Neutrophils, crucial players in the effector phase of the immune response, are recognized as important mediators of both innate and adaptive immune responses. Through the production of pro- and anti-inflammatory cytokines, they modulate the function of T and other lymphoid cells. Countless reports have highlighted the importance of these cells as efficient antimicrobial agents and annotated their involvement in the pathology of infectious and noninfectious diseases. The development of modern, sophisticated technologies has allowed the study of the functions of these cells in clinical settings. These advanced technologies include fluorescence-activated cell sorters, confocal microscopy, automated cell image analyzers, and live cell analysis instruments. Unfortunately, the cost of these modern instruments, maintenance, reagents, and the need for qualified technicians prohibit their use in low-income laboratories and universities in developing countries. With this in mind, we propose a series of basic tests that can be used in low-input clinical laboratories and universities to evaluate the function of neutrophils in health and disease. Our methodology allows us to assess in a practical and low-cost manner the functions of neutrophils in the phagocytic process, including opsonization, ingestion, ROI production (NBT reduction), myeloperoxidase content, phagosome-lysosome fusion, microbicidal activity, and NET production. Thus, under a disadvantageous ambiance, this may guide physicians in deciding whether a patient’s illness involves phagocytic defects without imposing a heavy financial burden.Graphical Abstract[-rId13-]
References
[1]
Fountain, A., Inpanathan, S., Alves, P., Verdawala, M.B. and Botelho, R.J. (2021) Phagosome Maturation in Macrophages: Eat, Digest, Adapt and Repeat. Advances in Biological Regulation, 82, Article 100832. https://doi.org/10.1016/j.jbior.2021.100832
[2]
Burn, G.L., Foti, A., Marsman, G., Patel, D.F. and Zychlinsky, A. (2021) The Neutrophil. Immunity, 54, 1377-1391. https://doi.org/10.1016/j.immuni.2021.06.006
[3]
Othman, A., Sekheri, M. and Filep, J.G. (2021) Roles of Neutrophil Granule Proteins in Orchestrating Inflammation and Immunity. The FEBS Journal, 289, 3932-3953. https://doi.org/10.1111/febs.15803
[4]
Rawat, K., Syeda, S. and Shrivastava, A. (2021) Neutrophil-Derived Granule Cargoes: Paving the Way for Tumor Growth and Progression. Cancer and Metastasis Reviews, 40, 221-244. https://doi.org/10.1007/s10555-020-09951-1
[5]
Futosi, K., Fodor, S. and Mócsai, A. (2013) Neutrophil Cell Surface Receptors and Their Intracellular Signal Transduction Pathways. International Immunopharmacology, 17, 638-650. https://doi.org/10.1016/j.intimp.2013.06.034
[6]
Sengeløv, H. (1995) Complement Receptors in Neutrophils. Critical Reviews in Immunology, 15, 107-31. https://doi.org/10.1615/CritRevImmunol.v15.i2.10
[7]
Metzemaekers, M., Gouwy, M. and Proost, P. (2020) Neutrophil Chemoattractant Receptors in Health and Disease: Double-Edged Swords. Cellular & Molecular Immunology, 17, 433-450. https://doi.org/10.1038/s41423-020-0412-0
[8]
Torres, M. and Coates, T.D. (1999) The Function of the Cytoskeleton in Human Neutrophils and Methods for Evaluation. Journal of Immunological Methods, 232, 89-109. https://doi.org/10.1016/S0022-1759(99)00168-4
[9]
