Figure 2.

LPS-mediated ROS production in human neutrophils depends on TLR4 signaling and JNK activation. Human neutrophils were treated with cytosolic ROS indicator dye DHR123 and activated with PMA (25 nM), LPS (25 μg/ml) or only media (-ve control) in the presence or absence of JNK inhibitor SP600125 or TCSJNK6o (TCS), or TLR4-TIRAP/TRAM inhibitor TAK242 (TAK). (A–C; E–G) R123 based ROS generation kinetics by plate reader assays show that both PMA and LPS induce ROS generation, over 40 min of post activation. JNK inhibition with both SP600125 and TCSJNK6o drastically reduces the generation of ROS in LPS treated Neutrophils, while inhibitors do not suppress ROS generation in neutrophils activated with PMA. The ROS values were calculated by considering the PMA-mediated ROS production as 100% at 40-minute time point (n = 3, *p-value < 0.05; Two-way ANOVA with Bonferroni’s multiple comparison post test). (D) Confirmation of generation and inhibition of ROS by confocal imaging. R123 (green) and DNA (blue). Confocal images confirm the inhibition of ROS in LPS activated neutrophils but not in neutrophils activated with PMA, at 60-minute time point (n = 3; scale bar 20 μm). (E–G) Measuring R123-based ROS generation kinetics by plate reader assays show that TLR4-TIRAP/TRAM inhibition with TAK242 significantly reduces the generation of ROS in LPS treated Neutrophils. However, the inhibitor does not suppress ROS generation in neutrophils activated with PMA. The ROS values were calculated by considering the PMA-mediated ROS production as 100%, at 40-minute time point (n = 3, *p-value < 0.05; One-way ANOVA with Tukey’s post test compared to negative control). See Supplementary Fig. S2 for relative changes in ROS production. Error bars in all the panels represent SEM.