Transformation of NETs under the effect of pathogens and IgG

Background. Many studies have shown that neutrophil extracellular traps (NETs) in the form of web-like structures are present in the peripheral blood of patients with inflammatory diseases. In our research, in addition to traditional web-like NET structures, several anomalous forms were identified, including NETs with cloud-like appearance.

Aim. To investigate morphological and functional transformation of NETs under the influence of Klebsiella pneumoniae and immunoglobulin G (IgG).

Materials and methods. The study included 42 patients of Moscow City Clinical Hospital No. 51: 28 patients with acute inflammation in the abdominal cavity (appendicitis, cholecystitis, pancreatitis, peritonitis), 6 patients diagnosed with ulcerative colitis, and 8 patients with hernias. Neutrophils were isolated using gradient-density centrifugation. To calculate NETs, we used SYBR Green I-induced fluorescence microscopy (Evrogen, Russia), with the dye specifically interacting with double-stranded DNA. The functional activity of NETs was determined in the Klebsiella pneumoniae (ATCC 700603) capture test.

Results. In patients with inflammatory diseases of the abdominal cavity in the postoperative period, the functional activity of NETs was several times lower than in healthy individuals. NETs in these patients capture and bind no more than 20 cells of the microorganism. Under the effect of IgG, neutrophil networks transform into loose cloud-like structures, which can hardly capture and bind the pathogen, binding only 8.46 ± 0.44 cells of the microorganism. Spontaneous enzymatic degradation of cloud like NETs may be accompanied by the production of secondary alteration factors.

Conclusion. The results of the study provide the grounds for the development of new approaches to elaborating vaccination regimens and using immunobiologics that require preliminary monitoring of the state of innate immunity, in particular, neutrophil networks in the patient’s body.

1. Chakraborty S., Tabrizi Z., Bhatt N.N., Franciosa S.A., Bracko O. A brief overview of neutrophils in neurological diseases. Biomolecules. 2023;13(5):743. DOI: 10.3390/biom13050743.

2. Казимирский А.Н., Салмаси Ж.М., Порядин Г.В. Антивирусная система врожденного иммунитета: патогенез и лечение COVID-19. Вестник РГМУ. 2020;(5):5–14. DOI: 10.24075/vrgmu.2020.054.

3. Казимирский А.Н., Салмаси Ж.М., Порядин Г.В., Панина М.И., Рогожина Л.С., Ступин В.А. и др. Нейтрофильные экстраклеточные ловушки при воспалительных заболеваниях брюшной полости. Патологическая физиология и экспериментальная терапия. 2024;68(1):15–25. DOI: 10.25557/0031-2991.2024.01.15-25.

4. Казимирский А.Н., Салмаси Ж.М., Порядин Г.В., Панина М.И., Ларина В.Н., Ступин В.А. и др. IgG активирует формирование нейтрофильных экстраклеточных ловушек и модифицирует их структуру. Бюллетень экспериментальной биологии. 2022;174(12):786–789. DOI: 10.47056/0365-9615-2022-174-12-786-789.

5. Новиков Д.Г., Золотов А.Н., Кириченко Н.А., Мордык А.В. Способ обнаружения нейтрофильных внеклеточных ловушек в суправитально окрашенном препарате крови. Патент RU 2768 152 С1, 2022.03.23. https://yandex.ru/patents/doc/RU2768152C1_20220323.

6. Новиков Д.Г., Золотов А.Н., Бикбавова Г.Р., Ливзан М.А., Телятникова Л.И. Исследование нейтрофильных внеклеточных ловушек у пациента с язвенным колитом. Доказательная гастроэнтерология. 2022;11(2):31–38. DOI: 10.17116/dokgastro20221102131.

7. Казимирский А.Н., Салмаси Ж.М., Порядин Г.В., Панина М.И. Новые возможности диагностики и исследования патогенеза различных видов воспаления. Патологическая физиология и экспериментальная терапия. 2022;66(2):34– 42. DOI: 10.25557/0031-2991.2022.02.34-42.

8. Казимирский А.Н., Салмаси Ж.М., Порядин Г.В., Панина М.И., Ступин В.А., Ким А.Э. и др. Противоинфекционная защита организма человека с участием нейтрофильных сетей. Бюллетень сибирской медицины. 2024;23(1):56–63. DOI: 10.20538/1682-0363-2024-1-56-63.

9. Demkow U. Molecular mechanisms of neutrophil extracellular trap (NETs) degradation. Int. J. Mol. Sci. 2023;24(5):4896. DOI: 10.3390/ijms24054896.

10. Farrera C., Fadeel B. macrophage clearance of neutrophil extracellular traps is a silent process. J. Immunol. 2013;191:2647–2656. DOI: 10.4049/jimmunol.1300436.

11. Chen L., Zhao Y., Lai D., Zhang P., Yang Y., Li Y. et al. Neutrophil extracellular traps promote macrophage pyroptosis in sepsis. Cell Death Dis. 2018;9(6):597. DOI: 10.1038/s41419-018-0538-5.

12. Zhou Y., Xu Z., Liu Z. Impact of neutrophil extracellular traps on thrombosis formation: new findings and future perspective. Front. Cell. Infect. Microbiol. 2022;12:910908. DOI: 10.3389/fcimb.2022.910908.

13. Lauková L., Konečná B., Janovičová Ľ., Vlková B., Celec P. Deoxyribonucleases and their applications in biomedicine. Biomolecules. 2020;10:1036. DOI: 10.3390/biom10071036.

14. Nakazawa D., Shida H., Kusunoki Y., Miyoshi A., Nishio S., Tomaru U. et al. The responses of macrophages in interaction with neutrophils that undergo NETosis. J. Autoimmun. 2016;67:19–28. DOI: 10.1016/j.jaut.2015.08.018.

15. Szturmowicz M., Barańska I., Skoczylas A., Jędrych M.E., Demkow U. Correlation of bronchoalveolar lavage lymphocyte count with the extent of lung fibrosis and with plethysmographic lung volumes in patients with newly recognized hypersensitivity pneumonitis. Cent. Eur. J. Immunol. 2020;45(3):276–282. DOI: 10.5114/ceji.2020.101246.

16. Connors J., Cusimano G., Mege N., Woloszczuk K., Konopka E., Bell M. et al. Using the power of innate immunoprofiling to understand vaccine design, infection, and immunity. Hum. Vaccin Immunother. 2023;19(3):2267295. DOI: 10.1080/21645515.2023.2267295.

17. De Michele M., Kahan J., Berto I., Schiavo O.G., Iacobucci M., Toni D. et al. Cerebrovascular complications of COVID-19 and COVID-19 vaccination. Circ. Res. 2022;130(8):1187– 1203. DOI: 10.1161/CIRCRESAHA.122.319954.

18. Sharma K., Tolaymat S., Yu H., Elkhooly M., Jaiswal S., Jena A. et al. Progressive multifocal leukoencephalopathy in anti-CD20 and other monoclonal antibody (mAb) therapies used in multiple sclerosis: A review. J. Neurol. Sci. 2022;443:120459. DOI: 10.1016/j.jns.2022.120459.

19. Faccini T., Dhesi Z., Shah S. Death by antibody. BMJ Case Rep. 2019;12(5):e225519. DOI: 10.1136/bcr-2018-225519.

20. Kirby C., Herlihy D., Clarke L., Mullan R. Sarcoidosis manifesting during treatment with secukinumab for psoriatic arthritis. BMJ Case Rep. 2021;14(2):e240615. DOI: 10.1136/bcr-2020-240615.