EXPERIENCE IN THE DEVELOPMENT AND APPLICATION OF A METHOD FOR INVESTIGATING THE ABILITY OF NEUTROPHILS TO FORM EXTRACELLULAR TRAPS

Abstract

The phenomenon of neutrophil extracellular trap (NETs) formation, or netosis, plays a key role in the pathogenesis of infectious and inflammatory diseases. However, the lack of standardized methods for assessing NETs makes it difficult to correctly interpret the results. The aim of the study was to develop a practical methodology to investigate the ability of neutrophils to form NETs, which would allow correct identification of netosis-associated phenomena.

Neutrophils were isolated from peripheral blood of 15 healthy donors by gradient centrifugation (Ficoll-Verographin, density gradient 1.077 - 1.105 g/mL). A mixture of bacteria (Lactobacillus reuteri, L. acidophilus, L. rhamnosus, Bifidobacterium longum) was used to stimulate netosis. Propidium iodide intercalating dye and mouse monoclonal antibodies to human CD15/FITC were used to visualize NETs and other luminescence-positive objects. Luminescence microscopy was performed at excitation wavelengths of 450-480 nm and emission wavelengths from 515 nm. Intact neutrophils (bright green objects without a stained nucleus), activated neutrophils (bright green with a red-orange, propidium iodide stained nucleus), early netosis cells (bright green cells with nuclear material in one or more locations outside the cell membrane), NETs: cloud-shaped (in the form of a «cloud» surrounding the neutrophil or its fragments on all sides and more than 1.5 times its size) and filamentous (in the form of filaments 2 or more times the size of the neutrophil) were recorded in the preparation. The proportion of each type of observed objects relative to all fluorescent-positive objects was determined. During the optimization of the method the key parameters were determined: the temperature of leukocytes incubation with the stimulant (37°C), its concentration (2.5x10⁹ bacteria/ml), stimulation time (30 min).

Evaluation of the reproducibility of the method showed an acceptable inter-series coefficient of variation: 13.8% for filamentous and 15.2% for cloud-shaped NETs. The study method was applied in different groups of patients with certain infectious (pulmonary tuberculosis) and non-infectious pathology (ulcerative colitis, aneurysmal bone cysts). The results of the assessment of neutrophils' ability to form NETs in the representatives of the examined cohorts allow us to conclude that the level of sensitivity and manifestability of the indicators registered with this technique is adequate.

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