Lysosomal cationic proteins as the basis of cellular and humoral immunity of animals: the role of neutrophil extracellular traps (NETs) in immune homeostasis (review)

Relevance. Lysosomal cationic proteins (LCP) of granulocytic leukocytes: elastase, cathepsin G, proteinase-3, calgranulin, cathelicidins, defensins, lactoferrin, protegrins are active against viruses, bacteria, fungi, protozoa. The issues of physiological regulatory, immune and pathological effects of LCP and their derivatives – neutrophil (heterophil) extracellular traps (NETs) on pathogens, healthy cellular and tissue structures of the body are noted.
Results. The increment of LCP granulocytes is realized by: 1. merocrine type — by degranulation; 2. exocytosis and false degranulation, that is, the process of decationization of lysosomes containing granules of cationic proteins with apocrine or holocrine type of secretion. Decationization implements exocytosis of LCP, extrusion of intact lysosomes from the cell with LCP, and diffusion of LCP through the lysosome membrane. Lysosome degranulation reactions with LCP form phagolysosomes and initiate phagocytosis, lysosome decationization reactions with LCP ensure the formation and functions of NETs. NETs is formed by non-lytic (non-lytic) and lytic (lyzed) pathways in septic and aseptic inflammation, with the ontogenetic development of immune links. NETs is stereotypically formed intravascular during aseptic inflammation, oxidative stress and in a physiological regime, when granulocytes are stimulated by products of oxidative metabolism. Using a cytochemical test with a highly sensitive acid-base bromophenol blue indicator, subcellular and cellular manifestations of the physiological age-related immune activity of cationic proteins accumulated in granulocyte lysosomes were studied on the avian model organism (Aves), and nonspecific adaptive reactions (NAR) of vertebrates in early postnatal ontogenesis were studied. The basis for the formation of NAR is the relationship of groups of leukocytes (lymphocytes, monocytes and granulocytes) with the dynamics of their lysosomal cationic proteins. The method for calculating the level of activity and potential capabilities of granulocytes in phagocytic reactions and in the formation of .NETs includes indices characterizing the directions and intensity of immune reactions of granulocytes, taking into account the processes: 1. degranulation of lysosomes with LCP — in the initiation of the cellular phagocytic link; 2. decationization of lysosomes with LCP — in the initiation of extracellular traps involved in the implementation of the humoral link of immunity.

1. Berezhnaya N.M. Neutrophils and immunological homeostasis. Kyiv: Naukova dumka. 1988; 187 (in Russian). ISBN 5-12-000251-X

2. Borregaard N., Cowland J.B. Granules of the Human Neutrophilic Polymorphonuclear Leukocyte. Blood. 1997; 89(10): 3503-3521. https://doi.org/10.1182/blood.V89.10.3503

3. Soehnlein O., Weber C., Lindbom L. Neutrophil granule proteins tune monocytic cell function. Trends in Immunology. 2009; 30(11): 538-546. https://doi.org/10.1016/j.it.2009.06.006

4. Pigarevsky V.E. Granular leukocytes and their properties. Moscow: Meditsina. 1978; 127 (in Russian).

5. Wu Z. et al. Fumonisin B1 induces chicken heterophil extracellular traps mediated by PAD4 enzyme and P2 x 1 receptor. Poultry Science. 2022; 101(1): 101550. https://doi.org/10.1016/j.psj.2021.101550

6. Chen Y et al . Citrinin stimulated heterophil extracellular trap formation in chickens. Molecular Immunology. 2022; 152: 27-34. https://doi.org/10.1016/j.molimm.2022.09.014

7. Wu H. et al. The release of FB1-induced heterophil extracellular traps in chicken is dependent on autophagy and glycolysis. Poultry Science. 2023; 102(4): 102511. https://doi.org/10.1016Zj.psj.2023.102511

8. Lima-Gomes Pd.S. et al. Chick heterophils release DNA extracellular traps (DETs) in vitro and in vivo upon Aspergillus fumigatus conidia exposure. Microbes and Infection. 2024; 26(3): 105261. https://doi.org/10.1016/j.micinf.2023.105261

