Neutrophil extracellular traps (NETs) are released by neutrophils during NETosis, a unique form of cell death triggered by various agents like phorbol myristate acetate (PMA), bacterial lipopolysaccharide (LPS), and certain bacteria. These agents activate NADPH oxidase (NOX), generating ROS and activating MAPKs. Previous research established that genome-wide transcriptional firing is crucial for chromatin decondensation in NETosis. However, the role of DNA repair in this process was unknown. ROS, a critical component of NETosis, is generated in massive amounts by NOX-dependent agonists. Inhibition of ROS production completely blocks NETosis. This study hypothesized that ROS induces DNA damage, and the subsequent DNA repair process, particularly the chromatin unwinding aspect, is a key driver of NETosis. ROS can oxidize DNA bases (e.g., converting guanine to 8-oxoguanine), causing transcription machinery to stall. The repair machinery, including OGG1, PCNA, APE1, PARP, DNA ligase, and DNA polymerases β and δ, then assembles at the damaged sites, opening the chromatin for repair. The initial steps of the base excision repair (BER) or nucleotide excision repair (NER) pathways were the focus of this investigation to determine their contribution to the chromatin decondensation and NET formation.

Existing literature highlights the role of ROS and genome-wide transcriptional firing in NETosis. Studies demonstrated the necessity of ROS production for NETosis, as its inhibition completely prevents the process. The importance of a genome-wide transcriptional response in chromatin decondensation during NETosis was already established in previous work by Khan and Palaniyar (2017). However, the precise mechanism by which ROS triggers NETosis remained elusive. This paper builds upon this foundation by exploring the previously unstudied role of DNA repair in mediating the effects of ROS on NETosis.

The study employed multiple methods to investigate the role of ROS-mediated DNA damage and DNA repair in NETosis. Human neutrophils were isolated from peripheral blood and treated with PMA or LPS to induce NETosis. The extent of DNA damage was assessed by measuring 8-oxoguanine (8-oxoG), a marker of oxidative DNA damage, using immunofluorescence and in-cell ELISA. The localization of PCNA, a key DNA repair protein, was examined using confocal microscopy. To assess the importance of DNA repair pathways in NETosis, SYTOX Green assays measured DNA release, a marker of NETosis, in neutrophils treated with various inhibitors of the BER/NER pathways (APE1, PARP1, DNA ligase, PCNA, and DNA polymerases β/δ inhibitors) before stimulation with PMA or LPS. The effects of these inhibitors on NETosis induced by Staphylococcus aureus and Pseudomonas aeruginosa were also investigated. For knockdown studies, siRNA was used to silence APE1 and PARP1 in differentiated HL-60 cells, and NETosis was again assessed using SYTOX Green assays. Confocal microscopy confirmed the results obtained from the plate reader assays. Statistical analysis was performed using GraphPad Prism 7, employing appropriate tests based on the data distribution.

The key findings of the study demonstrate a strong link between ROS-induced DNA damage and NETosis. Treatment with PMA and LPS significantly increased 8-oxoG levels in neutrophils, indicating substantial oxidative DNA damage. PCNA, typically cytoplasmic in resting neutrophils, translocated to the nucleus following NETosis induction. Inhibition of the early steps of the BER/NER pathway (APE1, PARP1, and DNA ligase) significantly suppressed NETosis induced by PMA, LPS, Staphylococcus aureus, and Pseudomonas aeruginosa. In contrast, inhibiting the later steps (PCNA-polymerase interaction or polymerase β activity) did not affect NETosis. Similarly, siRNA knockdown of APE1 and PARP1 in HL-60 cells reduced NETosis. These findings strongly suggest that the initial steps of DNA repair, leading to chromatin decondensation, are essential for ROS-mediated NETosis, while the subsequent steps are less critical.

The study's findings reveal a novel mechanism by which ROS induces NETosis. The results support the hypothesis that ROS initially triggers extensive DNA damage, and the subsequent activation of the DNA repair machinery is essential for chromatin decondensation and NET formation. The observed inhibition of NETosis upon blocking the initial steps of DNA repair, but not the later steps, suggests a specific role for the early steps in chromatin remodeling during NETosis. This is supported by the observation that the initial DNA repair steps up to DNA ligase are sufficient to induce extensive chromatin decondensation. The authors discuss the multifunctional nature of APE1 and explain why the observed NETosis reduction with APE1 inhibitors is likely due to the inhibition of its endonuclease activity rather than its redox activity. The study also confirms that DNA replication is not necessary for NETosis, emphasizing the specific role of DNA repair in this process. The observed coupling of DNA repair with transcription suggests that both contribute to chromatin unwinding in NETosis.

This study demonstrates that ROS induces NETosis through a mechanism involving DNA damage and subsequent DNA repair. The initial steps of the BER/NER pathway are crucial for chromatin decondensation and NET formation. These findings provide insights into the intricate process of NETosis and highlight potential therapeutic targets for NETosis-related diseases. Future research could investigate the relative contributions of different DNA repair pathways in NETosis and further explore the clinical implications of these findings.

The study primarily uses in vitro models (neutrophils and HL-60 cells). While these models offer valuable insights, the findings may not fully translate to the complex in vivo environment. The study focuses primarily on NOX-dependent NETosis; the mechanisms of NETosis induced by other pathways may differ. Finally, the study does not fully elucidate the specific molecular interactions and signaling pathways involved in the coupling of DNA repair and transcription during NETosis.