DIFFERENT RESPONSE OF HUMAN BLOOD NEUTROPHIL FCᵧRIIIB (CD16) TO EX VIVO MODELING OF BACTEREMIA BY PATHOGENIC AND OPPORTUNISTIC MICROORGANISMS

The presence of FcᵧRIIIb (CD16) on the surface of neutrophil granulocytes (NG) in human blood endows these cells, through interaction with IgG, with a key property of adaptive immunity: an antigen-specific cellular  response. The purpose of the work was to compare the ex vivo reaction of human neutrophil FcᵧRIIIb in response to the addition in blood the live cells of  opportunistic (Escherichia coli, Staphylococcus aureus) and pathogenic (Yersinia pestis, Brucella abortus) bacterial species. Materials and methods. The density of CD16 expression on NG was determined by flow cytometry in arbitrary fluorescence intensity units (MFI) after staining leukocytes with CD45-FITC and CD16-PE reagents (Backman Coulter, USA) when immunophenotyping them in the blood according to the Lyse/No-Wash protocol. To model  bacteremia, we used attenuated (vaccine) strains B. abortus 19BA and Y. pestis EV NIIEG, as well as strains S. aureus ATCC 6538 (209-P) and E. coli ATCC 25922. Blood was obtained from healthy donors (n= 10), who were not vaccinated against plague and brucellosis. Bacteria of 4 species were added to the blood samples of each donor in the same dose of 10⁸ mc/ml and the results were taken into account according to the studied indicator after 30 minutes, 1, 2 and 6 hours of incubation. Results. Opportunistic bacteria, in contrast to Y. pestis and B. abortus, caused a sharp decrease in the density of FcᵧRIIIb expression on human blood NG after 2 hours of incubation, which was a marker for the development of ex vivo IgG-mediated anaphylaxis, caused by the presence in the blood of all healthy adults IgG to specific antigens of E. coli and S.aureus. Changes in the CD16 marker induced in NG by opportunistic bacteria preceded degranulation and lysis of these cells under ex vivo conditions, while in the presence of Y. pestis and B. abortus neutrophils did not undergo degranulation and cytolysis within 6 hours. Conclusion. The experimental data obtained in the work reflect the known fact of the participation in the neutralization of E. coli and S. aureus of the phenomenon of extracellular antibody-dependent cytotoxicity of NG, which is realized in the bloodstream using the NETosis mechanism and plays a decisive role in the prevention of sepsis. The absence of a reaction of the molecular trigger CD16 NETosis to Y. pestis and B. abortus indicates that in the human body not vaccinated against plague or brucellosis, this protective antibody-dependent bactericidal mechanism does not work. Evaluation of FcᵧRIIIb reactivity by flow cytometry in an ex vivo bacteremia model is promising from the point of assessing the intensity of post-vaccination immunity in humans. 

1. Кравцов А.Л., Бугоркова С.А., Клюева С.Н., Кожевников В.А., Кудрявцева О.М. Определение экспрессии FcᵧRIIIB (CD16) на поверхности нейтрофилов крови привитых против чумы людей // Молекулярная медицина, 2020. Т.18, № 2. С.33-38. [Kravtsov A.L., Bugorkova S.A., Klyueva C.N., Kozhevnikov V.A., Kudryavtseva O.M. Determination of FcᵧRIIIB (CD16) expression on the surface of blood neutrophils in anti-plague vaccinated people. Molekulyarnaya meditsina = Molecular medicine, 2020, Vol.18, no.2, pp. 33-38. (In Russ.)] doi: 10.29296/24999490-2020-02-06.

2. Кравцов А.Л., Бугоркова С.А., Клюева С.А., Шмелькова Т.П., Кожевников В.А. Оценка изменений фенотипа, интенсивности дегрануляции, гибели и лизиса нейтрофилов при моделировании ex vivo стафилококковой бактериемии. Журнал Микробиологии, Эпидемиологии и Иммунобиологии, 2023. Т.100, №4. С.293-305. [Kravtsov A.L., Bugorkova C.A., Klyueva S.N., Shmelkova T.P., Kozhevnikov V.A. Assessment of changes in the phenotype, intensity of degranulation, death and lysis of neutrophils in ex vivo modeling of Staphylococcal bacteremia. Zhurnal mikrobiologii, epidemiologii i immunologii = Journal of Microbiology, Epidemiology and Immunobiology, 2023; Vol.100, no.4, pp. 293-305 (in Russ.] doi: https://doi.org/10.36233/0372-9311-384.

