Fig 2: 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

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: There are low levels of RANKL/RANK at the maternal–fetal interface during miscarriage. (a) Immunohistochemistry analysis of RANKL expression in villi and deciduas from women with normal pregnancy (n=12) or miscarriage (n=12) during the first trimester. RANKL expression was localized to the cell membrane and the cytoplasm (arrows) in the deciduas and villi. Original magnification: × 200. (b and c) FCM analysis of the percentage of RANK+ dMφ from women with normal pregnancy (n=11) or miscarriage (n=11) during the first-trimester. (d and e) FCM analysis of the percentage of CD163+, CD206+, CD80+ and CD86+dMφ from women with normal pregnancy (n=11) or miscarriage (n=11) during the first trimester. Normal: normal pregnant women; Miscarriage: SA women. Data are expressed as the mean±S.E.M. **P<0.01 and ***P<0.001 (Student’s t-test)

Fig 5: RANKL-mediated harmonious dialog between the fetus and mother guarantees a smooth gestation by inducing decidual M2 macrophage polarization. Together with blastocyst implantation during normal pregnancy, PAHs trigger ESCs to differentiate into DSCs, further inducing high levels of RANKL expression and CCL2 release. The latter recruits more peripheral monocytes to the decidua. Embryonic trophoblasts that are deeply implanted in decidua can closely contact DSCs and DLCs. This dialog not only further increases RANKL expression in trophoblasts and DSCs, but it also enhances the sensitivity of RANK to RANKL by upregulating RANK expression on Mφ. Subsequently, the RANKL–RANK interaction drives Mφ to M2 differentiation (low expression of CD80 and CD86, high secretion of IL-10, and low level of IL-12 and IL-23) by activating Akt/STAT6-Jmjd3/IRF4 signaling. After education, these Mφs will create and maintain a maternal immune tolerance microenvironment (increase in Th2 and decrease in Th1). After the development of abnormally low RANKL expression at the maternal–fetal interface, the dysfunction of Mφ, the imbalance of maternal–fetal immune regulation and then abortion will occur. Tro: trophoblasts