Supplementary Material for: Neutrophil Extracellular Traps Stimulate Proinflammatory Responses in Human Airway Epithelial Cells

Tissue injury leads to the release of uric acid (UA). At high local concentrations, UA can form monosodium urate crystals (MSU). MSU and UA stimulate neutrophils to release extracellular traps (NET). Here, we investigated whether these NET could be involved in the development of inflammation by stimulating cytokine release by airway epithelial cells. We found that NET significantly increased the secretion of CXCL8/IL-8 and IL-6 by alveolar and bronchial epithelial cells. These effects were not observed when NETosis was inhibited by Diphenyleneiodonium, elastase inhibitor, or Cl-amidine. Similar findings were made with NET induced by cigarette smoke extract, suggesting that NET proinflammatory capacity is independent of the inducing stimulus. Furthermore, NET affected neither the viability and morphology of epithelial cells nor the barrier integrity of polarized cells. The epithelial stimulatory capacity of NET was not affected by degradation of DNA with micrococcal nuclease, treatment with heparin, or inhibition of the elastase immobilized to DNA, but it was significantly reduced by pretreatment with an anti-HMGB-1 blocking antibody. Altogether, our findings indicate that NET exert direct proinflammatory effects on airway epithelial cells that might contribute in vivo to the further recruitment of neutrophils and the perpetuation of inflammation upon lung tissue damage.组织损伤可导致尿酸（uric acid, UA）释放。当局部浓度升高时，尿酸可形成单钠尿酸盐结晶（monosodium urate crystals, MSU）。单钠尿酸盐结晶与尿酸可刺激中性粒细胞释放中性粒细胞胞外陷阱（extracellular traps, NET）。本研究旨在探究此类中性粒细胞胞外陷阱是否可通过刺激气道上皮细胞释放细胞因子参与炎症发生发展。我们发现，中性粒细胞胞外陷阱可显著促进肺泡与支气管上皮细胞分泌趋化因子CXCL8/白细胞介素8（CXCL8/IL-8）及白细胞介素6（IL-6）。当使用二苯基碘鎓盐、弹性蛋白酶抑制剂或Cl-脒抑制中性粒细胞胞外陷阱形成（NETosis）时，未观察到上述效应。采用香烟烟雾提取物诱导生成的中性粒细胞胞外陷阱亦可得到相似结果，提示中性粒细胞胞外陷阱的促炎活性不依赖于其诱导刺激因素。此外，中性粒细胞胞外陷阱既未影响上皮细胞的存活活力与形态结构，亦未破坏极化上皮细胞的屏障完整性。中性粒细胞胞外陷阱对上皮细胞的刺激活性，不受微球菌核酸酶降解DNA、肝素处理或抑制结合于DNA的弹性蛋白酶的影响，但经抗高迁移率族蛋白B1（HMGB-1）封闭抗体预处理后，该刺激活性显著降低。综上，本研究结果表明，中性粒细胞胞外陷阱可对气道上皮细胞产生直接的促炎效应，该效应可能在体内参与肺组织损伤后中性粒细胞的进一步招募及炎症的持续维持。