Neutrophil extracellular traps and fibrocytes in ST-segment elevation myocardial infarction

Fig. 1. Neutrophil extracellular trap (NET) markers in patients with ST-segment elevation myocardial infarction and healthy controls, and correlation with enzymatic infarct size. a Double-stranded (ds)DNA was measured using PicoGreen. b Citrullinated histone H3 (citH3) was measured using ELISA. Data are provided as ng/ml. Correlations of NET surrogate markers c double-stranded (ds)DNA and d citrullinated histone H3 (citH3) at the culprit site with enzymatic infarct size. Infarct size is expressed as creatine-phosphokinase isoform MB area under the curve (CK-MB AUC, given as arbitrary units). For STEMI patients, n = 48; for healthy controls, n = 21. Lines in a and b indicate median and interquartile range. Significance was determined by paired Wilcoxon signed-rank and unpaired Mann–Whitney U test. Correlation coefficients were calculated by Spearman’s rank correlation. **p < 0.01, ****p < 0.0001

Fig. 2. Differentiation of monocytes to fibrocytes by neutrophil extracellular traps (NETs) in vitro. Isolated peripheral blood mononuclear cells containing monocytes were stimulated with vehicle control, 500 ng/ml isolated NETs, NETs plus DNase or NETs plus anti-TLR-4 blocking antibody for 72 h (n = 5). Representative hematoxylin stainings for b vehicle control; c NETs; d NETs plus DNase; e NETs plus anti-TLR-4 blocking antibody; and f NETs plus DNase + anti-TLR-4 blocking antibody are shown. Data in a are given as mean ± standard error of the mean. Significance was determined by one-way analysis of variance with Dunn’s multiple post hoc comparison. *p < 0.05, **p < 0.01

Fig. 3. Fibrocyte activation by neutrophil extracellular traps (NETs) in vitro. Fibrocytes were stimulated with vehicle control, 500 ng/ml isolated NETs from healthy donors or NETs plus DNase for 6 h (n = 6). Cells were characterized using flow cytometry for their surface expression of a collagen-I; b bone morphogenic protein receptor II (BMPRII); c CD34; and for their intracellular expression of d interleukin (IL)-6. Data are provided as mean ± standard error of the mean. Significance was determined by one-way analysis of variance with Dunn’s multiple post hoc comparison. *p < 0.05

Fig. 4. Circulating fibrocytes in ST-segment elevation myocardial infarction. Flow cytometry was used to analyze the frequency and activation of fibrocytes. Fibrocytes were identified by co-expression of CD45, CD34 and collagen-I (see Supplemental Fig. S2). Data are expressed as a number of fibrocytes/106 CD45+ cells (baseline n = 50, 72 h FU n = 21, healthy n = 21) or mean fluorescence intensity (MFI) of b collagen-I (baseline n = 50, 72 h FU n = 21, healthy n = 21); c CD34 (baseline n = 50, 72 h FU n = 21, healthy n = 21); d CD11b (baseline n = 49, 72 h FU n = 21, healthy n = 21); e Bone morphogenic protein receptor II (BMPRII, baseline n = 43, 72 h FU n = 19, healthy n = 18). Respective n, numerical MFI values and statistical analyses are provided in Supplemental Tables 2, 3, and 4. Lines indicate median and interquartile range. Significance was determined by paired Wilcoxon signed-rank and unpaired Mann–Whitney U test. 72 h FU, follow-up at 72 h; *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 5. Fibrocytes and neutrophil extracellular traps in coronary culprit site thrombi. Thrombi were stained for a DAPI (blue); b CD45 (purple); c CD34 (red) and d collagen-I (yellow). e The merged image of sections a–d. White arrows mark fibrocytes. f For detection of NETs, thrombi were stained for f DAPI (blue) and g DNA-Histone (red). h The merged image for sections f-g

Fig. 6. Fibrocytes in myocardium after myocardial infarction. Exemplary staining of tissue specimens from patients dying from STEMI using a a modified Trichrome stain and immunofluorescence. The white box indicates an area shown with immunofluorescence in b–f. Specimens were stained for b DAPI (blue); c CD45 (white); d CD34 (red) and e collagen-I (yellow). f represents the merged image. White arrow marks a fibrocyte

Fig. 7. NETs marker and echocardiographic Wall Motion Score Index (WMSI) at follow-up (24 ± 8 months). a dsDNA at the culprit site and WMSI (n = 23); b WMSI and bone morphogenic protein receptor II (BMPRII) expression on culprit site fibrocytes (n = 22); c WMSI and CD11b expression on culprit site fibrocytes (n = 24). Correlation coefficients were calculated by Spearman’s rank correlation

Fig. 8. Graphical summary of the main findings. At the culprit site during ST-segment elevation myocardial infarction (STEMI), neutrophils undergo neutrophil extracellular trap (NET) formation. NETs activate fibrocytes and promote the differentiation of monocytes into fibrocytes, which can be antagonized by deoxyribonuclease and toll-like receptor (TLR)-4 blockade. Activated fibrocytes, expressing CD11b, adhere to the endothelium via interaction with intercellular adhesion molecule 1 (ICAM-1) and migrate into the myocardium to contribute to adverse remodeling