Human blood granulocyte degranulation and lysis intensity during interaction with <i>Yersinia pestis</i> in the <i>ex vivo</i> model of bacteriemia

Introduction. Considering the decisive role of antibacterial strategies of secretory degranulation and NETosis in the prevention of sepsis, it is of interest to study the interaction of Yersinia pestis with human blood granulocytes using an ex vivo bacteremia model to assess the effectiveness of this antibacterial strategy of the host organism in plague. Purpose: evaluation of granulocyte degranulation and lysis in human whole blood samples in the presence of live Y. pestis. Materials and methods. Bacteremia was modeled by adding Y. pestis EV NIIEG cells grown at 37оC or 28оC to whole blood (with heparin) at a dose of 108 mc/mL. Strains Staphylococcus aureus ATCC 6538 (209-P) and Escherichia coli ATCC 25922 were used in experiments with blood from the same donors as a positive control. The bactericidal effect was determined at different time points during blood incubation at 37оC (for 6 hours) using a microbiological method. Using flow cytometry, immunophenotyping of leukocytes was performed in the blood according to the Lyse/No-Wash protocol to determine the expression of the main leukocyte antigen CD45 and the secretory azurophilic degranulation marker CD63 on the surface of the granulocytes. The intensity of granulocyte lysis was assessed by the decrease in the proportion of these cells in the total leukocyte population. Results. It has been established that live plague microbes, unlike E. coli and S. aureus, do not cause the development of azurophilic degranulation in human blood granulocytes and do not induce autolysis (NETosis) of these cells within 6 hours when bacteremia is modeled ex vivo. Conclusion. Information was obtained on the ability of the plague microbe to suppress the extracellular bactericidal mechanisms of granulocytes in the blood of people not vaccinated against plague, which effectively function under conditions of bacteremia against E. coli and S. aureus. An experimental and methodological basis has been prepared for further research with blood cells from donors vaccinated against plague in order to develop new effective tests for assessing the intensity of acquired cellular anti-plague immunity in humans.