In vitro phenotypic re-orientation of functionally important neutrophil subpopulations and their microbicidal activity in the children with purulent inflammatory diseases influenced by glucosaminil muramildipeptide

Numerous studies over last decade have shown that functional capacity of neutrophil granulocytes (NG) determines the course and outcome of many diseases. Identification of phenotypic variants of functionally significant NG subpopulations is a new approach allows us to assess the adequacy or deficiency of NG involvement into infectious inflammation processes at molecular level. An opportunity of reorienting a deficient NG subpopulational phenotype in purulent inflammatory diseases due to the rearrangement of the receptor set induced by various immunotropic substances may serve as a key to recovery of normal NG functioning.

Our aim was to study the effect of glucosaminylmuramyldipeptide (GMDP) under in vitro conditions upon the phenotypic profile of four functionally significant subpopulations, i.e., CD62L⁺CD63^-NG, CD62L⁺CD63⁺NG and CD64^-CD32⁺CD16⁺CD11b⁺NG, CD64⁺CD32⁺CD16⁺CD11b⁺NG, along with assessment of expression density of appropriate membrane molecules and NG microbicidal activity in the children with purulent inflammatory diseases. 90 samples of peripheral blood (PC) were taken from children 2 to 4 years old, including 12 children with minor purulent infection (MPI), and 7 children were studied as conditionally healthy controls. Their peripheral blood was incubated for 60 minutes at 37 °C with GMP (10^-6 g/l). Using flow cytometry technique, the relative numbers of some NG subpopulations, i.e., CD64^-CD16⁺CD32⁺CD11b⁺NG, CD64⁺CD16⁺CD32⁺CD11b⁺NG, CD62L⁺CD63^-NG, CD62L⁺CD63⁺NG were evaluated, and the phenotype features of each subpopulation were investigated according to the density of appropriate membrane molecule expression (MFI). In parallel, phagocytic and microbicidal activity of NG was tested in these study groups. The obtained data indicate for presence of for distinct NG subpopulations, both in healthy children and in children with MPI. We have revealed phenotypic transformation of the four studied NG subpopulations from MPI patients including disturbed phagocytic and microbicidal functions of the cells. Using of this in vitro system, we have shown that the transformed phenotype of the four functionally significant NG subpopulations of MPI patients was re-arranged under GMDP treatment. At the same time, the number of CD62L⁺CD63⁺NG and CD64^-CD32⁺CD16⁺CD11b⁺NG subpopulations was increased, along with decreased amounts of CD64⁺CD32⁺CD16⁺CD11b⁺NG and CD62L⁺CD63^-NG subpopulations, being accompanied by restoration of microbicidal activity of NGs.

The obtained data allow us to accomplish current understanding of immunotropic effects of GMDP, and to extend the potential scope of its experimental and clinical application. The new data on GMDP effects revealed by in vitro system, i.e. phenotype rearrangement of functionally significant NG subpopulations CD64^-CD16⁺CD32⁺CD11b⁺, CD64⁺CD16⁺CD32⁺CD11b⁺, CD62L⁺CD63^-, CD62L⁺CD63⁺ in atypical purulent inflammatory diseases in children, may be used in the future in order to develop innovative strategies of immunotherapy aiming for correction of NG dysfunction in children with MPI.

1. Abakumova T.V., Gening T.P., Dolgova D.R., Antoneeva I.I., Peskov A.B., Gening S.O. Phenotype of circulating neutrophils at different stages of cervical neoplasia. Meditsinskaya immunologiya = Medical Immunology (Russia), 2019, Vol. 21, no. 6, pp. 1127-1138. (In Russ.) doi: 10.15789/1563-0625-2019-6-1127-1138.

2. Abaturov A.E., Agafonova E.A., Abaturova N.I., Babich V.L. Evolution and age characteristics of the innate and adaptive immune system. Sovremennaya pediatriya = Modern Pediatrics, 2016, no. 3 (75), pp. 74-84. (In Russ.)

3. Nesterova I.V., Chudilova G.A., Lomtatidze L.V., Kovaleva S.V., Kolesnikova N.V., Avdeeva M.G., Rusinova T.V. Differentiation of variants subpopulations transformed phenotype CD16+CD11b+ neutrophils in acute viral and acute bacterial infections. Immunologiya = Immunology, 2016, Vol. 37, no. 4, pp. 199-204. (In Russ.)

4. Nesterova I.V., Malinovskaya V.V., Khaydukov S.V., Nguyen T.D.L., Chudilova G.A., Lomtatidze L.V. Differentiated effects of glucosaminyl muramil dipeptide on the non-transformed and experimentally transformed phenotype of CD62L+CD63+CD66d+ neutrophilic granulocytes in conventionally healthy people. Meditsinskaya immunologiya = Medical Immunology (Russia), 2018, Vol. 20, no. 6, pp. 847-854. (In Russ.) doi: 10.15789/1563-0625-2018-6-847-85410.17513/NP278.

