Assessment of changes in the phenotype, intensity of degranulation, death and lysis of neutrophils in <i>ex vivo</i> modeling of <i>Staphylococcal</i> bacteremia

Abstract

Introduction. At present, it is important to study the interaction of Staphylococcus aureus with human innate immunity cells under conditions of ex vivo bacteremia modeling, but not only in experiments with phagocytes previously isolated from the blood. Purpose: comparative assessment of changes in the phenotype, intensity of aurophilic degranulation, death and lysis of neutrophils in whole blood samples from apparently healthy donors following the addition of live and killed microbial cells of S. aureus ATCC 6538 (209-P). Materials and methods. Bacteremia was modeled by adding microbes to whole blood (with heparin) at doses ranging from 108 to 101 mc/ml. The bactericidal effect was determined at different time points of blood incubation at 37C (for 6 hours) by the microbiological method. The blood leukocyte immunophenotyping was performed using flow cytometry according to the Lyse/No-Wash protocol to determine the expression of markers of azurophilic degranulation (CD63), early apoptosis (CD95) on the surface of neutrophilic granulocytes (NG) and cell adhesion (CD62L), as well as to assess the surface expression density of the molecular trigger of netosis FcᵧRIIIb (CD16) and tyrosine protein phosphatase (CD45), which is a regulator of FcR-mediated cell signaling during phagocytosis and netosis. The presence of degenerative changes in NG was confirmed by microscopy in the analysis of blood smears stained according to RomanovskyGiemsa. Results. Significant differences were revealed in the response of human blood NG to live and killed S. aureus cells according to the studied phenotypic parameters. Only live microbes triggered azurophilic degranulation in NCs and stimulated, starting from 2 h incubation, the appearance of a large number of NG with the CD95+ phenotype in the blood. In addition, live bacteria induced in phagocytes twice as much functional rearrangement in terms of CD45 expression. They induced a rapid decrease in the density of CD16 expression on NG and activated NG lysis in the blood with an intensity depending on the initial microbial concentration. Following 6 h incubation at concentrations of more than 105 mc/ml, flow cytometry detected the presence in the blood of a population of low density neutrophils characteristic of sepsis (Low Density Neutrophils with the CD63+CD16low phenotype), whose cells are prone to spontaneous autolysis (netosis). Conclusion. Under the conditions of ex vivo modeling of bacteremia, various strategies of human blood neutrophils in the implementation of the mechanisms of protection of the macroorganism from infection were evaluated, and the prospects of using the obtained information to identify individuals at high risk of developing staphylococcal sepsis were determined.