Extended Data Fig. 1: Lesional neutrophils do not correlate with macrophage or endothelial phenotype.
From: Externalized histone H4 orchestrates chronic inflammation by inducing lytic cell death
a–f, Methodology used to analyse advanced atherosclerotic lesions. a, b, Representative pictures of H&E staining. a, Intima to media ratio was calculated as the quotient of intima area and media area. Red and black dotted lines delineate intima and media, respectively. b, Necrotic core area was quantified as the percentage of necrotic core area over the intima area. Red and green dotted lines delineate intima and necrotic core, respectively. c, d, Representative pictures of Sirius red staining. c, Sirius red pictures were converted into 8-bit images and segmented by thresholding hence generating binary images. Collagen area was quantified as the percentage of collagen area over the intima area. The black dotted line delineates the intima. d, Fibrous cap thickness was analysed as the average of lengths measurements in the positions overlapping with the lines of a square-shaped grid. Averaged fibrous cap thickness was corrected by the intima area. e, f, Representative pictures of smooth muscle cell actin (SMA, e) and CD68 (f) staining. Immunofluorescence images were segmented by thresholding to convert fluorescence signal into a binary image. SMA/CD68 area was quantified as the percentage of SMA/CD68 area over the intima area. The red dotted line delineates the intima. g–o, Advanced atherosclerotic lesions were generated in the carotid artery of Apoe−/− mice. Pearson correlation between lesional neutrophils and lesion size (n = 28 mice, g), fibrous cap thickness (n = 28 mice, h), collagen area (n = 28 mice, i), macrophage area (CD68+, n = 24 mice, j), pro-inflammatory macrophages (CD68+iNOS+, n = 24 mice, k), anti-inflammatory macrophages (CD68+CD206+, n = 23 mice, l), endothelial cell area (CD31+, n = 25 mice, m), VCAM1 expression on endothelial cells (n = 25 mice, n) and ICAM1 expression on endothelial cells (n = 25 mice, o). Dotted line represents 95% confidence interval. Note that data in g–i and j–o were generated in a different set of lesions due to a shortage of sections from mice displayed in g–i.
