Figure 9.
CO inhibits LPS-induced TLR4 trafficking by inhibiting NADPH oxidase activity. (A) RAW 264.7 cells were incubated with 100 ng/ml LPS, 100 ng/ml Pam, and 20 μg/ml poly(I:C) in the presence or absence of CO after preincubation with 10 μM CM-H2DCFDA for 30 min. Fluorescence intensity of the cells was measured as the amount of ROS accumulation (left). Representative images are shown from three independent experiments (right). *, P < 0.05 and **, P < 0.01 versus CO-treated cells. (B) RAW 264.7 cells were treated with LPS in the presence or absence of CO or DPI. Interaction of gp91phox and p47phox was analyzed by immunoprecipitation assay. (C) RAW 264.7 cells were treated with LPS for 30 min in the presence or absence of CO or DPI. Superoxide production was analyzed by determining the reduction rate of acetylated cytochrome C. ††, P < 0.01 versus LPS-treated control cell. (D) Cytochrome b558 fraction was isolated from bovine neutrophils and the spectra of cytochrome b558 were analyzed. The isolated oxidized cytochrome b558 was reduced by adding 5 mM of sodium dithionite and CO was bubbled for 30 s, followed by spectra analysis (left). The spectral difference between the control (Reduced form) and CO-treated sample (CO Reduced) was shown (right). (E) Peritoneal macrophages were isolated from gp91phox-deficient (gp91phox−/−) mice and wild-type (WT) mice. Cells were exposed to LPS for 1 h in the presence or absence of CO. TNF-α production in cell media was analyzed by ELISA. ††, P < 0.01 versus air/LPS (WT). **, P < 0.01 versus air/LPS (WT). (F and G) RAW 264.7 cells were stimulated with LPS in the presence or absence of DPI or CO, and the interaction of TLR4 and gp91phox was analyzed using immunoprecipitation assay. (H) Peritoneal macrophages from gp91phox-deficient and wild-type mice were stimulated with 100 ng/ml LPS for 5 min. Cell lysates were fractionated to 12 subfractions, followed by immunoblotting for TLR4 and GM1.