title: Synergistic effect of regulatory T cells and proinflammatory cytokines in angiogenesis in the endometriotic milieu.
abstract: Do regulatory T cells (Tregs) contribute to angiogenesis in endometriosis?
  
  
  
  High levels of CCL17 and CCL22 cause the recruitment of Tregs, upregulate the immunosuppression of Tregs and, in turn, may promote angiogenesis in endometrial cells in synergy with proinflammatory cytokines.
  
  
  
  The peritoneal fluid of patients with endometriosis has a higher percentage of Tregs than that of normal individuals; however, the regulatory role of Tregs in the disease remains unclear.
  
  
  
  This study used primary human endometrial stromal cells (ESCs), monocytes (Mo), Tregs and human umbilical vein endothelial cells (HUVECs). All experiments were performed at least three times.
  
  
  
  The migration of Tregs was evaluated by the transwell migration assay. The activation of extracellular signal-regulated kinase (ERK)1/2, c-Jun N-terminal kinase and p38 signaling pathways was examined using the In-Cell WesternTM (LI-COR®) western blot analysis system, as well as by traditional western blot analysis. Changes in the expression of CCL22, CCL17, transforming growth factor-beta 1 (TGF-β1), Interleukin (IL)-1β, tumor necrosis factor alpha (TNF-α), IL-8 and vascular endothelial growth factor (VEGF) in cell-culture supernatant were detected by ELISA. We analyzed the Tregs by multicolor flow cytometry to directly test the expression of CCR4, CD4, CD25, Foxp3, CTLA-4, CD39 and CD73.
  
  
  
  Our results showed that ESCs-Mo co-culture produced significantly higher levels of CCL22 and CCL17 than ESCs or Mo cultured alone, and that estradiol (E2) or progesterone (P) further promoted this upregulation, demonstrating stronger chemotaxis on Tregs. The co-culture of ESCs with Mo stimulated TGF-β1 secretion by Tregs, which could be inhibited by anti-CCL22 or/and anti-CCL17 neutralizing antibodies (Abs). The expression of CCR4 by Tregs was upregulated in ESCs-Mo co-culture, especially by treatment with E2 and/or P, and this effect could be abolished by anti-CCL22 and/or anti-CCL17-neutralizing Abs. The Treg-ESCs-Mo co-culture treated with E2 (10-8 mol/l) and P (10-8 mol/l) could enhance the immunosuppression of Tregs, as proved by the elevated expression of Foxp3, CTLA-4, CD39 and CD73 on Tregs. ESCs-Mo co-culture could significantly promote the secretion of IL-1β and TNF-α. TGF-β1 from Tregs could activate p38/ERK1/2 signaling pathways in ESCs, and IL-1β and TNF-α produced by ESCs-Mo co-culture had synergistic roles with TGF-β1. TGF-β1 and the proinflammatory cytokines IL-1β or TNF-α could synergistically promote IL-8 and VEGF expression in ESCs via the p38/ERK1/2 signaling pathways. The high levels of IL-8 and VEGF in the supernatant of ESCs stimulated the angiogenesis of HUVECs.
  
  
  
  None.
  
  
  
  This study was only performed in vitro using eutopic ESCs, instead of ectopic cells, from endometriosis patients. Therefore, it is necessary to do further experiments to determine whether Tregs promote angiogenesis in the endometriotic milieu in synergy with proinflammatory cytokines in vivo.
  
  
  
  Co-targeting Tregs and proinflammatory cytokines may be an effective treatment for endometriosis.
  
  
  
  This study was supported by Ministry of Science and Technology of China 2015CB943300 to L.D.-J.; National Natural Science Foundation of China, item number 81200425 to  W.X.-Q.; National Natural Science Foundation of China, item number 81471548 to L.D.-J.; and The Research Fund for the Doctoral Program of Higher Education of China to W.X.-Q. (20110071120093). The authors have no conflicts of interest to declare.
authors.affiliation: Laboratory for Reproductive Immunology, Key Laboratory of Reproduction Regulation of NPFPC, SIPPR, Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases, Hospital and Institute of Obstetrics and Gynecology, IRD, Fudan University Shanghai Medical College, Shanghai, China.
authors.forename: Xiao-Qiu
authors.initials: XQ
authors.lastname: Wang
authors.affiliation: Laboratory for Reproductive Immunology, Key Laboratory of Reproduction Regulation of NPFPC, SIPPR, Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases, Hospital and Institute of Obstetrics and Gynecology, IRD, Fudan University Shanghai Medical College, Shanghai, China.
authors.forename: Wen-Jie
authors.initials: WJ
authors.lastname: Zhou
authors.affiliation: Laboratory for Reproductive Immunology, Key Laboratory of Reproduction Regulation of NPFPC, SIPPR, Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases, Hospital and Institute of Obstetrics and Gynecology, IRD, Fudan University Shanghai Medical College, Shanghai, China.
authors.forename: Xue-Zhen
authors.initials: XZ
authors.lastname: Luo
authors.affiliation: Laboratory for Reproductive Immunology, Key Laboratory of Reproduction Regulation of NPFPC, SIPPR, Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases, Hospital and Institute of Obstetrics and Gynecology, IRD, Fudan University Shanghai Medical College, Shanghai, China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Tao
authors.affiliation: Laboratory for Reproductive Immunology, Key Laboratory of Reproduction Regulation of NPFPC, SIPPR, Shanghai Key Laboratory of Female Reproductive Endocrine Related Diseases, Hospital and Institute of Obstetrics and Gynecology, IRD, Fudan University Shanghai Medical College, Shanghai, China.
authors.forename: Da-Jin
authors.initials: DJ
authors.lastname: Li
mesh_terms: D000328:Adult; D015415:Biomarkers / Q000378:metabolism; D002450:Cell Communication; D002465:Cell Movement; D002478:Cells, Cultured; D054414:Chemokine CCL17 / Q000378:metabolism*; D054422:Chemokine CCL22 / Q000378:metabolism*; D018920:Coculture Techniques; D004715:Endometriosis / Q000276:immunology / Q000378:metabolism / Q000473:pathology*; D004717:Endometrium / Q000098:blood supply / Q000276:immunology / Q000378:metabolism / Q000473:pathology*; D004730:Endothelium, Vascular / Q000276:immunology / Q000378:metabolism / Q000473:pathology*; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D061307:Human Umbilical Vein Endothelial Cells / Q000166:cytology; D006801:Humans; D007165:Immunosuppression Therapy; D018836:Inflammation Mediators / Q000378:metabolism; D020935:MAP Kinase Signaling System; D009000:Monocytes / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D009389:Neovascularization, Pathologic / Q000276:immunology / Q000378:metabolism / Q000473:pathology*; D017154:Stromal Cells / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D050378:T-Lymphocytes, Regulatory / Q000276:immunology / Q000378:metabolism / Q000473:pathology*
chemical_list: D015415:Biomarkers; C106230:CCL17 protein, human; C515482:CCL22 protein, human; D054414:Chemokine CCL17; D054422:Chemokine CCL22; D018836:Inflammation Mediators
keywords: angiogenesis; ectopic milieu; endometriosis; proinflammatory cytokines; regulatory T cells