Pick, R., Brechtefeld, D. and Walzog, B. (2013) Intraluminal Crawling Versus Interstitial Neutrophil Migration During Inflammation. Molecular Immunology, 55, 70-75. https://doi.org/10.1016/j.molimm.2012.12.008
[10]
Mickael, M-E., Kubick, N., Klimovich, P., Flournoy, P.H., Bieńkowska, I. and Sacharczuk, M. (2021) Paracellular and Transcellular Leukocytes Diapedesis Are Divergent but Interconnected Evolutionary Events. Genes, 12, Article 254. https://doi.org/10.3390/genes12020254
[11]
Mylvaganam, S., Freeman, S.A. and Grinstein, S. (2021) The Cytoskeleton in Phagocytosis and Macropinocytosis. Current Biology, 31, R619-R632. https://doi.org/10.1016/j.cub.2021.01.036
[12]
Elsbach, P. and Weiss, J. (1985) Oxygen-Dependent and Oxygen-Independent Mechanisms of Microbicidal Activity of Neutrophils. Immunology Letters, 11, 159-163. https://doi.org/10.1016/0165-2478(85)90163-4
[13]
Rosen, H., Crowley, J.R. and Heinecke, J.W. (2002) Human Neutrophils Use the Myeloperoxidase-Hydrogen Peroxide-Chloride System to Chlorinate but Not Nitrate Bacterial Proteins during Phagocytosis. Journal of Biological Chemistry, 277, 30463-30468. https://doi.org/10.1074/jbc.M202331200
[14]
Klebanoff, S.J. (2005) Myeloperoxidase: Friend and Foe. Journal of Leukocyte Biology, 77, 598–625. https://doi.org/10.1189/jlb.1204697
[15]
Scribner, D.J. and Fahrney, D. (1976) Neutrophil Receptors for IgG and Complement: Their Roles in the Attachment and Ingestion Phases of Phagocytosis. The Journal of Immunology, 116, 892-897. https://doi.org/10.4049/jimmunol.116.4.892
Lee, H.J., Woo, Y., Hahn, T.W., Jung, Y.M. and Jung, Y.J. (2020) Formation and Maturation of the Phagosome: A Key Mechanism in Innate Immunity Against Intracellular Bacterial Infection. Microorganisms, 8, Article 1298. https://doi.org/10.3390/microorganisms8091298
[18]
Uribe-Querol, E. and Rosales, C. (2022) Phagocytosis. Encyclopedia of Infection and Immunity, 1, 99-109. https://doi.org/10.1016/B978-0-12-818731-9.00049-5
[19]
Dąbrowska, D., Jabłońska, E., Iwaniuk, A. and Garley, M. (2019) Many Ways-One Destination: Different Types of Neutrophils Death. International Reviews of Immunology, 38, 18-32. https://doi.org/10.1080/08830185.2018.1540616
[20]
Rojas-Espinosa, O., Arce-Mendoza, A.Y., Islas-Trujillo, S., Muñiz-Buenrostro, A., Arce-Paredes, P., Popoca-Galván, O., Moreno-Altamirano, B. and Rivero Silva, M. (2023) Necrosis, Netosis, and Apoptosis in Pulmonary Tuberculosis and Type-2 Diabetes Mellitus. Clues from the Patient’s Serum. Tuberculosis, 143, Article 102426. https://doi.org/10.1016/j.tube.2023.102426
[21]
Kumar, V. (2020) Phagocytosis: Phenotypically Simple Yet a Mechanistically Complex Process. International Reviews of Immunology, 39, 118-150. https://doi.org/10.1080/08830185.2020.1732958
[22]
Bassøe, C.F. and Bjerknes, R. (1984) The Effect of Serum Opsonins on the Phagocytosis of Staphylococcus Aureus and Zymosan Particles, Measured by Flow Cytometry. Acta Pathologica Microbiologica Scandinavica Series C: Immunology, 92, 51-58. https://doi.org/10.1111/j.1699-0463.1984.tb00051.x
[23]
Oikonomopoulou, Z., Shulman, S., Mets, M. and Katz, B. (2022) Chronic Granulomatous Disease: An Updated Experience, with Emphasis on Newly Recognized Features. Journal of Clinical Immunology, 42, 1411-1419. https://doi.org/10.1007/s10875-022-01294-6
[24]
