Fig. 3. Neutrophil NETosis is the main source of post-stroke cfDNA.

a, Tissue-of-origin analysis using cell-type-specific methylation markers for cfDNA isolated from n = 10 patients with stroke within 24 h after symptom onset. NK, natural killer; T_reg cell, regulatory T cell. b, Flow cytometry histograms (left) and quantification (right) of citrullinated histone 3 (citH3⁺) Ly6G⁺ neutrophils 4 h after sham or stroke (U-test; n = 5–6 per group). c, Microphotographs of mouse neutrophils stimulated with 25% sham or stroke serum for 4 h. The arrowheads indicate DNA⁺citH3⁺ NET formations. Scale bars, 50 µm. d, Quantification of phorbol 12-myristate 13-acetate (PMA)-stimulated, sham serum-stimulated or stroke serum-stimulated neutrophils, shown as citH3⁺ neutrophils of total cultured neutrophils (K-test; n = 6–23 replicates per group from 3 independent experiments). e, BMDMs from ASC–citrine reporter mice were incubated with 250 ng exogenous DRAQ5-labelled NET–DNA. Immunohistochemistry was used to visualize cytoplasmatic citrine⁺DRAQ5⁺ ASC–DNA specks. The cytoplasm and nucleus of the BMDMs were visualized using β-tubulin and Hoechst. Arrowheads indicate colocalization of ASC specks and DRAQ5⁺ NET DNA. Scale bars, 5 µm. f, Experimental design for anti-Ly6G neutrophil depletion and PAD4 inhibition before stroke. g, cfDNA concentration (shown as percentage normalized to sham) of mice with control, anti-Ly6G or PAD4 inhibitor treatment (left; K-test; n = 6–8 per group) and caspase-1 activity in blood monocytes (percentage of sham, indicated by dotted line) by flow cytometry (right; K-test; n = 5–8 per group). h, NETosis was inhibited using a PAD4 inhibitor before stroke in HCD-fed ApoE^−/− with tandem stenosis. i, Quantification of citH3⁺Ly6G⁺ neutrophils 24 h after stroke surgery in blood (U-test; n = 4–6 per group). j, Flow cytometry analysis of caspase-1 activity in CD11b⁺ CCA monocytes 24 h after stroke (U-test; n = 4–6 per group). b,d,g,i,j, Bars indicate the mean.