HMGB1 induces NET formation trough RAGE in DVT. (A-I) Quantification of neutrophils and NETs. Results are mean ± SEM. (A) BoxA (n = 5) compared with control (n = 5). (B) Immunofluorescence staining for Ly6G (red) and MPO (green) from cross-sections of the IVC 48 hours after flow reduction of BoxA-treated (bottom) or control mice (top). Nuclei are counterstained with DAPI (blue); bar, 200 µm. (C) Hmgb1^−/− (n = 3) fetal liver cell chimeras compared with Hmgb1^+/+ chimeras (n = 5). (D) Immunofluorescence staining for Ly6G (red) and MPO (green) from cross-sections of the IVC 48 hours after flow reduction of Hmgb1^+/+ (top) or Hmgb1^−/− fetal liver cell chimeras (bottom). Nuclei are counterstained with DAPI (blue); bar, 200 µm. (E) Quantification of NETs in Rage^−/− mice compared with control (n = 5 each). (F) Quantification of NETs in Tlr2^−/− mice compared with control (n = 3 each). (G) Quantification of NETs in Tlr4^−/− mice compared with control (n = 3 each). (H) Quantification of NETs in Myd88^−/− mice compared with control (n = 3 each). (I) Left, NET formation capacity shown as NETs/neutrophil (n = 5 each) in Hmgb1^−/− chimeras compared with Hmgb1^−/− chimeras receiving wild-type platelets or wild-type neutrophils. Dotted line indicates mean in Hmgb1^+/+ bone marrow chimeras. Right, Immunofluorescence staining for Ly6G (red) and MPO (green) from cross-sections of the IVC 48 hours after flow reduction in Hmgb1^−/− chimeras compared with Hmgb1^−/− chimeras receiving wild-type platelets, Hmgb1^−/− platelets or wild-type neutrophils. (n = 3 each). Nuclei are counterstained with DAPI (blue); bar, 200 µm. The Student t test was used to compare results between 2 groups, 1-way ANOVA followed by LSD–post hoc test for 3 groups.