL-10+Th17 differentiation via c-MAF/PRDM1/IL-10 pathwayNaive T cells were cultured under Th polarization conditions, respectively. Subsequently, transcription factor c-MAF was detected by flow cytometry. We discovered c-MAF expression in IL-10+Th17 was higher than that of IL-10"Thl7 cells. In order to elucidate the molecular mechanism in IL-10+Th17 cell differentiation, Th17 cell and IL-10 associated genes, including BLIMP1 (PRDM1), RORC, c-MAF, IL-17A and LAG3, were as candidate genes according to reports in the literatures. Recombinant luciferase reporter plasmids were constructed by inserting the promoter segments of candidate genes into the pGL(3)-promoter plasmid immediately upstream of the luciferase gene. The IL-27-IRES2-EGFP expression plasmid was constructed in the IRES2-EGFP vector (Invitrogen, USA) by standard molecular biology techniques and then confirmed by sequencing. Similarly, the RORC-3FLAG and c-MAF-3FLAG expression plasmid were respectively constructed in the CMV-MCS-3FLAG-SV40-Neomycin vector (Invitrogen, USA) and transfected into the human HEK 293T cell line. It was found that transfection of IL-27 recombinant over-expression plasmid transactivated BLMP1 (PRDM1), LAG3, IL-17A, c-MAF promoter expression, but inhibited RORC expression; c-Maf induced a strong BLIMP1 (PRDM1) and LAG3 reporter gene response, RORC transactivated BLIMP1 (PRDM1), LAG3, IL-17A, c-MAF promoter expression. Taken together, these results demonstrate that during Thl7 polarization, Thl7 cell related RORC and IL-17A expression significantly upregulated, negative factor c-MAF also were simultaneously transactivated. IL-27 and c-MAF strongly induce BLIMP1 expression.Afterwards, we verified the relationship candidate genes by quantitative real-time PCR. It was showed that Rorc was strongly induced under Th17 polarization condition, but il10 was suppressed; IL-27 alone or Th17 polarization condition added by IL-27 unaffected Rorc expression, but promoted c-MAF, prdml and il10 expression. In brief, when T cells were in Th17 polarizing conditions, il10 was suppressed, but ill 7a was induced via Rorc. When IL-27 emerged Th17 polarizing conditions, c-MAF, Prdml and il10 was greatly induced, whereas Rorc expression was unaffected. This finding suggests that IL-27 could increase IL-10+Thl7 differentiation during Th17 polarization. Moreover, IL-27 cannot inhibit IL-17A production after initiation of the Th 17 program but could still regulate pathogenicity of Th17 cells through induction of IL-10 production.Based on several databases, we also modeled the regulatory networks of IL-27 related genes It has been known that RORC induced by IL-6 and TGFβ could promote IL-17A expression under Th17 polarizing conditions. RORC partially activated c-MAF, which were associated with IL-10 expression. When IL-27 polarized naive T cells, gp130 and WSX-1 mainly inhibited RORC expression via activating STAT1, or partially inhibited RORC expression through c-MAF, whereas inhibited Thl7 cell differentiation. Meanwhile, IL-27 also phosphorylated MAPK and induced PRDM1 through AP-1/c-MAF transcription factors, which strongly promoted IL-10 secretion and Trl differentiation. If IL-6, TGFβ and IL-27 coexisted, IL-27 might enhance the c-MAF/PRDM1 pathway, but unaffect IL-17A secretion.This finding suggests that IL-27 cannot inhibit IL-17A production after initiation of the Th17 program but could still induction of IL-10 production through induction of c-MAF/PRDMl pathway or STAT1 pathway.8. IL-10+ Th17 cells play a vital role in the progress of endometriosisIL-17AR expression was analyzed by flow cytometry. Data show that different ESCs express high level of EL-17AR. Normal ESCs, eutopic ESCs and ectopic ESCs were respectively treated with human recombinant IL-17A. The pro-inflammary cytokine rhIL-17A enhanced cell viability and invasive ability of all ESCs. Furthermore, rhIL-17A promoted the apoptosis of normal ESCs, while it reduced the apoptosis of eutopic and ectopic ESCs. On the contrary, rhIL-17A significantly decreased the adhesive ability of the three types of ESCs, which might promote ESCs dissemination in peritoneal cavity.The pro-inflammatory cytokine IL-17A and immune tolerant cytokine IL-10 could further promote the cell viability and invasive ability of normal ESCs, which suggests that pro-inflammatory cytokine in the I-II stage of endometriosis mainly promoted the implantation and growth of ESCs. With the progress of the disease, increased IL-10 worked synergistically with IL-17A to boost the invasive ability and cell viability of ESCs. In other words, IL-10+ Th17 cell associated cytokines promoted the formation of ectopic lesion and progression of endometriosis.We built mice model of i.p. endometriosis. Th17 cell from endometriotic lesion were analyzed by flow cytometry, and we found that antagonism of IL-10 and IL-17 specifically reduced the endometriotic lesion volume.