9. Brinkmann V., Zychlinsky A. Beneficial suicide: why neutrophils die to make NETs. Nature Reviews Microbiology. 2007; 5(8): 577-582. https://doi.org/10.1038/nrmicro1710

10. Horwitz M., Benson K.F., Duan Z., Li F.-Q., Person R.E. Hereditary neutropenia: dogs explain human neutrophil elastase mutations. Trends in Molecular Medicine. 2004; 10(4): 163-170. https://doi.org/10.1016/j.molmed.2004.02.002

11. Brinkmann V. et al. Neutrophil Extracellular Traps Kill Bacteria. Science. 2004; 303(5663): 1532-1535. https://doi.org/10.1126/science.1092385

12. Averhoff P, Kolbe M., Zychlinsky A., Weinrauch Y Single Residue Determines the Specificity of Neutrophil Elastase for Shigella Virulence Factors. Journal of Molecular Biology. 2008; 377(4): 1053-1066. https://doi.org/10.1016/jJmb.2007.12.034

13. Urban C.F. et al. Neutrophil Extracellular Traps Contain Calprotectin, a Cytosolic Protein Complex Involved in Host Defense against Candida albicans. PLoS Pathogens. 2009; 5(10): e1000639. https://doi.org/10.1371/journal.ppat.1000639

14. Bianchi M., Niemiec M.J., Siler U., Urban C.F., Reichenbach J. Restoration of anti-Aspergillus defense by neutrophil extracellular traps in human chronic granulomatous disease after gene therapy is calprotectin-dependent. The Journal of Allergy and Clinical Immunology. 2011; 127(5): 1243-1252.e7. https://doi.org/10.1016/jJaci.2011.01.021

15. Chen C.X.-J., Soto I., Guo Y-L., Liu Y Control of Secondary Granule Release in Neutrophils by Ral GTPase. Journal of Biological Chemistry. 2011; 286(13): 11724-11733. https://doi.org/10.1074/jbc.M110.154203

16. Serov V.V., Shekhter A.B. Connective tissue: functional morphology and general pathology. Moscow: Meditsina. 1981; 312 (in Russian).

17. Witko-Sarsat V., Rieu P, Descamps-Latscha B., Lesavre P, Halbwachs-Mecarelli L. Neutrophils: Molecules, Functions and Pathophysiological Aspects. Laboratory Investigation. 2000; 80(5): 617-653. https://doi.org/10.1038/labinvest.3780067

18. Soehnlein O., Lindbom L., Weber C. Mechanisms underlying neutrophil-mediated monocyte recruitment. Blood. 2009; 114(21): 4613-4623. https://doi.org/10.1182/blood-2009-06-221630

19. Nesterova I.V., Kolesnikova N.V., Chudilova G.A., Lomtatidze L.V., Kovaleva S.V, Evglevsky A.A. Neutrophilic granulocytes: a new look at “old players” on the immunological field. Immunologiya. 2015; 36(4): 257-265 (in Russian). https://www.elibrary.ru/umhqkj

20. Mocsai A. Diverse novel functions of neutrophils in immunity, inflammation, and beyond. Journal of Experimental Medicine. 2013; 210(7): 1283-1299. https://doi.org/10.1084/jem.20122220

21. Sheshachalam A., Srivastava N., Mitchell T., Lacy P, Eitzen G. Granule protein processing and regulated secretion in neutrophils. Frontiers in Immunology. 2014; 5: 448. https://doi.org/10.3389/fimmu.2014.00448

22. Bystrom J., Amin K., Bishop-Bailey D. Analysing the eosinophil cationic protein — a clue to the function of the eosinophil granulocyte. Respiratory Research. 2011; 12: 10. https://doi.org/10.1186/1465-9921-12-10

23. Clark R.A., Olsson I., Klebanoff S.J. Cytotoxicity for tumor cells of cationic proteins from human neutrophil granules. Journal of Cell Biology. 1976; 70(3): 719-723. https://doi.org/10.1083/jcb.70.3J19

24. Tal T., Sharabani M., Aviram I. Cationic proteins of neutrophil azurophilic granules: protein-protein interaction and blockade of NADPH oxidase activation. Journal of Leukocyte Biology. 1998; 63(3): 305-311. https://doi.org/10.1002/jlb.63.3.305

