Heme stimulates NET formation in neutrophils in vivo. (A) Quantification of plasma heme concentrations of TNF-α–stimulated SA mice pretreated with vehicle (gray circle, n = 8) or heme (red circle, n = 11) (mean ± SEM, ****P < .0001). (B) Quantification of NETs in the lungs of TNF-α–stimulated SA mice, vehicle pretreated (gray bar, n = 4) or heme pretreated (red bar, n = 6). Results presented are the average values of independent experiments with sex- and age-matched mice (mean ± SEM, **P < .01). (C) Representative histological images of NETs in the lungs of heme pretreated-, TNF-α–stimulated SA mice (arrows indicate NETs). Scale bar, 10 μm. (D) Representative immunofluorescence images of the lung sections of heme pretreated-, TNF-α–stimulated SA mice showing colocalization of extracellular DNA (green/SYTO13) and NE (red/Alexa Fluor 568, i-ii) or H3Cit (red/Alexa Fluor 568, iii-iv). CD31 (blue/Alexa Fluor 647) labeled the endothelial cells. Scale bar, 10 μm. (E) Quantification of NET biomarkers, plasma DNA (left), and plasma nucleosome (right) of TNF-α–challenged SA mice, vehicle pretreated (gray circle, n = 7) or heme pretreated (red circle, n = 6) (Mann-Whitney test, median [IQR], **P < .01). (F) Quantification of plasma MPO activity of TNF-α–challenged SA mice, vehicle pretreated (gray circle, n = 4) or heme pretreated (red circle, n = 6) (Mann-Whitney test, median [IQR], *P < .05). (G) Decrease in rectal temperature of TNF-α–stimulated SA mice, pretreated with vehicle (gray circle, n = 12) or heme (red circle, n = 12) (mean ± SEM, **P < .01).