Fig 1: FABP4 promotes Th17 differentiation of naive T cells via increasing IL-17A/IL-23 release(A) The mRNA levels of cytokines were determined by qRT-PCR. (B) The protein level of FABP4 was detected by western blot. (C) The IL-17A and IL-23 levels in cell culture supernatant were assessed by ELISA. (D) The protein levels of NLRP3 inflammasome components were determined by western blot. Naive T cells were cultured under Treg or Th17 cell-polarizing conditions with or without primary macrophages-CM and neutralizing antibody. (E–H) The percentage of Tregs (E and F) or Th17 cells (G and H) was assessed by FACS. GAPDH and ß-actin were used for normalization in qRT-PCR and western blot, respectively. Data are expressed as representative images or the mean ± SD of n = 3 experiments. *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 2: Knockdown of FABP4 suppresses Th17 differentiation of naive T cells through paracrine mechanismsNaive T cells were cultured under Treg or Th17 cell-polarizing conditions with or without primary macrophages-CM. (A and B) The percentage of Tregs (A) or Th17 cells (B) was assessed by FACS. Naive T cells were cultured under Treg or Th17 cell-polarizing conditions with or without rhFABP4. (C and D) The percentage of Tregs (C) or Th17 cells (D) was assessed by FACS. Data are expressed as representative images or the mean ± SD of n = 3 experiments. ns, not significant, **p < 0.01, ***p < 0.001.
Fig 3: Elevation of FABP4 is accompanied with Treg/Th17 imbalance in PE(A) The serum level of FABP4 was determined by ELISA (normal pregnant women, n = 10; PE, n = 20). (B) The immunoreactivities of FABP4, IL-17A, and FOXP3 in placental tissues were monitored by IHC analysis. (C and D) The percentages of Treg (C) and Th17 cells (D) in peripheral blood were assessed by FACS (normal pregnant women, n = 10; PE, n = 20). (E) The serum level of IL-17A was determined by ELISA (normal pregnant women, n = 10; PE, n = 20). (F) Pearson’s correlation analysis between IL-17A and FABP4 expression. Data were representative images. *p < 0.05.
Fig 4: Silencing of FABP4 attenuates LPS-induced NLRP3 inflammasome activation and IL-17A production(A) The mRNA level of FABP4 was determined by qRT-PCR. (B) The protein level of FABP4 was determined by western blot. (C) The protein levels of NLRP3 inflammasome components were determined by western blot. (D) The mRNA levels of IL-1ß, IL-18, and IL-17A were detected by qRT-PCR. (E) The secretion of IL-1ß, IL-18, and IL-17A were assessed by ELISA. GAPDH and ß-actin were used for normalization in qRT-PCR and western blot, respectively. Data are expressed as the mean ± SD of n = 3 experiments. *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 5: IL-17A upregulates FABP4 and activates the NLRP3 inflammasome(A) The protein levels of FABP4 and NLRP3 inflammasome components were determined by western blot. (B) The protein levels of NF-?B signaling components were detected by western blot. (C) The protein levels of ERK and p38 MAPK signaling components were determined by western blot. (D) The protein levels of ER stress-related proteins were examined by western blot. ß-actin served as a loading control. (E) IF staining of NLRP3 (green) and ASC (red). Nuclei was visualized by DAPI (blue). (F) The protein levels of FABP4 and NLRP3 inflammasome components in placental tissues were determined by western blot. (G) The immunoreactivities of FABP4 in placental tissues were monitored by IHC analysis. Data are expressed as representative images or the mean ± SD of n = 3 experiments.
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