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Bioluminescence Imaging of NADPH Oxidase Activity in Different Animal Models
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Regulating NETosis: Increasing pH Promotes NADPH Oxidase-Dependent NETosis
Meraj A Khan1,2, Lijy M Philip1, Guillaume Cheung1
1Program in Translational Medicine, Peter Gilgan Centre for Research and Learning, The Hospital for Sick Children, Toronto, ON, Canada.
Frontiers in Medicine
|March 1, 2018
Summary
Higher extracellular pH enhances neutrophil extracellular trap (NET) formation by boosting reactive oxygen species (ROS) and protease activity. This suggests pH modulation, particularly with Tris, could manage NET-related diseases.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Neutrophils migrate to tissues with varying extracellular pH (pHe).
- Neutrophil activation via NADPH oxidase 2 (Nox) lowers intracellular pH (pHi) due to H+ production.
- The precise regulation of neutrophil extracellular trap (NET) formation (NETosis) by pHe remains unclear.
Purpose of the Study:
- To investigate how extracellular pH influences neutrophil extracellular trap (NET) formation.
- To determine if elevated pH enhances reactive oxygen species (ROS) production and protease activity, thereby stimulating NETosis.
Main Methods:
- Measured intracellular pH using Seminaphtharhodafluor dual fluorescence.
- Assessed ROS production via R123 generation using flow cytometry, plate reader assay, and imaging.
- Quantified NETosis using Sytox Green assays and immunoconfocal microscopy; analyzed histone H4 cleavage by Western blots.
Main Results:
- Increased pHe (6.6-7.8) raised pHi in both resting and activated neutrophils.
- Higher pH stimulated Nox-dependent ROS production and NETosis during spontaneous and phorbol myristate acetate-induced NETosis.
- Elevated pH enhanced histone H4 cleavage and NETosis induced by bacterial components (LPS, P. aeruginosa, S. aureus).
Conclusions:
- Higher extracellular pH promotes Nox-dependent ROS production, protease activity, and NETosis.
- pH modulation, using buffers like Tris (tromethamine), can regulate NETosis.
- Tris may be more effective than bicarbonate for managing NET-related diseases due to its buffering capacity.
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