Fig 2: Evidence of a potential reciprocal interaction between FASN and CEACAM1 in the placenta of RPL patients and in HTR8/SVneo cells. (A) GeneMANIA Cytoscape program predicted that FASN might interact with CEACAM1. (B) The protein level of CEACAM1 in HTR8/SVneo cells treated with a range of concentrations of FK506 for 48 hours were examined by Western blot. β-tubulin (TUBB) was used as an internal control. (C) The protein level of CEACAM1 in the placental villus from NP and RPL examined by Western blot. β-actin (ACTB) was used as an internal control. (D) Flow cytometric analysis and quantitation of CEACAM1 expression of HTR8/SVneo cells after transfected with FASN overexpression plasmid or FASN-specific siRNA for 48h. NC, negative control. oe, overexpression. si, small interfering RNA. Images are representatives of three independent experiments. Flow cytometry plot is from one representative experiment. Data represent mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Fig 3: Schematic diagram of FK506-mediated positive effects on the placentation and maternal-fetal tolerance through modulating FASN-CEACAM1 pathway. (A) FK506 promoted invasion, tube formation and proliferation, but inhibited apoptosis of HTR8/SVneo cells. Based on the integrated metabolomics and transcriptomics analyses, FK506 might regulate biological behaviors of HTR8/SVneo cells via inhibiting FASN, which might interact with CEACAM1. FK506 increased CEACAM1 expression on HTR8/SVneo cells. In turn, CEACAM1 rescued the adverse effects of FASN on HTR8/SVneo cells. (B) The SA mouse model gave further evidence that FK506 exerted a protective effect on pregnancy by promoting placentation and inducing Th2/Treg bias in the pregnant uterus to maintain maternal-fetal tolerance through regulating the FASN-CEACAM1 axis.

Fig 4: FK506 alleviated fetal loss of SA models in a FASN-CEACAM1 dependent way. (A, B) Representative images of uterus (A), statistics of absorption rate, fetal weight and placental weight (B) of SA mice with the indicated treatments. (C) Representative images of placental hemi-sections (H&E-stained) of SA mice with the indicated treatments. (D) Immunofluorescence analysis and quantitation of placental FASN and CEACAM1 expression of SA mice with the indicated treatments. (E) Flow cytometric analysis and quantitation of IFN-γ, TNF-α, IL-4 and TGF-β1 expression on decidual CD4+T cells of SA mice with the indicated treatments. Flow cytometry plot and images are representative of three individual experiments. Data represent mean ± SEM. *p<0.05, **p<0.01, ***p< 0.001, ****p< 0.0001.