Plasma neutrophil extracellular trap level is modified by disease severity and inhaled corticosteroids in chronic inflammatory lung diseases

Abstract

www.nature.com/scientificreports www.nature.com/scientificreports/ a background. Measurements were conducted by BD FACSCalibur (BD Biosciences). Optimization of cytometer settings and gating strategy was carried out as described earlier for extracellular vesicle quantification [30][31][32] . For the quantification only the number of MPO/Sytox Red/histone triple positive events were considered.Human samples. Whole blood samples were collected to sodium citrate tubes from all individuals. During the blood collection no patients were in exacerbation. Platelet free plasma samples were prepared by centrifugation two times at 2500 RPM for 15 minutes. For in vivo NET quantification the platelet free plasma samples were processed as described above.Confocal laser-scanning microscopy. Platelet free plasma samples (10 µL) were attached to MilliCell EZ slides (Merck-Millipore), dried for 10 minutes at room temperature (RT) and were fixed with 4% 0.22 µm filtered PFA for 20 minutes at RT. Following three washes with 1% PBS, samples were blocked with 5% BSA and incubated for 30 minutes at RT. Because of NETs occurrence we used BSA instead of fetal bovine serum (FBS). Primary antibodies (mouse monoclonal anti-MPO FITC (BD Biosciences) and rabbit polyclonal anti-histone H3 antibody (ab5103, Abcam) in 5% BSA solution, both same as before) were used in 1:100 dilution for 1 hour at RT in dark. After three times washes with 5% BSA, secondary antibodies were applied such as F(ab')2-goat anti-mouse IgG (H + L) conjugated with eFluor 570 (eBioscience) in 1:200 dilution and goat anti-rabbit IgG conjugated with ATTO647N (Sigma-Aldrich) in 1:500 dilution for 1 hour at RT in dark. Washed twice with 1% PBS and twice with distillated water. Slides were mounted with DAPI containing Prolong Diamond (Invitrogen). Samples were examined by Lecia SP8 laser-confocal microscope (Leica Microsystems, Wetzlar, Germany). Statistical analysis.All statistical evaluations were performed with R statistical computing program (R Foundation for Statistical Computing, Vienna, Austria). Differences between experimental groups were analysed by one-way ANOVA. Differences between subgroups of patients were analysed by Student's t-test (unpaired, two-tailed). P-values were adjusted with Benjamini Hochberg method to correct for the false discovery rate, and adjusted p-values <0.05 were considered to be statistically significant. The relationship between the level of NET and FEV1 was assessed by calculating Pearson's correlation coefficient. R statistical computing program was used for graphic illustrations. Data availabilityAll datasets and protocols of the measurements are available in request.

Complement C3 vs C5 inhibition in severe COVID-19: Early clinical findings reveal differential biological efficacy

“…Of note, the increase of NETosis in a fraction of ecu-treated patients with high neutrophil counts, despite the concomitant use of corticosteroids, likely argues against a steroid-driven effect on neutrophilia. This notion is consistent with evidence that corticosteroids can dampen neutrophil responses and NET release in lung patients [ 38 ].…”

Neutrophils in COVID-19

“…Corticosteroids are a currently recommended treatment for severe COVID-19 ( 104 ) shown to reduce neutrophils’ respiratory burst and recruitment to the inflamed tissue at system level ( 105 ) and inhaled glucocorticoids have been shown to reduce NET formation in asthma patients ( 106 ). Additionally, they might have a pro-inflammatory and anti-apoptotic effect on neutrophils by reducing the levels of both, the secreted IL-1 receptor antagonist (IL-1RA) and the membrane-exposed Fas ( 105 ).…”

IL‐33/ST2 axis deficiency exacerbates neutrophil‐dominant allergic airway inflammation

“…NETs play an important role in host defence, and these structures are also associated with tissue damage 5 . Several recent studies have revealed that NETs, which are associated with disease severity, are increased in a subset of patients with severe asthma 6,7 …”