Figure 3

TLR-4 mediates NET induction by extracellular superoxide. (A) Flow cytometry analysis of citrullinated Histone H3 in WT neutrophils after treatment with media alone (control), eritoran TLR-4 antagonist (8 ng/mL), xanthine oxidase (10 mU/mL) and hypoxanthine (500 nM) to yield superoxide, or superoxide and eritoran, for 8 h. (B) Western blot of citrullinated Histone H3 in WT or TLR-4 KO neutrophils after treatment with media alone (negative control), PMA (100 nM), LPS (10 ug/mL), or xanthine oxidase (10 mU/mL) and hypoxanthine (500 nM) to yield superoxide, for 1–8 h at 37°C. β-actin is shown as a loading control. Densitometric quantification chart shows mean ± SEM cit-H3 to β-actin, expressed as fold over control. (C) Assay of NET MPO-DNA complexes formed after treatment as above. Shown as mean fold over control. #p < .05 vs respective uninhibited treatment. (D) Neutrophils were fixed after treatment as above, immunolabeled with antibody to citrullinated Histone H3, detected with a conjugated fluorescent secondary antibody (green), then stained for DNA with Hoechst (blue). All figures represent at least 3 independent experiments for each stimulus.