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Immunology and Infection

Real-Time Quantification of Reactive Oxygen Species in Neutrophils Infected with Meningitic Escherichia Coli

Published: April 20th, 2021

DOI:

10.3791/62314

1Department of Developmental Cell Biology, Key Laboratory of Cell Biology, Ministry of Public Health, and Key Laboratory of Medical Cell Biology, Ministry of Education, China Medical University
* These authors contributed equally

Escherichia coli is the leading cause of neonatal Gram-negative bacterial meningitis. During the bacterial infection, reactive oxygen species produced by neutrophils play a major bactericidal role. Here we introduce a method to detect the reactive oxygen species in neutrophils in response to meningitis E. coli.

Escherichia coli (E. coli) is the most common Gram-negative bacteria causing neonatal meningitis. The occurrence of bacteremia and bacterial penetration through the blood-brain barrier are indispensable steps for the development of E. coli meningitis. Reactive oxygen species (ROS) represent the major bactericidal mechanisms of neutrophils to destroy the invaded pathogens. In this protocol, the time-dependent intracellular ROS production in neutrophils infected with meningitic E. coli was quantified using fluorescent ROS probes detected by a real-time fluorescence microplate reader. This method may also be applied to the assessment of ROS production in mammalian cells during pathogen-host interactions.

Neonatal bacterial meningitis is a common pediatric infectious disease. Escherichia coli (E. coli) with a K1 capsule is the most common Gram-negative pathogen causing neonatal bacterial meningitis, accounting for about 80% of the total incidence1,2,3. Despite the advances in the antimicrobial chemotherapy and supportive care, bacterial meningitis is still one of the most devastating conditions with high morbidity and mortality4.

The occurrence of neonatal bacterial meningitis usually begins with bacteremia ....

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Peripheral blood from volunteers applied in this research was approved by the Institutional Review Board of the first Hospital of China Medical University (#2020-2020-237-2).

1. Preparation of reagents and culture medium

  1. Prepare the red blood cell lysis buffer by adding 8.29 g of NH4Cl, 1 g of KHCO3, 37.2 mg of Na2EDTA into 1 L of double distilled water and adjust the pH to 7.2-7.4. Remove the bacteria by filtration using 0.22 µm filters.
  2. .......

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Using the protocol outlined in this article, the neutrophils were isolated from human peripheral blood and loaded with fluorescence probe DHE to detect the changes of ROS levels in response to E44 infection. Here, we provide representative data demonstrating the ROS production evoked by E44 strain determined by a microplate reader in real-time. By adding E44 strains at a MOI of 100, the ROS levels increased immediately and showed a continuous upward trend with a time-dependent manner (Figure 1

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Neutrophils act as the most abundant component of white blood cells in human blood circulation. They are important effector cells in the innate human immune system, which builds the first line of defense against the invasion of pathogens11. The generation of ROS represents one of the major bactericidal mechanisms of neutrophils following phagocytosis11. Recent studies have shown that a net-like structure released by a neutrophil called neutrophil extracellular trap (NET) is.......

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This work was supported by the National Natural Science Foundation of China (31670845, 31870832, 32000811) and the Program of Distinguished Professor of Liaoning Province (LJH2018-35).

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Name Company Catalog Number Comments
15 mL polypropylene conical centrifuge tubes KIRGEN KG2611
96-well plate Corning 3025
Agar DINGGUO DH010-1.1
Autuomated cell counter Bio-rad 508BR03397
Biological Safety Carbinet Shanghai Lishen Hfsafe-1200Lcb2
Brain heart infusion BD 237500
CD16 Microbeads, human Miltenyi Biotec 130-045-701
Centrifuge Changsha Xiangyi TDZ5-WS
Columns Miltenyi Biotec 130-042-401
Dihydroethidium (DHE) MedChemExpress 104821-25-2
Fetal bovine serum Cellmax SA211.02
Incubator Heraeus Hera Cell
MACS separation buffer Miltenyi Biotec 130-091-221
Microplate Reader Molecular Devices SpectraMax M5
Phorbol 12-myristate 13-acetate (PMA) Beyoitme S1819-1mg
QuadroMACS separation Unit Miltenyi Biotec 130-090-976
Rifampicin Solarbio 13292-46-1
RPMI1640 medium Sangon Biotech E600027-0500
Thermostatic shaker Shanghai Zhicheng ZWY-100D
Trypton OXOID LP0042
Yeast extract OXOID LP0021

