Neutrophil extracellular traps and monocyte subsets at the culprit lesion site of myocardial infarction patients

Figure 1. Stimulation of monocytes with NETs in vitro. (A) Monocyte subset shift as % of total monoctes, expression of (B) CD11b, (C) CD142 and (D) CX3CR1 of classical, intermediate and non-classical monocytes. Monocyte subsets in percent (%) or mean fluorescence intensity (MFI) levels are displayed. Monocytes were stimulated in whole blood from healthy donors (n = 5) with NETs derived from isolated neutrophils from a healthy donor (n = 1) or vehicle control for 60 minutes. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 2. Surrogate markers of neutrophil extracellular traps. (A) Double-stranded DNA (dsDNA, n = 83, femoral 581.7 [519.8–672.7] versus culprit site 692.7 [552.7–888.0] ng/ml) and (B) citrullinated histone 3 levels (citH3, n = 87, femoral 233 [156.5–545.6] versus culprit site 380.5 [173.8–820.3] ng/ml) were significantly increased at the culprit site of STEMI patients. Enzymatic infarct size expressed as the creatine phosphokinase isoform MB area under the curve (CK-MB AUC) correlated with (C) double-stranded DNA (dsDNA, n = 65, rs = 0.409, p < 0.001) and (D) citrullinated histone 3 (citH3, n = 65, rs = 0.315, p < 0.01). Significance ****p < 0.0001.

Figure 3. Monocyte subsets at the culprit lesion site of STEMI patients. (A) Classical monocytes (femoral 91.4 [85.2–94.0] versus culprit site 89.2 [82.8–92.9] %), (B) intermediate monocytes (femoral 4.5 [2.9–7.9] versus culprit site 5.2 [3.1–8.9] %) and (C) non-classical monocytes (femoral 3.6 [2.0–7.19] versus culprit site 4.9 [2.5–8.2] %) were determined by flow cytometry as % of total monoctes in femoral and culprit site whole blood samples of STEMI patients (n = 91). The gating strategy is depicted in Supplementary Fig. 2. (D) Culprit site dsDNA correlates with non-classical monocyte percentage (n = 63, rs = −0.224, p < 0.05). ****p < 0.0001.

Figure 4. Expression markers of monocyte subsets in STEMI patients. Expression markers were determined by flow cytometry in femoral and culprit site whole blood samples (n = 36). (A) CX3CR1, (B) CD11b, (C) CD142 and (D) HLA-DR on classical monocytes, intermediate monocytes and non-classical monocytes. Data are presented as mean fluorescence intensity (MFI) of monocyte subsets. All expression marker data are listed in Supplementary Table 1. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5. Correlation of non-classical monocyte CX3CR1 expression with NET surrogate markers and infarct size. Low expression of CX3CR1 on non-classical monocytes correlates with higher (A) double-stranded DNA (dsDNA, n = 31, rs = −0.423, p < 0.05), (B) citrullinated histone 3 (citH3, n = 33, rs = −0.359, p < 0.05) and (C) enzymatic infarct size expressed as the creatine phosphokinase isoform MB area under the curve (CK-MB AUC, n = 30, rs = −0.556, p < 0.01).