Figure 3

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