DataSheet_3_ROS and DNA repair in spontaneous versus agonist-induced NETosis: Context matters.pdf

Reactive oxygen species (ROS) is essential for neutrophil extracellular trap formation (NETosis). Nevertheless, how ROS induces NETosis at baseline and during neutrophil activation is unknown. Although neutrophils carry DNA transcription, replication and repair machineries, their relevance in the short-lived mature neutrophils that carry pre-synthesized proteins has remained a mystery for decades. Our recent studies show that (i) NETosis-inducing agonists promote NETosis-specific kinase activation, genome-wide transcription that helps to decondense chromatin, and (ii) excess ROS produced by NADPH oxidase activating agonists generate genome-wide 8-oxy-guanine (8-OG), and the initial steps of DNA repair are needed to decondense chromatin in these cells. These steps require DNA repair proteins necessary for the assembly and nicking at the damaged DNA sites (poly ADP ribose polymerase PARP, apurinic endonuclease APE1 and DNA ligase), but not the enzymes that mediate the repair DNA synthesis (Proliferating cell nuclear antigen (PCNA) and DNA Polymerases). In this study, we show that (i) similar to agonist-induced NETosis, inhibition of early steps of oxidative DNA damage repair proteins suppresses spontaneous NETosis, but (ii) the inhibition of late stage repair proteins DNA polymerases and PCNA drastically promotes baseline NETosis. Hence, in the absence of excessive ROS generation and neutrophil activation, DNA repair mediated by PCNA and DNA polymerases is essential to prevent chromatin decondensation and spontaneous NETosis. These findings indicate that ROS, oxidative DNA damage, transcription and DNA repair differentially regulate spontaneous and agonist-induced NETosis. Therefore, context matters.

活性氧簇（ROS）对于中性粒细胞胞外陷阱形成（NETosis）至关重要。然而，ROS在基线水平和中性粒细胞激活过程中如何诱导NETosis尚属未知。尽管中性粒细胞携带DNA转录、复制和修复机制，但其对于携带预合成蛋白的短暂存活成熟中性粒细胞的相关性数十年来一直是个谜。我们最近的研究表明：（i）NETosis诱导的激动剂可促进NETosis特异性激酶的激活，全基因组转录有助于解旋染色质；（ii）由NADPH氧化酶激活激动剂产生的过量ROS在细胞中产生全基因组8-氧鸟嘌呤（8-OG），并需要DNA修复的初始步骤来解旋染色质。这些步骤需要DNA修复蛋白，如聚ADP核糖聚合酶PARP、无碱基末端核酸酶APE1和DNA连接酶，以在受损DNA位点进行组装和切割，而不需要介导DNA合成修复的酶（如增殖细胞核抗原PCNA和DNA聚合酶）。在本研究中，我们发现：（i）与激动剂诱导的NETosis相似，抑制氧化DNA损伤修复的早期步骤可抑制自发性NETosis；（ii）抑制晚期修复蛋白DNA聚合酶和PCNA则极大地促进基线NETosis。因此，在没有过量ROS生成和中性粒细胞激活的情况下，PCNA和DNA聚合酶介导的DNA修复对于防止染色质解旋和自发性NETosis至关重要。这些发现表明，ROS、氧化DNA损伤、转录和DNA修复在调节自发性NETosis和激动剂诱导NETosis方面存在差异调节作用。因此，环境因素至关重要。