Yu, H.H., Yang, Y.H. and Chiang, B.L. (2021) Chronic Granulomatous Disease: A Comprehensive Review. Clinical Reviews in Allergy & Immunology, 61, 101-113. https://doi.org/10.1007/s12016-020-08800-x
[25]
Valenta, H., Erard, M., Dupré-Crochet, S. and Nüβe, O. (2020) The NADPH Oxidase and the Phagosome. Advances in Experimental Medicine and Biology, 1246, 153-177. https://doi.org/10.1007/978-3-030-40406-2_9
[26]
Naish, E., Wood, A.J., Stewart, A.P., Routledge, M., Morris, A.C., Chilvers, E.R. and Lodge, K.M. (2023) The Formation and Function of the Neutrophil Phagosome. Immunological Reviews, 314, 158-180. https://doi.org/10.1111/imr.13173
[27]
Pérez-Figueroa, E., Álvarez-Carrasco, P., Ortega, E. and Maldonado-Bernal, C. (2021) Neutrophils: Many Ways to Die. Frontiers in Immunology, 12, Article 631821. https://doi.org/10.3389/fimmu.2021.631821
[28]
Thiam, H.R., Wong, S.L., Wagner, D.D. and Waterman, C.M. (2020) Cellular Mechanisms of NETosis. Annual Review of Cell and Developmental Biology, 36, 191-218. https://doi.org/10.1146/annurev-cellbio-020520-111016
[29]
Vorobjeva, N.V. and Chernyak, B.V. (2020) NETosis: Molecular Mechanisms, Role in Physiology and Pathology. Biochemistry (Moscow), 85, 1178-1190. https://doi.org/10.1134/S0006297920100065
[30]
Zhai, W., Wu, F., Zhang, Y., Fu, Y. and Liu, Z. (2019) The Immune Escape Mechanisms of Mycobacterium tuberculosis. International Journal of Molecular Sciences, 20, Article 340. https://doi.org/10.3390/ijms20020340
[31]
Simeone, R., Bobard, A., Lippmann, J., Bitter, W., Majlessi, L., Brosch, R. and Enninga, J. (2012) Phagosomal Rupture by Mycobacterium tuberculosis Results in Toxicity and Host Cell Death. PLOS Pathogens, 8, e1002507. https://doi.org/10.1371/journal.ppat.1002507
[32]
Frehel, C. and Rastogi, N. (1987) Mycobacterium Leprae Surface Components Intervene in the Early Phagosome-Lysosome Fusion Inhibition Event. Infection and Immunity, 55, 2916-2921. https://doi.org/10.1128/iai.55.12.2916-2921.1987
[33]
Rojas-Espinosa, O., Camarena-Servin, V., Estrada-Garcia, I., Arce-Paredes, P. and Wek-Rodriguez, K. (1998) Mycobacterium lepraemurium, a Well-Adapted Parasite of Macrophages: I. Oxygen Metabolites. International Journal of Leprosy and Other Mycobacterial Diseases, 66, 365-373.
[34]
Smith, C.C., Barr, R.M. and Alexander, J. (1979) Studies on the Interaction of Mycobacterium microti and Mycobacterium lepraemurium with Mouse Polymorphonuclear Leucocytes. The Journal of General Microbiology, 112, 185-189. https://doi.org/10.1099/00221287-112-1-185
[35]
Block, M.S. and Rowan, B.G. (2020) Hypochlorous Acid: A Review. Journal of Oral and Maxillofacial Surgery, 78, 1461-1466. https://doi.org/10.1016/j.joms.2020.06.029
[36]
Schultz, B.M., Acevedo, O.A., Kalergis, A.M. and Bueno, S.M. (2022) Role of Extracellular Trap Release during Bacterial and Viral Infection. Frontiers in Microbiology, 13, Article 798853. https://doi.org/10.3389/fmicb.2022.798853
[37]
Brinkmann, V., Reichard, U., Goosmann, C., Fauler, B., Uhlemann, Y., Weiss, D.S., Weinrauch, Y. and Zychlinsky, A. (2004) Neutrophil Extracellular Traps Kill Bacteria. Science, 303, 1532-1535. https://doi.org/10.1126/science.1092385
[38]
Demkow, U. (2023) Molecular Mechanisms of Neutrophil Extracellular Trap (NETs) Degradation. International Journal of Molecular Sciences, 24, Article 4896. https://doi.org/10.3390/ijms24054896