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

Postępy Higieny i Medycyny Doświadczalnej
Volume 77 (2023): Issue 1 (January 2023)
By:
Open Access
|Dec 2023

Figures & Tables

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

VariablePatients with AS (n=13)Controls (n=9)
Age, years66 [60–70]59 [58–65]0.148
Male, n (%)10 (76.9)7 (77.8)0.991
Body mass index, kg m−231.2 ±5.424 ±6.50.002
Risk factors, n (%)
Arterial hypertension12 (92.3)0ND
Current smoking1 (7.7)0ND
Medications, n (%)
Beta-blockers11 (84.6)0ND
Acetylsalicylic acid7 (53.8)0ND
Angiotensin converting enzyme inhibitors9 (69.2)0ND
Statins10 (76.9)0ND
Echocardiographic parameters
Aortic valve area, cm20.86 [0.8–0.9]ND
Mean gradient, mmHg48 ±10.0ND
Maximum gradient, mmHg80 ±15.4ND
Peak transvalvular velocity, m/s4.5 ± 0.4ND
Left ventricular ejection fraction, %52.3 ±12.5ND
Laboratory investigation
White blood cells, 103/µl6.2 ±1.46.5 ±1.70.655
Neutrophils, 103/µl3.5 [2.9–4.0]3.2 [2.3–3.6]0.189
Creatinine, µmol/L96 [86–100]67 [48–76]0.003
C-reactive protein, mg/L1.5 [1.0–2.5]0.99 [0.99–1.46]0.015
Glucose, mmol/L5.2 [5.0–5.6]5.0 [4.7–5.4]0.115
Total cholesterol, mmol/L4.2±1.14.9±0.90.131
Low density lipoprotein cholesterol, mmol/L2.9±1.03.0±1.10.827
High density lipoprotein cholesterol, mmol/L1.1±0.31.4±0.30.032
Triglycerides, mmol/L1.7±0.71.5±0.70.518
DOI: https://doi.org/10.2478/ahem-2023-0018 | Journal eISSN: 1732-2693
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Language: English
Page range: 163 - 169
Submitted on: Oct 14, 2022
Accepted on: Mar 21, 2023
Published on: Dec 29, 2023
Published by: Hirszfeld Institute of Immunology and Experimental Therapy
In partnership with: Paradigm Publishing Services
Publication frequency: 1 times per year
Keywords:
neutrophil extracellular traps,
aortic stenosis,
flow cytometry
Related subjects:
Life sciences,
Molecular biology,
Microbiology and virology,
Medicine,
Basic medical science,
Immunology

© 2023 Piotr Kopiński, Magdalena Kopytek, Michał Ząbczyk, Anetta Undas, Joanna Natorska, published by Hirszfeld Institute of Immunology and Experimental Therapy
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 License.

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