Uncovering the Mechanisms of how ROS drive NETosis: The Crucial Role of DNA Repair Pathway

Neutrophil extracellular traps (NETs) are cytotoxic protein-coated, DNA-based web-like structures implicated in antimicrobial defense, autoimmune diseases and tissue injury. NETotic cell death (NETosis) is defined as a reactive oxygen species (ROS)-dependent cell death, restricted to cells of hematopoietic origin, and associated with NET extrusion. In response to different agonists, neutrophils produce ROS via either NADPH oxidase (NOX-dependent) or mitochondria (NOX-independent) pathways. However, the role of ROS in NETosis was unclear. Additionally, whether NETosis and apoptosis could happen in the same neutrophil was unknown. It was also unknown whether ultraviolet (UV) radiation, a classical inducer of apoptosis and mitochondrial ROS, could induce NETosis. Here, I uncovered that: (i) High UV doses induce both NETosis and apoptosis in the same neutrophil (ApoNETosis); (ii) Base excision repair (BER), the pathway that repairs oxidative DNA damage, is necessary for UV-induced mitochondrial ROS-mediated NETosis. Specifically, inhibiting early, but not late, stages of BER leading to nick formation inhibited UV-induced NETosis; (iii) Unlike in UV-induced NETosis, mitochondrial ROS-induced DNA damage repair and histone citrullination both contribute to calcium ionophore (A23187, ionomycin from Streptomyces conglobatus)-induced NOX-independent NETosis; (iv) In response to bacteria and bacterial components, neutrophils produce large quantities of NOX-derived ROS. Early, but not late, stages of BER were also involved in NETosis induced by Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli lipopolysaccharide, and phorbol 12-myristate 13-acetate; (v) Typically, neutrophils undergo NETosis in serum-free conditions (e.g., lung airways). Baseline NETosis in untreated neutrophils, under normothermic and hyperthermic conditions, is driven by NOX-derived ROS. Under normothermic conditions, early stage BER inhibition abolishes NETosis while late stage BER inhibition promotes it, contrasting agonist-induced NETosis. In mechanistic terms, I identified a novel NETosis agonist, UV, capable of inducing both NETosis and apoptosis (ApoNETosis). We have determined how ROS drives NETosis: through base oxidation, ROS drives NETosis by activating DNA repair machinery, resulting in DNA nicking and chromatin unwinding. This mechanism is consistently observed across all forms of NETosis we investigated. My findings provide new insights into the mechanisms of NETosis, and suggest that some of the DNA repair genes may be relevant to NET-related diseases and candidates for therapeutic studies.

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