Fig. 4

PAD4, NE and ROS are required for the formation of NETs induced by ferritin. Ferritin-injected mice were treated with PAD4, NE, and ROS inhibitors. A DNA concentration and (B) MPO-DNA complex were measured in cell culture supernatants after exposing healthy human neutrophils to indicated concentrations of ferritin. C ROS levels in healthy human neutrophils after ferritin stimulation for 12 h in vitro. D ROS levels in mouse BMDNs after 12 h of ferritin stimulation. E and F the concentration of MPO-DNA in human neutrophils’ culture supernatants was measured after being stimulated with ferritin alone or in combination with Cl-amidine, sivelestat, and DPI. G IF images of the effect of ferritin alone and combined with inhibitors on the formation of neutrophil NETs. (MPO, red; CitH3, green; DAPI, blue. Bar = 100 μm). H and I Neutrophil counts and frequencies in peripheral blood and lung tissue of mice after application of ferritin alone and in combination with various inhibitors. J HE staining of mouse lung tissue (Scale bar = 50 μm). K and L Lung injury score and wet-to-dry ratio. M, N and O Plasma concentrations of inflammatory factors IL-6, TNF-α and MCP-1 in mice. P, Q and R Lung tissue mRNA levels of inflammatory factors including Il-6, Tnf and Il-b. S IF-stained images of NETs markers in lung tissue. (MPO, green; CitH3, red; DAPI, blue. Bar = 30 μm)