Figure 5. Palmitic acid-induced IL-1β secretion in THP-1 cells is mediated through activation of TLR2.

(A) THP-1 cells were transfected with negative control or TLR2 siRNA. After 48 hours cells were serum starved in 0.25% FBS-RPMI-1640 for 12 hours then treated with C16:0 (150 µM) or Pam₃CSK₄ (10 ng/ml) for 24 hours. Supernatants were analyzed for IL-1β by ELISA. Data are presented as mean percentages of control siRNA treated cells ± SEM and were calculated from three independent experiments. (***P < 0.001) Significance was determined by two-tailed, unpaired t-test. (B) TLR2 cell surface expression on THP-1 cells was measured 60 hours after transient transfection with siRNAs by flow cytometry. Data is representative of at least 3 independent experiments. (C) Schematic of the TR-FRET assays performed to detect dimerization of TLR2 with TLR1 using anti-TLR2 antibodies labeled with europium cryptate as the donor fluorophore and anti-TLR1 antibodies labeled with d2 as the acceptor fluorophore. (D) THP-1 cells were serum starved in 0.25% FBS-RPMI-1640 for 12 hours with TLR2 and TLR1 antibodies then incubated with DHA (10 µM) for one hour and treated with C16:0 (150 µM) or Pam₃CSK₄ (100 ng/ml) for 10 minutes. TR-FRET data are presented as mean percentages of untreated cells ± SEM and were calculated from at least five independent experiments. Significance was determined by ANOVA (#P < 0.001 significantly different from untreated, *P < 0.05, ***P < 0.001 significantly different from C16:0 and Pam3, respectively).