  1. Kim, K. S. Acute bacterial meningitis in infants and children. Lancet Infectious Diseases. 10 (1), 11 (2010).
  2. Woll, C., et al. Epidemiology and Etiology of Invasive Bacterial Infection in Infants </=60 Days Old Treated in Emergency Departments. Journal of Pediatrics. 200, 210-217 (2018).
  3. Xu, M., et al. Etiology and Clinical Features of Full-Term Neonatal Bacterial Meningitis: A Multicenter Retrospective Cohort Study. Frontiers in Pediatrics. 7, 31 (2019).
  4. Kim, K. S. Human Meningitis-Associated Escherichia coli. EcoSal Plus. 7 (1), (2016).
  5. Rosales, C. Neutrophils at the crossroads of innate and adaptive immunity. Journal of Leukocyte Biology. 108 (1), 377-396 (2020).
  6. Kolaczkowska, E., Kubes, P. Neutrophil recruitment and function in health and inflammation. Nature Reviews: Immunology. 13 (3), 159-175 (2013).
  7. Winterbourn, C. C., Kettle, A. J., Hampton, M. B. Reactive Oxygen Species and Neutrophil Function. Annual Review of Biochemistry. 85, 765-792 (2016).
  8. Witko-Sarsat, V., Descamps-Latscha, B., Lesavre, P., Halbwachs-Mecarelli, L. Neutrophils: Molecules, Functions and Pathophysiological Aspects. Laboratory Investigation. 80 (5), 617-653 (2000).
  9. Zorov, D. B., Juhaszova, M., Sollott, S. J. Mitochondrial reactive oxygen species (ROS) and ROS-induced ROS release. Physiological Reviews. 94 (3), 909-950 (2014).
  10. Zeng, M. Y., Miralda, I., Armstrong, C. L., Uriarte, S. M., Bagaitkar, J. The roles of NADPH oxidase in modulating neutrophil effector responses. Molecular Oral Microbiology. 34 (2), 27-38 (2019).
  11. Liew, P. X., Kubes, P. The Neutrophil's Role During Health and Disease. Physiological Reviews. 99 (2), 1223-1248 (2019).
  12. Brinkmann, V., et al. Neutrophil Extracellular Traps Kill Bacteria. Science. 303 (5), 1532-1535 (2004).
  13. Lam, G. Y., Huang, J., Brumell, J. H. The many roles of NOX2 NADPH oxidase-derived ROS in immunity. Seminars in Immunopathology. 32 (4), 415-430 (2010).
  14. Panday, A., Sahoo, M. K., Osorio, D., Batra, S. NADPH oxidases: an overview from structure to innate immunity-associated pathologies. Cellular & Molecular Immunology. 12 (1), 5-23 (2015).
  15. Nunes, P., Demaurex, N., Dinaue, C. Regulation of the NADPH Oxidase and Associated Ion Fluxes During Phagocytosis. Traffic. 14, 1118-1131 (2013).
  16. Dahlgren, C., Karlsson, A., Bylund, J. Intracellular Neutrophil Oxidants: From Laboratory Curiosity to Clinical Reality. Journal of Immunology. 202 (11), 3127-3134 (2019).
  17. Stoiber, W., Obermayer, A., Steinbacher, P., Krautgartner, W. D. The Role of Reactive Oxygen Species (ROS) in the Formation of Extracellular Traps (ETs) in Humans. Biomolecules. 5 (2), 702-723 (2015).
  18. Haynes, A. P., Fletcher, J. neutrophil function test. Clinical Haematology. 3 (4), 871-887 (1990).
  19. Eichelberger, K. R., Goldman, W. E. Human Neutrophil Isolation and Degranulation Responses to Yersinia pestis Infection. Methods in Molecular Biology. 2010, 197-209 (2019).
  20. Siano, B., Oh, H., Diamond, S. Neutrophil isolation protocol. Journal of Visualized Experiments. (17), (2008).
  21. Chen, X., Zhong, Z., Xu, Z., Chen, L., Wang, Y. 2',7'-Dichlorodihydrofluorescein as a fluorescent probe for reactive oxygen species measurement: Forty years of application and controversy. Free Radical Research. 44 (6), 587-604 (2010).
  22. Woolley, J. F., Stanicka, J., Cotter, T. G. Recent advances in reactive oxygen species measurement in biological systems. Trends in Biochemical Sciences. 38 (11), 556-565 (2013).
  23. Dikalov, S. I., Harrison, D. G. Methods for detection of mitochondrial and cellular reactive oxygen species. Antioxidants and Redox Signaling. 20 (2), 372-382 (2014).
  24. Puleston, D. Detection of Mitochondrial Mass, Damage, and Reactive Oxygen Species by Flow Cytometry. Cold Spring Harbor Protocols. 2015 (9), (2015).

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