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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 caused by the entry of pathogenic bacteria into the peripheral circulation from the local lesions of the newborns, followed by penetration through the blood-brain barrier (BBB) into the brain, resulting in the inflammation of the meninges4. The onset of bacteremia depends on the interaction between bacteria and host immune cells including neutrophils and macrophages, etc. Neutrophils, which account for ~50-70% of white blood cells, are the first line of defense against bacterial infections5,6. During the invasion of bacteria, the activated neutrophils are recruited to the infectious sites and release reactive oxygen species (ROS) including the superoxide anion, hydrogen peroxide, hydroxyl radicals, and singlet oxygen7. The ROS undergo redox reactions with the cell membrane, nucleic acid molecules and proteins of the bacteria, resulting in the injury and death of the invading bacteria8. The mitochondria is the main site of ROS production in eukaryotic cells, and various oxidases (e.g., nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex, lipoxygenase system, protein kinase C and cyclooxygenase system) mediate the production of ROS9,10. The real-time measurement of the production of ROS, representing the primary antimicrobial mechanism in neutrophils, is a useful method for studying host defense during the bacteria-host interaction.
In this protocol, the time-dependent ROS production in neutrophils infected with meningitic E. coli was quantified with a fluorescent ROS probe DHE, detected by a real-time fluorescence microplate reader. This method may also be applied to the assessment of ROS production in other mammalian cells during the pathogen-host interaction.
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
2. Preparation of E44 bacteria strain
NOTE: E44 is a mutant strain of meningitic E. coli with rifampicin resistance.
3. Isolation of neutrophils from human peripheral blood
4. Measurement of ROS
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
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...
The authors declare no competing financial interests or other conflicts of interest.
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).
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 |
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