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Neutrophil extracellular traps (NETs) in aortic stenosis: Comparison of methods for assessment of NETs formation

Piotr Kopiński
Piotr Kopiński
, 
Magdalena Kopytek
Magdalena Kopytek
, 
Michał Ząbczyk
Michał Ząbczyk
, 
Anetta Undas
Anetta Undas
 oraz 
Joanna Natorska
Joanna Natorska
   | 29 gru 2023
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Article Category: Original Study
Data publikacji: 29 gru 2023
Zakres stron: 163 - 169
Otrzymano: 14 paź 2022
Przyjęty: 21 mar 2023
DOI: https://doi.org/10.2478/ahem-2023-0018
Słowa kluczowe
© 2023 Piotr Kopiński et al., published by Sciendo
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Figure 1.

Serum levels of NET-specific biomarkers in AS patients and healthy controlsSingle dot represents individual result. Horizontal central lines express median values. Boxes express interquartile (Q1–Q3) range
Serum levels of NET-specific biomarkers in AS patients and healthy controlsSingle dot represents individual result. Horizontal central lines express median values. Boxes express interquartile (Q1–Q3) range

Figure 2.

Representative microphotograph of NETosis generated after ionomycin-stimulation of isolated neutrophils from healthy volunteersImmunolabelling for citH3 (green). The red arrows indicate the NET-releasing neutrophils. Original magnification 40×
Representative microphotograph of NETosis generated after ionomycin-stimulation of isolated neutrophils from healthy volunteersImmunolabelling for citH3 (green). The red arrows indicate the NET-releasing neutrophils. Original magnification 40×

Figure 3.

NETosis examination in flow cytometrya. Healthy person from the control group, ionomycin-stimulated neutrophils. Density plot, FSC lin/SSC log (SSC data presented in logarithmic scale) display. Neutrophils gated as elliptic gate. Arrows indicate: eosinophils (green arrow), disintegrated neutrophils (debris, red arrow) and mononuclears (blue arrow).b. The same sample as a. Gated neutrophils displayed as MPO FITC / citH3 Alexa Fluor700 dot plot. Markers inserted according to the control sample (stained with secondary antibodies, ionomycin-stimulated). NET-releasing neutrophils are MPO+citH3+ events in the upper right quadrant (6.7%).c. In comparison to the control, ionomycin stimulated neutrophils in AS patient. High percentage of NET-releasing neutrophils (22.1%).d. NET-releasing neutrophils in unstimulated (ionomycin-free) sample (baseline NETs formation in 3.7% neutrophils) from AS patient.e. The cellular debris (disintegrated neutrophils) gated. They contain the major part of positive MPO+citH3+ staining in the sample but they are not NET-releasing neutrophils.f. The percentage of NET-releasing (MPO+citH3+) neutrophils in peripheral blood of AS patients compared to healthy controls. Single dot represents individual result. Horizontal central lines express median values. Boxes express interquartile (Q1–Q3) range.
NETosis examination in flow cytometrya. Healthy person from the control group, ionomycin-stimulated neutrophils. Density plot, FSC lin/SSC log (SSC data presented in logarithmic scale) display. Neutrophils gated as elliptic gate. Arrows indicate: eosinophils (green arrow), disintegrated neutrophils (debris, red arrow) and mononuclears (blue arrow).b. The same sample as a. Gated neutrophils displayed as MPO FITC / citH3 Alexa Fluor700 dot plot. Markers inserted according to the control sample (stained with secondary antibodies, ionomycin-stimulated). NET-releasing neutrophils are MPO+citH3+ events in the upper right quadrant (6.7%).c. In comparison to the control, ionomycin stimulated neutrophils in AS patient. High percentage of NET-releasing neutrophils (22.1%).d. NET-releasing neutrophils in unstimulated (ionomycin-free) sample (baseline NETs formation in 3.7% neutrophils) from AS patient.e. The cellular debris (disintegrated neutrophils) gated. They contain the major part of positive MPO+citH3+ staining in the sample but they are not NET-releasing neutrophils.f. The percentage of NET-releasing (MPO+citH3+) neutrophils in peripheral blood of AS patients compared to healthy controls. Single dot represents individual result. Horizontal central lines express median values. Boxes express interquartile (Q1–Q3) range.

Baseline characteristics of patients with aortic stenosis (AS) and healthy controls

Variable Patients with AS (n=13) Controls (n=9)
Age, years 66 [60–70] 59 [58–65] 0.148
Male, n (%) 10 (76.9) 7 (77.8) 0.991
Body mass index, kg m−2 31.2 ±5.4 24 ±6.5 0.002
Risk factors, n (%)
Arterial hypertension 12 (92.3) 0 ND
Current smoking 1 (7.7) 0 ND
Medications, n (%)
Beta-blockers 11 (84.6) 0 ND
Acetylsalicylic acid 7 (53.8) 0 ND
Angiotensin converting enzyme inhibitors 9 (69.2) 0 ND
Statins 10 (76.9) 0 ND
Echocardiographic parameters
Aortic valve area, cm2 0.86 [0.8–0.9] ND
Mean gradient, mmHg 48 ±10.0 ND
Maximum gradient, mmHg 80 ±15.4 ND
Peak transvalvular velocity, m/s 4.5 ± 0.4 ND
Left ventricular ejection fraction, % 52.3 ±12.5 ND
Laboratory investigation
White blood cells, 103/µl 6.2 ±1.4 6.5 ±1.7 0.655
Neutrophils, 103/µl 3.5 [2.9–4.0] 3.2 [2.3–3.6] 0.189
Creatinine, µmol/L 96 [86–100] 67 [48–76] 0.003
C-reactive protein, mg/L 1.5 [1.0–2.5] 0.99 [0.99–1.46] 0.015
Glucose, mmol/L 5.2 [5.0–5.6] 5.0 [4.7–5.4] 0.115
Total cholesterol, mmol/L 4.2±1.1 4.9±0.9 0.131
Low density lipoprotein cholesterol, mmol/L 2.9±1.0 3.0±1.1 0.827
High density lipoprotein cholesterol, mmol/L 1.1±0.3 1.4±0.3 0.032
Triglycerides, mmol/L 1.7±0.7 1.5±0.7 0.518
eISSN:
1732-2693
Język:
Angielski
Częstotliwość wydawania:
Volume Open
Dziedziny czasopisma:
Life Sciences, Molecular Biology, Microbiology and Virology, Medicine, Basic Medical Science, Immunology
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