Neutrophil extracellular traps (NETs) occur during the development of autoimmune diseases, cancer and diabetes. A novel form of cell death that is induced by NETs is called NETosis. Although these diseases are known to have an epigenetic component, epigenetic regulation of NETosis has not previously been explored. In the present study, we investigated the effects of epigenetic change, especially DNA demethylation, on NETosis in neutrophil-like cells differentiated from HL-60 cells, which were incubated for 72 h in the presence of 1.25% DMSO. DMSO-differentiated neutrophil-like cells tended to have increased methylation of genomic DNA. NETosis in the neutrophil-like cells was induced by the treatment with A23187, calcium ionophore, and increased by the addition of the DNMT inhibitor 5-azacytidine (Aza) during differentiation. Interestingly, Aza-stimulated neutrophil-like cell induced NETosis without treatment with A23187. Although reactive oxygen species (ROS), especially superoxide and hypochlorous acid, are important in NETosis induction, treatment with Aza decreased production of ROS, while mitochondria ROS scavenger tended to decrease Aza-induced NETosis. Moreover, the genomic DNA in Aza-stimulated neutrophil-like cell was demethylated, and the expression of peptidylarginine deiminase4 (PAD4) and citrullinated histone H3 (R2+R8+R17) was increased, but myeloperoxidase expression was unaffected. Additionally, PAD4 inhibition tended to decrease Aza-induced NETosis. The DNA demethylation induced by the DNMT inhibitor in neutrophil-like cells enhanced spontaneous NETosis through increasing PAD4 expression and histone citrullination. This study establishes a relationship between NETosis and epigenetics for the first time, and indicates that various diseases implicated to have an epigenetic component might be exacerbated by excessive NETosis also under epigenetic control.

中性粒细胞胞外陷阱（NETs）发生在自身免疫性疾病，癌症和糖尿病的发展过程中。NETs引起的一种新型细胞死亡形式称为NETosis。尽管已知这些疾病具有表观遗传成分，但以前尚未探讨过NETosis的表观遗传调控。在本研究中，我们研究了表观遗传变化（尤其是DNA脱甲基）对从HL-60细胞分化出的嗜中性白细胞样细胞中NETosis的影响，该细胞在1.25％DMSO存在下孵育72小时。DMSO分化的嗜中性粒细胞样细胞倾向于增加基因组DNA的甲基化。中性粒细胞样细胞的NETosis通过用A23187，钙离子载体处理诱导，并在分化过程中通过添加DNMT抑制剂5-氮杂胞苷（Aza）来增加。有趣的是 不用A23187治疗的Aza刺激的中性粒细胞样细胞诱导的NETosis。尽管活性氧（ROS），尤其是超氧化物和次氯酸在NETosis的诱导中很重要，但是用Aza处理会降低ROS的产生，而线粒体ROS清除剂则倾向于减少Aza引起的NETosis。此外，Aza刺激的中性粒细胞样细胞中的基因组DNA被去甲基化，并且肽酰精氨酸脱亚氨酶4（PAD4）和瓜氨酸化的组蛋白H3（R2 + R8 + R17）的表达增加，但是髓过氧化物酶的表达不受影响。另外，PAD4抑制作用倾向于减少Aza诱导的NETosis。DNMT抑制剂在嗜中性粒细胞样细胞中诱导的DNA去甲基化通过增加PAD4表达和组蛋白瓜氨酸化来增强自发性NETosis。