Department of Biochemistry, Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science, 3500-3, Minamitamagaki, Suzuka-city, Mie, 513-8670, Japan.
Department of Anesthesiology, Osaka City University Medical School, 1-5-7, Asahi-machi, Abeno, Osaka, 545-8586, Japan.
Arch Biochem Biophys. 2020 Aug 15;689:108465. doi: 10.1016/j.abb.2020.108465. Epub 2020 Jun 17.
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.
中性粒细胞胞外诱捕网（NETs）在自身免疫性疾病、癌症和糖尿病的发展过程中发生。NETs 诱导的一种新型细胞死亡形式称为 NETosis。尽管这些疾病已知具有表观遗传成分，但 NETosis 的表观遗传调控尚未得到探索。在本研究中，我们研究了表观遗传变化，特别是 DNA 去甲基化，对 HL-60 细胞分化的中性粒细胞样细胞 NETosis 的影响，这些细胞在存在 1.25% DMSO 的情况下孵育 72 小时。DMSO 分化的中性粒细胞样细胞的基因组 DNA 甲基化倾向增加。NETosis 在中性粒细胞样细胞中通过 A23187 处理诱导，并在分化过程中添加 DNA 甲基转移酶抑制剂 5-氮杂胞苷（Aza）而增加。有趣的是，Aza 刺激的中性粒细胞样细胞在没有 A23187 处理的情况下诱导 NETosis。虽然活性氧（ROS），特别是超氧阴离子和次氯酸，在 NETosis 诱导中很重要，但 Aza 处理降低了 ROS 的产生，而线粒体 ROS 清除剂则倾向于降低 Aza 诱导的 NETosis。此外，Aza 刺激的中性粒细胞样细胞中的基因组 DNA 去甲基化，肽基精氨酸脱亚氨酶 4（PAD4）和瓜氨酸化组蛋白 H3（R2+R8+R17）的表达增加，但髓过氧化物酶的表达不受影响。此外，PAD4 抑制倾向于降低 Aza 诱导的 NETosis。DNMT 抑制剂在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增强自发性 NETosis。本研究首次建立了 NETosis 与表观遗传学之间的关系，并表明各种被认为具有表观遗传成分的疾病可能在表观遗传控制下通过过度 NETosis 而加剧。