25. Papayannopoulos V. Neutrophil extracellular traps in immunity and disease. Nature Reviews Immunology. 2018; 18(2): 134-147. https://doi.org/10.1038/nri.2017.105

26. Thiam H.R., Wong S.L., Wagner D.D., Waterman C.M. Cellular Mechanisms of NETosis. Annual Review of Cell and Developmental Biology. 2020; 36: 191-218. https://doi.org/10.1146/annurev-cellbio-020520-111016

27. Cochrane C.G. The participation of cells in the inflammatory injury of tissue. The Journal of Investigative Dermatology. 1975; 64(5): 301-306. https://doi.org/10.1111/1523-1747.ep12512255

28. Ranadive N.S., Cochrane C.G. Mechanism of Histamine Release from Mast Cells by Cationic Protein (Band 2) from Neutrophil Lysosomes. The Journal of Immunology. 1971; 106(2): 506-516. https://doi.org/10.4049/jimmunol.106.2.506

29. Nagoyev B.S. Essays on the neutrophilic granulocyte. Nalchik: Elbrus. 1986; 142 (in Russian).

30. Shubich M.G. Detection of cationic proteins in the cytoplasm of leukocytes with the use of bromphenol blue. Tsitologiya. 1974; 16(10): 1321-1322 (in Russian). https://www.elibrary.ru/qbthxd

31. Nagoyev B.S. Qualitative and quantitative indices of lysosomal cationic leukocyte protein in healthy persons. Laboratornoye delo. 1983; (6): 6-9 (in Russian).

32. Drobot G.P, Zabiyakin VA., Stepanova A.E., Smolentsev S.Yu. Dynamics of cytochemical indicators of psevdoeozinofilov [sic!] blood of guinea fowl. Rossiyskaya sel'skokhozyaystvennaya nauka. 2017; (1): 42-44 (in Russian). https://www.elibrary.ru/xtdnvf

33. Kolesnik E.A., Derkho M.A., Lebedeva I.A. Comprehensive morphophysiological description of the immune lysosomal cationic protein of leukocytes in the early ontogeny of broiler chickens. Uchenye Zapiski Kazanskogo Universiteta . Series: Estestvennye Nauki. 2019; 161(3): 440-458 (in Russian). https://doi.org/10.26907/2542-064X.2019.3.440-458

34. Pourtabrizi M., Shahtahmassebi N., Sharifmoghadam M.R. Bromophenol blue doped in nano-droplet: spectroscopy, nonlinear optical properties and Staphylococcus aureus treatment. Optical and Quantum Electronics. 2021; 53: 1. https://doi.org/10.1007/s11082-020-02634-9

35. Plaza-Garrido M., Salinas-Garcia M.C., Alba-Elena D., Martinez J.C., Camara-Artigas A. Lysozyme crystals dyed with bromophenol blue: where has the dye gone?. Acta Crystallographica Section D: Structural Biology. 2020; 76(9): 845-856. https://doi.org/10.1107/S2059798320008803

36. Barron A.J., Agrawal S., Lesperance D.N.A., Doucette J., Calle S., Broderick N.A. Microbiome-derived acidity protects against microbial invasion in Drosophila. Cell Reports. 2024; 43(4): 114087. https://doi.org/10.1016/j.celrep.2024.114087

37. Pastore A., Badocco D., Cappellin L., Pastore P Modeling the Dichromatic Behavior of Bromophenol Blue to Enhance the Analytical Performance of pH Colorimetric Sensor Arrays. Chemosensors. 2022; 10(2): 87. https://doi.org/10.3390/chemosensors10020087

38. Mazing Yu.A. Functional morphology of lysosomal cationic proteins in neutrophilic granulocytes. Voprosy meditsinskoy khimii. 1990; 36(6): 8-10 (in Russian).

