Fig. 2.
Effect of various Toll-like receptor ligands on intracellular H2O2 and mitochondrial O2- production in peritoneal leukocytes. (A, B) Effect of TLR ligands on intracellular H2O2 generation. Thioglycollate-elicited peritoneal leukocytes were treated with TLR ligands as indicated: Pam3Cys, LTA, or LPS (20 μg/ml) for 1 h, incubated with 10 μM of DCF and analyzed for intracellular H2O2 with flow cytometry. Representative histograms are presented in A and combined MFI data in B. n = 3 in each group. ***P < 0.001 vs. control. The experiments were performed twice with the similar results. (C, D) Effect of TLR ligands on mitochondrial O2- production. Cells were treated with TLR ligands as indicated: Pam3Cys, LTA, LPS, poly (I:C) or CpG (20 μg/ml) for 1 h, incubated with 2.5 μM of MitoSOX and analyzed for mitochondrial O2- production. Representative histograms are presented in C and combined MFI data in D. n = 3 in each group. *P < 0.05, ***P < 0.001 vs. control. The experiments were performed four times. (E) TLR2 activation induces a dose-dependent mitochondrial O2- production. n = 3 in each group. *P < 0.05, ***P < 0.001 vs. untreated control. The experiments were performed twice. (F, G) TLR2 mediates Pam3Cys-induced mitochondrial O2- production. Peritoneal leukocytes harvested from WT or TLR2-/- mice were treated with Pam3Cys (20 μg/ml) for 1 h and analyzed for mitochondrial O2– production with flow cytometry. Representative histograms are presented in F and combined MFI data in G. n = 3 in each group. ***P < 0.001 vs. control. ## P < 0.01 vs. WT. Each error bar represents mean ± SEM. DCF = dichlorodihydrofluorescein diacetate; H2O2 = hydrogen peroxide; LPS = lipopolysaccharides; LTA = lipoteichoic acid; MFI = mean fluorescence intensity; Mito O2– = mitochondrial superoxide; TLR2KO = TLR2 knockout; WT = wild type. The experiments were performed twice with similar results.