Fig 2: RANKL induces dMφ differentiation by activating the Akt/STAT6-Jmjd3/IRF4 signaling pathway. (a-c) dMφ were co-cultured with RANKL+D+J or Ctrl-D+J at a 1 : 1 : 1 ratio, and treated with or without 10 μM Akt signaling inhibitor (Akti) Ly294002 for 24 h. The intracellular phosphorylation level of Akt and or STAT6 in dMφ (n=5) was then analyzed by FCM. (d) The secretion level of IL-10, IL-12p40 and IL-23 in the co-culture of dMφ and RANKL+-D+J or Ctrl-D+J (at a 1 : 1 : 1 ratio), which was treated with or without Akti (10 μM) or the STAT6 signaling inhibitor (STAT6i) AS1517499 (21 nM) for 48 h. (One-way ANOVA). (e) After intraperitoneal injection of STAT6i (200 μl at the concentration of 2 mg/kg) in pregnant C57BL/6 mice (n=5 mice per group) at day 4, the levels of CD206, CD209, IL-10, CD80 and CD86 in uMφ, and the ratios of GATA-3 to T-bet and IL-4 to IFN-γ in uCD4+T cells at day 10 were detected by FCM. (Student’s t-test). (f) The transcription levels of IRF4, Jmjd3 and IRF5 in dMφ treated as described in Figure 3d. (One-way ANOVA). (g) After intraperitoneal injection of STAT6i or the Jmjd3 selective inhibitor (JMJD3i, 200 μl at a concentration of 4.48 mg/kg) GSK J4 HCl in pregnant C57BL/6 mice (n=5 mice per group) at day 4, the IRF4 level in uMφ cells at day 10 was detected. (h and i) After intraperitoneal injection of JMJD3i in pregnant C57BL/6 mice (n=5 mice per group) at day 4, the levels of CD206, CD209, IL-10, CD80 and CD86 in uMφ, and the ratios of GATA-3 to T-bet and IL-4 to IFN-γ in uCD4+T cells at day 10 were detected by FCM. (Student’s t-test). dMφ: human dMφ; uMφ: mouse uterus macrophages. Data are expressed as the mean±S.E.M. *P<0.05, **P<0.01 and ***P<0.001

Fig 3: 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

Fig 4: The crosstalk between fetus and mothers leads to high levels of RANKL/RANK expression at the maternal–fetal interface. (a) RANKL expression in villi and decidua of normal pregnancy (n=12) by immunohistochemistry. Original magnification: × 200. (b) RANKL secretion by primary trophoblasts (1 × 105 cells per well) and DSCs (1 × 105 cells per well) (n=6) from normal pregnant women by ELISA after culture for 24–96 h (left). RANKL production by trophoblasts alone, DSCs alone and the co-culture of trophoblasts and DSCs (n=6) for 48 h (right). (One-way ANOVA). (c) We isolated primary PBMCs from peripheral blood (n=6) and DLC from deciduas of normal pregnant women (n=6), and then analyzed RANK expression on pMo and dMφ from normal pregnant women by labeling with anti-CD14, RANK and CD45 antibodies. (Student’s t-test). (d) Further analysis of the phenotype of RANK+ and RANK− pMo and dMφ from normal pregnant women (n=24) by FCM. (One-way ANOVA). (e and f) Trophoblasts, DSCs and/or dMφ (n=6) were co-cultured at a 1 : 1 : 1 ratio for 48 h, and then RANKL expression on CK7+ trophoblasts and CK7− DSCs, and RANK on CD14+ dMφ were evaluated by FCM, respectively. (One-way ANOVA). Tro: human trophoblasts. pMo: human peripheral blood monocytes; dMφ: human dMφ. Data are expressed as the mean±S.E.M. *P<0.05, **P<0.01 and ***P<0.001. NS: no statistical difference

Fig 5: Absence of RANKL expression leads to mouse uMφ dysfunction and fetal loss. (a) RANK expression on uMφ from CBA/J♀ × DBA/2♂ matings (the abortion-prone model) and CBA/J♀ × BALB/c♂ matings (normal pregnancy model) at days 5 and 9 of gestation (n=6 mice per group). Moreover, the expression of CD80, CD86, CD206 and MHCII on F4/80+uMφ from CBA/J♀ × DBA/2♂ matings (the abortion-prone model) and CBA/J♀ × BALB/c♂ matings (normal pregnancy model) at days 5 and 9 of gestation (n=6 mice per group); (adjusted t-test). (b) FCM analysis of CD206, CD209, IL-10, CD80 and CD86 in uMφ of wild-type and RANKL knockout pregnant mice at day 10 (n=6 mice per group); (Student’s t-test). (c) FCM analysis of GATA-3, T-bet, IL-4, IL-10, IFN-γ and TNF-α in uCD4+T cells of WT and RANKL−/− pregnant mice at day 10 (n=5 mice per group); (Student’s t-test). (d) FCM analysis of the phosphorylation level of Akt and STAT6 in uMφ cells of WT and RANKL−/− pregnant mice at day 10 (n=6 mice per group); (Student’s t-test). (e) uMφ were isolated from mouse uterus (n=20 mice per group) from WT and RANKL−/− mice at day 10 of gestation by MACS, and then used to analyze the transcription of Jmid3 and IRF4 in uMφ. (Student’s t-test). (f) FCM analysis of IRF4 levels in uMφ cells of WT and RANKL−/− pregnant mice at day 10 (n=6 mice per group); (Student’s t-test). (g and h) The embryo absorption rate in WT and RANKL−/− pregnant mice (n=6 mice per group) was determined on day 14 of gestation. Fetal loss sites could be identified as hemorrhagic spots and necrosis (red arrows, left); (adjusted t-test). uMφ: Mφ from mouse uterus; uCD4+T cells: CD4+T cells from mouse uterus; Normal: normal pregnant mouse model; Abortion: abortion mouse model. D5: day 5 of gestation; D9: day 9 of gestation. WT: wild-type pregnant mice; RANKL−/−: RANKL knockout pregnant mice. Data are expressed as the mean±S.E.M. *P<0.05, **P<0.01 and ***P<0.001