Cytometric assessment of blood granulocyte damage intensity in vaccinated guinea pigs upon interaction with Yersinia pestis in an ex vivo bacteremia model

Kravtsov A.L., Goncharova A.Yu., Klyueva S.N., Bugorkova S.A.

Russian Research Anti-Plague Institute "Microbe", Saratov, Russia

Neutrophil granulocytes (NG) play an important role in the mechanisms of immunoallergic reaction and are capable of destroying extracellular pathogens through NETosis. The aim of the work was to evaluate by flow cytometry the intensity of NG damage in the blood of plague-immunized and intact guinea pigs when modeling plague bacteremia ex vivo.
Materials and methods. Guinea pigs (26 individuals) were immunized subcutaneously with the vaccine strain Y. pestis EV NIIEG and three of its isogenic derivatives; the comparison group consisted of intact animals. On the 21st day of immunogenesis, blood was taken from the heart in a volume of 4-5 ml from guinea pigs in the experimental and comparison groups. Bacteremia was modeled ex vivo by adding 108 mc/ml of Y.pestis grown at 37°С (Yp37), 28°С (Yp28) or cultures inactivated by heating at 60°С – DYp28 and DYp37 – to blood samples. For each group, blood samples without added bacteria served as controls. After 4 h of ex vivo incubation, the samples were fixed and disinfected using the BD FACS Lysing solution reagent, and the NG reaction was taken into account by flow cytometry. When assessing the NG reaction, cells were considered damaged if they had lost their initially high degree of granularity in the process of secretory degranulation and cytolysis. The neutrophil damage index (NDI) was calculated as Dk -Do/Dk, where Dk and Do are the relative content of NG with the initial degree of granularity, respectively, in the control and experiment.
Results. Degenerative changes in granulocytes were recorded only in blood samples from pre-immunized animals in response to the ex vivo addition of Yp37 to the blood. It was noted that the NG response in animals immunized with the vaccine strain was 3 times higher than in guinea pigs immunized with isogenic variants of this strain, which were 200 times less effective in protecting guinea pigs from plague.
Conclusion. The results of cytometric assessment of NDI in an ex vivo model of plague bacteremia can be used to characterize the severity of post-vaccination anti-plague immunity.