Figure 2

RANKL from trophoblasts and DSCs triggers M2 differentiation of dMϕ and Th2 bias. (a and b) We co-cultured dMϕ (n=6) with RANKL-overexpressed or control DSCs and JEG-3 cells at a 1 : 1 : 1 ratio for 48h, and then the expression levels of CD206, CD209, CD163, IL-10, CD80, CD86, IFN-γ and IL-12/23p40 were assessed in dMϕ. (Student’s t-test). (c-g) After 48 h of culture with or without trophoblasts and DSCs and treatment with or without recombinant human OPG protein (rhOPG, 100 ng/ml) or anti-human RANKL neutralizing antibody (α-RANKL, 5 μg/ml), CD14+ dMϕ (n=6) were collected and co-cultured with autologous decidual naive T cells at ratios of 1 : 1 (c). The decidual naive T cells had been previously activated with anti-CD3 (5 μg/ml), anti-CD28 (1 μg/ml) and rhIL-2 (20 U/ml) for 3 days, and then collected. After 5 days of co-culture, the expression of GATA-3 and T-bet in CD4+T cells (e-g) were analyzed by FCM; alternately, these CD4+T cells were collected and reactivated with anti-CD3 and anti-CD28 alone for another 24 h, and then the secretion levels of IL-4, IL-10, TNF-α and IFN-γ (d) were analyzed by ELISA. (One-way ANOVA). dMϕ: human dMϕ; Ctrl-D+J: control DSCs and JEG-3 cells; RANKL+D+J: RANKL-overexpressed DSCs and JEG-3 cells; Mϕ-CD4+T: co-culture of ctrl dMϕ and naive T cells; D+T+CD4+T: co-culture of dMϕ pre-cultured with DSCs and trophoblasts and naive T cells. Data are expressed as the mean±S.E.M. *P<0.05, **P<0.01 and ***P<0.001. #P<0.05, ##P<0.01 and ###P<0.001 versus ctrl Mϕ-CD4+T