Fig 2: ESCs and macrophages from endometriotic lesions highly expressed IL-27. (a) We co-cultured ESCs (normal ESCs or ectopic ESCs) with monocytes (n=5) from peripheral blood for 48 h. In addition, ESCs alone and monocytes alone were cultured as controls. Then, the secretion levels of IL-27, IL-6 and TGF-β1 were analyzed by ELISA. nESCs, ESCs from normal endometrium; eESCs, ectopic lesion with endometriosis; nESCs+Mo, co-culture of nESCs and monocytes; eESCs+Mo, co-culture of eESCs and monocytes (one-way ANOVA). (b) IL-27 expression in normal endometrium (n=6) and ectopic endometrium from women with endometriosis (n=8) by immunohistochemistry. Normal, normal endometrium; ectopic lesion from women with endometriosis. Original magnification: × 200. (c) The percentage of IL-27+ESCs (normal ESCs or ectopic ESCs, n=6) by flow cytometry (Student's t-test). (d) The percentage and median fluorescence intensity (MFI) of IL-27+monocytes of peripheral blood (pMo, n=6) and IL-27+macrophages of normal endometrium (nEMϕ, n=6) and ectopic lesions (eEMϕ, n=6) by flow cytometry (one-way ANOVA). (e) The IL-27 level in PF from women with (n=17, I–II: 8; III–IV: 9) and without (n=24) endometriosis by ELISA (one-way ANOVA). The data are expressed as the mean±S.E.M. *P<0.05, **P<0.01 and ***P<0.001

Fig 3: IL-10+Th17 cells induced by IL-27 promoted the ectopic growth and implantation of ESCs. (a–d) After treatment with rhIL-10 (100 ng/ml), rhIL-17A (10 ng/ml) or rhIL-10 plus rhIL-17A for 48 h, the viability (a), apoptosis (b), CD29 level (c) and invasiveness (d) of ESCs (n=5) were evaluated by CCK8 assay, apoptosis assay, flow cytometry and Matrigel invasion assay, respectively. Original magnification: × 200 (one-way ANOVA). (e–h) The human naive T cells were polarized to Th17 in vitro, and stimulated with or without rhIL-27. Then, these cells were collected and indirectly co-cultured with ESCs for 48 h, and αIL-10 and or αIL-17 were added to the co-culture system (e). Then, the viability (f), apoptosis (g) and the MFI of CD29 and CD82 (h) of ESCs (n=5) were evaluated by CCK8 assay, apoptosis assay and flow cytometry, respectively (one-way ANOVA). (i) The size of endometriosis-like lesions from the C57BL/6 endometriosis mouse model was measured after treatment with αIL-27 (50 μg/mouse), αIL-10 (50 μg/mouse) and/or αIL-17A (50 μg/mouse). (j) The number and weight of endometriosis-like lesions from the nude mouse endometriosis model was measured after transferring WSX-1−CD4+ T (n=10) or WSX-1+CD4+ T (n=9) cells. Ctrl, PBS treatment; WSX−, transferred WSX-1−CD4+ T cells; WSX+, transferred WSX-1+CD4+ T cells (one-way ANOVA). (k) The expression of Ki-67 and MMP2 in endometriosis-like lesions from the nude mouse endometriosis model was analyzed by flow cytometry (one-way ANOVA). Original magnification: × 200. The data are expressed as the mean±S.E.M. *P<0.05, **P<0.01 and ***P<0.001

Fig 4: IL-27 induces IL-10 production of Th17 cells in the endometriotic milieu. (a) The percentage of IL-27 receptors (WSX-1 and gp130)+CD4+ T, Treg and Th17 cells from peripheral blood (n=6), normal endometrium (n=6) and ectopic lesion (n=5) by flow cytometry. The right panel is from an ectopic lesion (one-way ANOVA). (b and c) ESCs were co-cultured with monocytes from peripheral blood (n=10) for 48 h, and then naive T cells were added to the co-culture system and further treated with or without rhIL-27 (100 ng/ml) or anti-human IL-27 neutralizing antibody (αIL-27, 5 μg/ml) for 5 days. Then, Th17 differentiation and IL-10 and IFN-γ levels in Th17 cells were detected by flow cytometry. Before co-culture, naive T cells were activated with anti-CD3 (5 μg/ml), anti-CD28 (1 μg/ml), and rhIL-2 (20 U/ml) for 2 days (one-way ANOVA). (d and e) We constructed an allotransplantation of intraperitoneal endometriosis model (n=10/group). On day 0, the uterus of female C57BL/6 mice was minced, and the tissue debris was intraperitoneally injected into nude mice. On day 5, the WSX-1−CD4+ T cells or WSX-1+CD4+ T cells from the uterus of female C57BL/6 mice collected by cell sorting were labeled with PKH-67 and transferred to the abdominal cavity in endometriosis nude mice. In addition, PBS was used as the control. On day 14, the endometriosis-like lesions and PF were collected and detected. (f) The characterization of transferred PKH-67-WSX-1−CD4+ T cells and PKH-67-WSX-1+CD4+ T cells in the PF from endometriosis mice by flow cytometry. (g and h) The Th17 differentiation, IL-10+Th17 and IFN-γ+Th17 cells in the PF from endometriosis mice by flow cytometry. WSX-1-CD4+T, transferred WSX-1−CD4+ T cells; WSX-1+CD4+T, transferred WSX-1+CD4+ T cells (Student's t-test). The data are expressed as the mean±S.E.M. *P<0.05, **P<0.01 and ***P<0.001

Fig 5: The schematic roles of IL-27 in the progression of endometriosis by inducing IL-10+Th17 differentiation. In the early stage of endometriosis, cytokines (such as IL-6 and TGF-β), high estrogen and TCDD exposure significantly promote macrophages in the local microenvironment of ectopic foci to secrete high levels of IL-27. Owing to the interaction between macrophages and ESCs in ectopic lesions, there is an accumulation of IL-27 in the microenvironment of ectopic foci. IL-27 inhibits Th17 differentiation, and promotes the production of IL-10 in Th17 cells via the c-Maf/RORC/Blimp-1 complex, participating in the formation of an immune tolerance pattern in the late stage of endometriosis. These IL-10-produced Th17 cells promote the growth, adhesion, invasion and deep infiltration of ESCs, thus accelerating the progression of endometriosis. With the progression of this disease, the growth of ectopic ESCs may result in increased recruitment and proliferation of macrophages and IL-27 levels in the microenvironment of ectopic foci. These changes will form a vicious circle and accelerate the development of endometriosis