39. Chuammitri P, Ostojic J., Andreasen C.B., Redmond S.B., Lamont S.J., Palic D. Chicken heterophil extracellular traps (HETs): Novel defense mechanism of chicken heterophils. Veterinary Immunology and Immunopathology. 2009; 129(1-2): 126-131. https://doi.org/10.1016/j.vetimm.2008.12.013

40. Jones M.P Avian Hematology. Clinics in Laboratory Medicine. 2015; 35(3): 649-659. https://doi.org/10.1016/j.cll.2015.05.013

41. Kolesnik E.A. Cytophysiological and mathematical criteria for assessing phagolysosomes and neutrophil extracellular traps in the cellular and humoral immunity. 2nd Congress of the International Society for Clinical Physiology and Pathology (ISCPP2024). Moscow. 2024; 24-26 (in Russian). https://doi.org/10.5281/zenodo.13739047

42. Kolesnik E.A., Derkho M.A. About participation of pituitary-adrenocortical hormones in regulation of blood cellular pool in chicken-broilers. Problems of Productive Animal Biology. 2018; (1): 64-74 (in Russian). https://doi.org/10.25687/1996-6733.prodanimbiol.2018.1.64-74

43. Rada B. Neutrophil Extracellular Traps. Knaus U., Leto T. (eds.). NADPH Oxidases. Methods in Molecular Biology. New York, NY: Humana. 2019; 1982: 517-528. https://doi.org/10.1007/978-1-4939-9424-3_31

44. Metzler K.D., Goosmann C., Lubojemska A., Zychlinsky A., Papayannopoulos V. A Myeloperoxidase-Containing Complex Regulates Neutrophil Elastase Release and Actin Dynamics during NETosis. Cell Reports. 2014; 8(3): 883-896. https://doi.org/10.1016/j.celrep.2014.06.044

45. Kharisma V.D. et al. Garcinoxanthones from Garcinia mangostana L. tackle SARS-CoV-2 infection and cytokine storm pathway inhibition: A viroinformatics study. Journal of Pharmacy and Pharmacognosy Research. 2023; 11(5): 743-756. https://doi.org/10.56499/jppres23.1650_11.5J43

46. Kolesnik E.A., Derkho M.A., Rebezov M.B. Forms of degeneration of blood cells, their physiological and clinical significance, mechanisms of formation, shadows of cells in blood smears of birds. Agrarian science. 2024; (1): 65-74 (in Russian). https://doi.org/10.32634/0869-8155-2024-378-1-65-74

47. Branzk N. et al. Neutrophils sense microbe size and selectively release neutrophil extracellular traps in response to large pathogens. Nature Immunology 2014; 15(11): 1017-1025. https://doi.org/10.1038/ni.2987

48. Delgado-Rizo V., Martinez-Guzman M.A., Iniguez-Gutierrez L., Garcia-Orozco A., Alvarado-Navarro A., Fafutis-Morris M. Neutrophil Extracellular Traps and Its Implications in Inflammation: An Overview. Frontiers in Immunology. 2017; (8): 81. https://doi.org/10.3389/fimmu.2017.00081

49. Pilsczek F.H. et al. A Novel Mechanism of Rapid Nuclear Neutrophil Extracellular Trap Formation in Response to Staphylococcus aureus. The Journal of Immunology. 2010; 185(12): 7413-7425. https://doi.org/10.4049/jimmunol.1000675

50. de Bont C.M., Koopman W.J.H., Boelens W.C., Pruijn G.J.M. Stimulus-dependent chromatin dynamics, citrullination, calcium signalling and ROS production during NET formation. Biochimica et Biophysica Acta (BBA) — Molecular Cell Research. 2018; 1865(11-A): 1621-1629. https://doi.org/10.1016/j.bbamcr.2018.08.014

51. Gupta A.K., Giaglis S., Hasler P, Hahn S. Efficient Neutrophil Extracellular Trap Induction Requires Mobilization of Both Intracellular and Extracellular Calcium Pools and Is Modulated by Cyclosporine A. PloS ONE. 2014; 9(5): e97088. https://doi.org/10.1371/journal.pone.0097088

52. Rossaint J. et al. Synchronized integrin engagement and chemokine activation is crucial in neutrophil extracellular trap-mediated sterile inflammation. Blood. 2014; 123(16): 2573-2584. https://doi.org/10.1182/blood-2013-07-516484

53. Keshari R.S. et al. Cytokines Induced Neutrophil Extracellular Traps Formation: Implication for the Inflammatory Disease Condition. PloS ONE. 2012; 7(10): e48111. https://doi.org/10.1371/journal.pone.0048111

