Extended Data Fig. 5: NET-derived histone H4 induces cell toxicity.
From: Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death
a–c, Analysis of cell death (propidium iodide uptake). a, NETs were pre-incubated with indicated antibodies for 1 h before addition to SMCs. MPO, myeloperoxidase; NE, neutrophil elastase; CG, cathepsin G; PR3, proteinase 3. n = 79 IgG, n = 23 MPO, n = 60 LL37, n = 60 NE, n = 58 CG and n = 60 PR3 fields. One-way ANOVA with Dunnet’s correction. P = 0.105 (MPO), P = 0.219 (LL37), P = 0.270 (NE), P = 0.925 (CG), P = 0.999 (PR3). All conditions were compared against control (ctrl). b, NETs were pre-incubated with inhibitors to myeloperoxidase (MPO), neutrophil elastase (NE), or secretory leukocyte protease (SLP) for 1 h before their addition to SMCs. n = 96 ctrl, n = 35 MPO, n = 58 NE, n = 58 SLP fields. One-way ANOVA with Dunnet’s correction. P = 0.299 (MPO), P = 0.085 (NE), P = 0.978 (SLP). All conditions were compared against control (ctrl). c, SMCs, endothelial cells (ECs) and peritoneal macrophages (PMs) were incubated with recombinant histone H4. Cell death was assessed by PI uptake. n = 36 and n = 36 for SMCs, n = 35 and n = 36 for ECs, n = 47 and n = 39 for PMs. Two-sided unpaired t-test, *P = 0.029; **P = 3.847 × 10−5; ***P = 8.775 × 10−6. d, Representative confocal immunofluorescence of advanced atherosclerotic lesions to visualize DNA (DAPI, blue), neutrophils (Ly6G, red), SMCs (SMA, green), histone H4 (magenta), and citrullinated histone H3 (white). Scale bar, 20 µm. e, Quantification of extranuclear histone H4 per section of indicated treatments. n = 17 ctrl, n = 8 anti-Ly6G, n = 9 AMD3100. One-way ANOVA with Dunnet’s correction, *P = 0.02; **P = 0.0002. Data are mean ± s.d. ctrl, control; SMA, smooth muscle actin.