5. Nesterova I.V., Chudilova G.A., Kovaleva S.V., Lomtatidze L.V., Kolesnikova N.V., Yevlevsky A.A. Methods for comprehensive assessment of the functional activity of neutrophil granulocytes in health and disease (guidelines). Krasnodar, 2017. 52 p.

6. Chudilova G.A., Nesterova I.V. Phenotypic profile subset CD64-CD16+CD32+CD11b+, CD64+CD16+CD32+CD11b+ neutrophil granulocytes in healthy newborns, conditionally healthy children of different age groups and conditionally healthy adult individuals. Rossiyskiy immunologicheskiy zhurnal = Russian Journal of Immunology, 2019, Vol. 13, no. 1, pp. 53-61. (In Russ.)

7. Shmagel K.V., Zubareva N.A., Renzhin A.V. Local immunity patterns in purulent wounds. Meditsinskaya immunologiya = Medical Immunology (Russia), 2010, Vol. 12, no. 4-5, pp. 393-398. (In Russ.) doi: 10.15789/1563-0625-2010-4-5-393-398.

8. Bartlett J.G. Methicillin-resistant Staphylococcus aureus infections. Topics in HIV medicine: a publication of the International AIDS Society, USA, 2008, Vol. 16, no. 5, pp. 151-155.

9. Boer K., Vogelsang H., Deufel T., Pfister W., Kiehntopf M. CD62L on neutrophil granulocytes, a useful, complementary marker for the prediction of ventriculitis in blood-containing CSF. Clin. Biochem., 2010, Vol. 43, no. 16-17, pp. 1351-1355.

10. Boyle J.P., Parkhouse R., Monie T.P. Insights into the molecular basis of the NOD2 signalling pathway. Open Biol., 2014, Vol. 4, no. 12, pp. 140-178.

11. Cortjens B., Ingelse S.A., Calis J.C., Valar A.P., Koendetman L., Bem R.A., van Woensel J.B. Neutrophil subset responses in infants with severe viral respiratory infection. Clin. Immunol., 2017, Vol. 176, pp. 100-106.

12. Chen K., Nishi H., Travers R., Tsuboi N., Martinod K., Wagner D.D., Stan R., Croce K., Mayadas T.N. Endocytosis of soluble immune complexes leads to their clearance by FcγRIIIB but induces neutrophil extracellular traps via FcγRIIA in vivo. Blood, 2012, Vol. 120, no. 22, pp. 4421-4431.

13. Dal Ponte S., Alegretti A., Pilger D., Rezende G., Andrioli G., Ludwig H. Diagnostic accuracy of CD64 for sepsis in emergency department. J. Glob. Infect. Dis., 2018, Vol. 10, no. 2, pp. 42-46.

14. de Jong E., de Lange D.W., Beishuizen A., van de Ven P.M., Girbes A.R., Huisman A. Neutrophil CD64 expression as a longitudinal biomarker for severe disease and acute infection in critically ill patients. Int. J. Lab. Hematol., 2016, Vol. 38, no. 5, pp. 576-584.

15. Garley M., Jabłońska E. Heterogeneity among neutrophils. Arch. Immunol. Ther. Exp. (Warsz), 2018, Vol. 66, no. 1, pp. 21-30.

16. Hoffmeyer F., Witte K., Schmidt R.E. The high-affinity FcyRI on PMN: regulation of expression and signal transduction. Immunology, 1997, no. 92, pp. 544-552.

17. Kobayashi Y. Neutrophil biology: an update. EXCLI J., 2015, no. 14, pp. 220-227.

18. Laman A.G., Lathe R., Shepelyakovskaya A.O., Gartseva A., Brovko F.A., Guryanova S., Alekseeva L., Meshcheryakova E.A., Ivanov V.T. Muramyl peptides activate innate immunity conjointly via YB1 and NOD2. Innate Immunity, 2016, Vol. 22, no. 8, pp. 1-8.

19. Rollet-Labelle E., Gilbert C., Naccache P.H. Modulation of human neutrophil responses to CD32 cross-linking by serine/threonine phosphatase inhibitors: cross-talk between serine/threonine and tyrosine phosphorylation. J. Immunol., 2000, Vol. 164, no. 2, pp. 1020-1028.

20. Scapini P., Marini O., Tecchio C., Cassatella M.A. Human neutrophils in the saga of cellular heterogeneity: insights and open questions. Immunol. Rev., 2016, Vol. 273, no. 1, pp. 48-60.

21. van der Heijden J., Nagelkerke S., Zhao X., Geissler J., Rispens T., van den Berg T.K., Kuijpers T.W. Haplotypes of FcγRIIa and FcγRIIIb polymorphic variants influence IgG-mediated responses in neutrophils. J. Immunol., 2014, Vol. 192, no. 6, pp. 2715-2721.

22. Yang F., Feng C., Zhang X., Lu J., Zhao Y. The diverse biological functions of neutrophils, beyond the defense against infections. Inflammation, 2017, Vol. 40, no. 1, pp. 311-323.