54. Schappe M.S. et al. Chanzyme TRPM7 Mediates the Ca2+ Influx Essential for Lipopolysaccharide-Induced Toll-Like Receptor 4 Endocytosis and Macrophage Activation. Immunity. 2018; 48(1): 59-74.e5. https://doi.org/10.1016/j.immuni.2017.11.026

55. Wang Y et al. Histone hypercitrullination mediates chromatin decondensation and neutrophil extracellular trap formation. Journal of Cell Biology. 2009; 184(2): 205-213. https://doi.org/10.1083/jcb.200806072

56. Kenny E.F. et al. Diverse stimuli engage different neutrophil extracellular trap pathways. eLife. 2017; 6: e24437. https://doi.org/10.7554/eLife.24437

57. Fuchs T.A. et al. Novel cell death program leads to neutrophil extracellular traps. Journal of Cell Biology. 2007; 176(2): 231-241. https://doi.org/10.1083/jcb.200606027

58. Sun B. et al. Citrullination of NF-kB p65 promotes its nuclear localization and TLR-induced expression of IL-1 в and TNFa. Science Immunology. 2017; 2(12): eaal3062. https://doi.org/10.1126/sciimmunol.aal3062

59. Li Y, Werth V.P., Mall M., Liu M.-L. Nuclear lamin B is crucial to the nuclear envelope integrity and extracellular trap release in neutrophils. bioRxiv. 2019; 647529. https://doi.org/10.1101/647529

60. Ruan J., Xia S., Liu X., Lieberman J., Wu H. Cryo-EM structure of the gasdermin A3 membrane pore. Nature. 2018; 557(7703): 62-67. https://doi.org/10.1038/s41586-018-0058-6

61. Chen K.W. et al. Noncanonical inflammasome signaling elicits gasdermin D-dependent neutrophil extracellular traps. Science Immunology. 2018; 3(26): eaar6676. https://doi.org/10.1126/sciimmunol.aar6676

62. Sollberger G. et al. Gasdermin D plays a vital role in the generation of neutrophil extracellular traps. Science Immunology. 2018; 3(26): eaar6689. https://doi.org/10.1126/sciimmunol.aar6689

63. Renganathan B. et al. Transport and Organization of Individual Vimentin Filaments Within Dense Networks Revealed by Single Particle Tracking and 3D FIB-SEM. bioRxiv. 2024; 2024.06.10.598346. https://doi.org/10.1101/2024.06.10.598346

64. Neubert E. et al. Chromatin swelling drives neutrophil extracellular trap release. Nature Communications. 2018; 9: 3767. https://doi.org/10.1038/s41467-018-06263-5

65. Giridharan S.S.P, Caplan S. MICAL-Family Proteins: Complex Regulators of the Actin Cytoskeleton. Antioxidants & Redox Signaling. 2014; 20(13): 2059-2073. https://doi.org/10.1089/ars.2013.5487

66. Petretto A. et al. Neutrophil extracellular traps (NET) induced by different stimuli: A comparative proteomic analysis. PloS ONE. 2019; 14(7): e0218946. https://doi.org/10.1371/journal.pone.0218946

67. Deng W. et al. MICAL1 controls cell invasive phenotype via regulating oxidative stress in breast cancer cells. BMC Cancer. 2016; 16: 489. https://doi.org/10.1186/s12885-016-2553-1

68. Chang Y-C. et al. Group B Streptococcus Engages an Inhibitory Siglec through Sialic Acid Mimicry to Blunt Innate Immune and Inflammatory Responses In Vivo. PLoS Pathogens. 2014; 10(1): e1003846. https://doi.org/10.1371/journal.ppat.1003846

69. Secundino I. et al. Host and pathogen hyaluronan signal through human siglec-9 to suppress neutrophil activation. Journal of Molecular Medicine. 2016; 94(2): 219-233. https://doi.org/10.1007/s00109-015-1341-8

70. Khatua B., Bhattacharya K., Mandal C. Sialoglycoproteins adsorbed by Pseudomonas aeruginosa facilitate their survival by impeding neutrophil extracellular trap through siglec-9. Journal of Leukocyte Biology. 2012; 91(4): 641-655. https://doi.org/10.1189/jlb.0511260