3. Нестерова И.В., Колесникова Н.В., Чудилова Г.А., Ломтатидзе Л.В., Ковалева С.В., Евглевский А.А., Нгуен Т.Л. Новый взгляд на нейтрофильные гранулоциты: переосмысление старых догм. Часть 2 // Инфекция и иммунитет, 2018. Т.8, № 1, С.7-18. [Nesterova I.V., Kolesnikova N.V., Chudilova G.A., Lomtatidze L.V., Kovaleva S.V., Evglevsky A.A., Nguyen T.L. The new look at neutrophilic granulocytes: rethinking old dogmas. Part 2. Infektsiya I immunitet = Russian Journal of Infection and Immunity, 2018, Vol.8, no.1, pp.7-18. (In Russ.)] doi: 10.15789/2220-7619-2018-1-7-18.

4. Фрадкин В.А. Диагностика аллергии реакциями нейтрофилов крови. М: Медицина, 1985,175с. [Fradkin V.A. Diagnosis of allergies by reactions of blood neutrophils. M: Medicine, 1985, 175 p (in Russ.)].

5. Barquero-Calvo E., Mora-Cartín R., Arce-Gorvel V., de Diego J.L., Chacón-Díaz C., Chaves-Olarte E., Gazman-Verri C., Buret A.G., Gorvel J-P., Moreno E. Brucella abortus induces the premature death of human neutrophils through the action of its lipopolysaccharide. PLoS Pathog., 2015, Vol.11, no.5, e1004853. https://doi.org/10.1371/journal.ppat.1004853.

6. Granger V., Peyneau M., Chollet-Martin S., de Chaisemartin L. Neutrophil extracellular traps in autoimmunity and allergy: immune complexes at work. Front. Immunology. 2019, 10: 2824. doi: 10.3389/fimmu.2019.02824.

7. Jönsson F., Mancardi D.A., Kita Y., Karasuyama Y., Iannascoli B., Van Rooijen N., Simizu T., Daeron M., Bruhns P. Mouse and human neutrophils induce anaphylaxis. J. Clin. Invest. 2011, Vol.121, no.4, pp.1484-1496. doi: 10.1172/JCI.145232.

8. Khodoun M.V., Strait R., Armstrong L., Yanase N., Frankelman F.D. Identification of markers that distinguish IgE-from IgG-mediated anaphylaxis. Proc. Natl. Acad. Sci., 2011, Vol.108, no.30, pp.12413-12418. doi:10.1073/pnas.1105695108.

9. МсDonald B., Urrutia R., Yipp B.G., Jenne C.N., Kubes P. Intravascular neutrophil extracellular traps capture bacteria from bloodstream during sepsis. Cell Host Microbe, 2012, 12(3):324-333. doi:10.1016/j.chom.2012.06.011.

10. Rosales C., Uribe-Querol E. Neutrophil activation by antibody receptors. In book: Neutrophis. Ed. Khajah M. London: Intechopen limited, 2019, 85p. doi: 10.5772/intechopen.80666.

11. Santos L.A., Grisolia J.C., Chavasco J.K., Coelho L.F.L., Malaquias L.C.C. Detection of serum antibodies and peripheral blood mononuclear cells response to Escherichia coli antigens in humans. Brazilian Archives of Biology and Technology. 2021, 64: e21200258. https://doi.org/10.1590/1678-4324-2021200258.

12. Stasulli M.N., Eichelberger K.R., Price P.A., Pechous R.D., Montgomery S.A., Parler J.S., Gildman W.E. Spatially distinct neutrophil responses within the inflammatory lesion of pneumonic plague. mBio, Vol.6, no.5, e01530-15. doi: 10.1128/mBio.01530-15.

13. Thomer L., Emolo C., Thammavongsa V., Lim H.K., McAdow M.E., Yu W., Kieffer M., Scheewind O., Missiakas D. Antibodies against a secreted product of Staphylococcus aureus trigger phagocytic killing. J. Exp. Med. 2016, Vol. 213, no.3, pp. 293-301.

14. Vera E.J., Chew Y.V., Nicholson L., Bruns H., Anderson P., Chen H.T. et al. Standartization of flow cytometry for whole blood immunophenotyping of islet transplant and transplant clinical trial recipients. PLoS One, 2019, Vol. 14, no.5, e0217163. doi: 10.1371/journal pone.0217163.

15. Wang Y., Jönsson F. Expression, role, and regulation of neutrophil Fcᵧ receptors. Front. Immunology, 2019, 10:1958. doi: 10.3389/fimmu.2019.01958.

16. Won D.I., Kim S., Lee E.H. Neutrophil oxidative burst as a diagnostic indicator of IgG-mediated anaphylaxis. Blood Res., 2018, Vol.53, no.4, pp.299-306. doi: 10.5045/br.2018.53.4.299.