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Visualizing and Quantifying Endonuclease-Based Site-Specific DNA Damage
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Mitochondrial ROS and base excision repair steps leading to DNA nick formation drive ultraviolet induced-NETosis
Dhia Azzouz1,2, Nades Palaniyar1,2,3
1Translational Medicine, Peter Gilgan Center for Research and Learning, The Hospital for Sick Children, Toronto, ON, Canada.
Frontiers in Immunology
|June 23, 2023
Summary
Ultraviolet (UV) radiation causes mitochondrial reactive oxygen species (ROS) to damage DNA, initiating neutrophil extracellular trap formation (NETosis) through DNA repair pathways. This reveals a novel mechanism for UV-induced NETosis independent of NADPH oxidases.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Reactive oxygen species (ROS) are crucial for neutrophil extracellular trap formation (NETosis).
- Mitochondria and NADPH oxidases are primary sources of ROS in neutrophils.
- Ultraviolet (UV) radiation induces mitochondrial ROS and promotes NETosis, but the underlying mechanism is unclear.
Purpose of the Study:
- To elucidate the mechanism by which ROS executes UV-induced NETosis.
- To investigate the role of oxidative DNA damage and repair pathways in UV-mediated NETosis.
Main Methods:
- Mitochondrial ROS detection using Mitosox.
- NETosis assessment via SYTOX staining and immunocytochemistry.
- Confocal imaging of 8-oxyguanine (8-oxoG) for DNA oxidation.
- Immunofluorescence microscopy for DNA repair protein localization.
- Inhibition of base excision repair (BER) pathway components.
Main Results:
- UV radiation generated mitochondrial ROS, and its inhibition suppressed NETosis.
- UV irradiation caused extensive DNA oxidation, evidenced by 8-oxoG accumulation in NETs.
- The base excision repair (BER) pathway was active, with early steps (up to DNA nicking) being critical for UV-induced NETosis.
Conclusions:
- High mitochondrial ROS levels from UV irradiation induce significant oxidative DNA damage.
- Early stages of the BER pathway, leading to DNA nicking, trigger chromatin decondensation and subsequent NETosis.
- This study reveals a novel NOX-independent pathway for ROS-mediated NETosis induced by UV radiation and mitochondrial ROS.
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