71. Beiter K., Wartha F, Albiger B., Normark S., Zychlinsky A., Henriques-Normark B. An Endonuclease Allows Streptococcus pneumoniae to Escape from Neutrophil Extracellular Traps. Current Biology 2006; 16(4): 401-407. https://doi.org/10.1016/j.cub.2006.01.056

72. Juneau R.A., Stevens J.S., Apicella M.A., Criss A.K. A Thermonuclease of Neisseria gonorrhoeae Enhances Bacterial Escape From Killing by Neutrophil Extracellular Traps. The Journal of Infectious Diseases. 2015; 212(2): 316-324. https://doi.org/10.1093/infdis/jiv031

73. Wartha F et al. Capsule and D-alanylated lipoteichoic acids protect Streptococcus pneumoniae against neutrophil extracellular traps. Cellular Microbiology. 2007; 9(5): 1162-1171. https://doi.org/10.1111/j.1462-5822.2006.00857.x

74. Vorobjeva N.V., Pinegin B.V. Neutrophil extracellular traps: mechanisms of formation and role in health and disease. Biochemistry (Moscow). 2014; 79(12): 1286-1296. https://doi.org/10.1134/S0006297914120025

75. Kawabata K., Hagio T., Matsuoka S. The role of neutrophil elastase in acute lung injury. European Journal of Pharmacology. 2002; 451(1): 1-10. https://doi.org/10.1016/s0014-2999(02)02182-9

76. Xu J. et al. Extracellular histones are major mediators of death in sepsis. Nature Medicine. 2009; 15(11): 1318-1321. https://doi.org/10.1038/nm.2053

77. Thomas G.M. et al. Extracellular DNA traps are associated with the pathogenesis of TRALI in humans and mice. Blood. 2012; 119(26): 6335-6343. https://doi.org/10.1182/blood-2012-01-405183

78. Abrams S.T. et al. Circulating Histones Are Mediators of Trauma-associated Lung Injury. American Journal of Respiratory and Critical Care Medicine. 2013; 187(2): 160-169. https://doi.org/10.1164/rccm.201206-1037OC

79. Poon I.K.H. et al. Phosphoinositide-mediated oligomerization of a defensin induces cell lysis. eLife. 2014; 3: e01808. https://doi.org/10.7554/eLife.01808

80. Horwitz D.A., Fahmy T.M., Piccirillo C.A., La Cava A. Rebalancing Immune Homeostasis to Treat Autoimmune Diseases. Trends in Immunology. 2019; 40(10): 888-908. https://doi.org/10.1016/j.it.2019.08.003

81. Saba H.I., Roberts H.R., Herion J.C. The Anticoagulant Activity of Lysosomal Cationic Proteins from Polymorphonuclear Leukocytes. Journal of Clinical Investigation. 1967; 46(4): 580-589. https://doi.org/10.1172/JCI105559

82. Fuchs T.A. et al. Extracellular DNA traps promote thrombosis. Proceedings of the National Academy of Sciences. 2010; 107(36): 15880-15885. https://doi.org/10.1073/pnas.1005743107

83. Brill A. et al. von Willebrand factor-mediated platelet adhesion is critical for deep vein thrombosis in mouse models. Blood. 2011; 117(4): 1400-1407. https://doi.org/10.1182/blood-2010-05-287623

84. Etulain J., Martinod K., Wong S.L., Cifuni S.M., Schattner M., Wagner D.D. P-selectin promotes neutrophil extracellular trap formation in mice. Blood. 2015; 126(2): 242-246. https://doi.org/10.1182/blood-2015-01-624023

85. Monfregola J., Johnson J.L., Meijler M.M., Napolitano G., Catz S.D. MUNC13-4 Protein Regulates the Oxidative Response and Is Essential for Phagosomal Maturation and Bacterial Killing in Neutrophils. Journal of Biological Chemistry. 2012; 287(53): 44603-44618. https://doi.org/10.1074/jbc.M112.414029

86. Hakkim A. et al. Impairment of neutrophil extracellular trap degradation is associated with lupus nephritis. Proceedings of the National Academy of Sciences. 2010; 107(21): 9813-9818. https://doi.org/10.1073/pnas.0909927107