Val Stéphanie, Krueger Anna, Hussain Arman, Tomney Amarel, Chen Yajun, Lazarski Christopher, Preciado Diego
Sheikh Zayed Institute for Pediatric Surgical Innovation Children's National Health System Washington District of Columbia USA.
Center for Cancer and Immunology Research Children's National Health System Washington District of Columbia USA.
Laryngoscope Investig Otolaryngol. 2020 May 23;5(3):536-545. doi: 10.1002/lio2.396. eCollection 2020 Jun.
Chronic otitis media (COM) is characterized by middle ear fluid predominantly containing cytokines, (NTHi), the mucin MUC5B, and neutrophil extracellular traps (NETs). NETs consist of extracellular DNA coated with antibacterial proteins such as myeloperoxidase (MPO) and citrullinated histone 3 (CitH3). NETs can damage tissues and sustain inflammation. Our study aimed to develop an in vitro model of NETosis, testing COM inductors.
NETosis was evaluated in fresh blood human neutrophils attached to collagen-coated plates and in suspension exposed to phorbol myristate acetate (PMA) as a control, and COM relevant mediators. Confocal microscopy, DNA fluorescence assay and flow cytometry were used to quantify NETosis.
PMA exposure induced DNA, MPO, and CitH3 by immunofluorescence (IF) most significantly at 3 hours (3.8-fold for DAPI, 7.6-fold for MPO, and 6.9-fold for CitH3, all < .05). IL-8 and TNF-α cytokines showed milder increases of DAPI, MPO, and CitH3 positive cells. NTHi had no effect on these NETs markers. Purified salivary MUC5B (10 to 40 μg/mL) produced potent increases, comparable to PMA. A composite NET score summing the fold-increases for DAPI, MPO, and CitH3 demonstrated PMA at 13.6 to 19 relative to control set at 1; and MUC5B at 8.6 to 16.3 (all < .05). IL-8 and TNF-α showed scores of 5.4 and 3, respectively, but these were not statistically significant.
We developed a reliable in vitro assay for NETosis which demonstrated that salivary MUC5B is a potent inductor of NETs whereas IL-8, TNF-α, live and lyzed NTHi demonstrated minimal to no NETosis.
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慢性中耳炎（COM）的特征是中耳积液主要包含细胞因子、非典型流感嗜血杆菌（NTHi）、粘蛋白MUC5B和中性粒细胞胞外诱捕网（NETs）。NETs由包被有抗菌蛋白（如髓过氧化物酶（MPO）和瓜氨酸化组蛋白3（CitH3））的细胞外DNA组成。NETs会损害组织并维持炎症。我们的研究旨在建立一种NETosis的体外模型，测试COM诱导剂。
在附着于胶原包被板的新鲜人血中性粒细胞以及暴露于佛波酯肉豆蔻酸酯乙酸酯（PMA）作为对照和COM相关介质的悬浮液中评估NETosis。共聚焦显微镜、DNA荧光测定法和流式细胞术用于量化NETosis。
PMA暴露在3小时时通过免疫荧光（IF）诱导DNA、MPO和CitH3最为显著（DAPI增加3.8倍，MPO增加7.6倍，CitH3增加6.9倍，均<0.05）。白细胞介素-8（IL-8）和肿瘤坏死因子-α（TNF-α）细胞因子使DAPI、MPO和CitH3阳性细胞的增加较为轻微。NTHi对这些NETs标志物没有影响。纯化的唾液MUC5B（10至40μg/mL）产生了与PMA相当的显著增加。将DAPI、MPO和CitH3的增加倍数相加得到的综合NET评分显示，PMA相对于设定为1的对照组为13.6至19；MUC5B为8.6至16.3（均<0.05）。IL-8和TNF-α的评分分别为5.4和3，但这些无统计学意义。
我们建立了一种可靠的NETosis体外检测方法，该方法表明唾液MUC5B是NETs的有效诱导剂，而IL-8、TNF-α、活的和裂解的NTHi显示出极少至无NETosis。
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