Interindividual variation of NETosis in healthy donors: introduction and application of a refined method for extracellular trap quantification
Interindividual variation of NETosis in healthy donors: introduction and application of a refined method for extracellular trap quantification
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DOI：
10.1111/exd.13125
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发表时间：
2016-11-01
影响因子：
3.6
通讯作者：
Hadaschik, Eva N.
中科院分区：
文献类型：
--
作者：
Hoffmann, Jochen H. O.;Schaekel, Knut;Hadaschik, Eva N.
中性粒细胞胞外陷阱(Net)的形成是一种先天免疫防御机制，中性粒细胞(PMN)通过这种机制产生带有抗菌肽的去凝集的染色质网状结构，以捕获并可能杀死微生物。如果这一过程导致细胞死亡，则称为网织红细胞增多症。据报道，这种特殊机制的改变与包括银屑病和红斑狼疮在内的慢性炎症性疾病的发病机制有关。尽管如此，NETsis的量化仍然构成了一个相当大的挑战。我们报告并测试了一种在健康人类捐赠者中进行形态网络量化的改进方案，该方案包括隔离、刺激、DNA染色、实时成像和半自动离线分析。结果重复性好，与人工计数结果吻合较好。供体内对佛波酯(PMA)刺激的净化率的平均变异系数低于供体间的变异系数(如果在同一天重复，分别为10%和82%，n=4；如果实验平均间隔42+/-34d，则分别为38%和74%，n=6)。总体来说，供体间变异系数为67%(n=10)。这些发现共同支持了不同供者的中性粒细胞易患NETsis的不同倾向。Picogreen荧光与细胞死亡的相关性比与形态网织红细胞增殖率的相关性更强(r(2)=0.89，P
Neutrophil extracellular trap (NET) formation is a mechanism of innate immune defence by which neutrophil (polymorphonuclear) granulocytes (PMN) produce net-like structures of decondensed chromatin decorated with antimicrobial peptides for trapping and possibly killing microorganisms. If this process leads to cell death, it is termed NETosis. Alterations of this particular mechanism have been reported to be involved in the pathogenesis of chronic inflammatory diseases including psoriasis and lupus erythematosus. Still, quantification of NETosis poses a considerable challenge. We report and test a refined protocol for morphological NET quantification in healthy human donors that encompasses isolation, stimulation, DNA staining, live imaging and semi-automated offline analysis. The results were highly reproducible and in good agreement with manual counting. The average intra-donor coefficient of variation of NETosis rates to phorbol myristate acetate (PMA) stimulation was low compared to the respective interdonor coefficient of variation (10% vs 82%, n=4, respectively, if experiments were repeated on the same day, and 38% vs 74%, n=6, respectively, if experiments were repeated on average 42 +/- 34days apart). Overall, the interdonor coefficient of variation was 67% (n=10). These findings altogether support the existence of a distinct predisposition of PMN from different donors for undergoing NETosis. Picogreen fluorescence correlated stronger to cell death than to morphological NETosis (r(2)=.89, P