Li Marina, Lin Cindy, Leso Aubrey, Nefedova Yulia
Immunology, Microenvironment, and Metastasis Program, The Wistar Institute, Philadelphia, PA 19104, USA.
Cancers (Basel). 2020 Nov 18;12(11):3424. doi: 10.3390/cancers12113424.
Formation of neutrophil extracellular traps (NETs) has been associated with multiple pathologies including cancer. While the visualization of NETs by microscopy is a routine technique, their quantification presents a number of challenges. Commonly, as citrullination of histone H3 is required for NET formation, the presence of this modified histone along with DNA is considered to be a hallmark of NETs. Here, we describe and validate a novel assay for the quantification of NETs based on the detection of citrullinated histone H3 bound to DNA (CitH3DNA binding assay). Using this assay, we investigated the effect of phorbol 12-myristate 13-acetate (PMA) on NET formation by neutrophils isolated from the bone marrow of control and myeloma-bearing mice. We found that PMA induced citrullination of histone H3, an increase in the level of CitH3DNA, and NET formation in neutrophils from both tumor-free and myeloma-bearing mice. The levels of CitH3DNA in the NET fractions, as measured by our assay, directly correlated with the citrullination of histone H3 in neutrophils, as detected by Western blotting, and were significantly higher in PMA-stimulated compared to unstimulated neutrophils. Neutrophils from tumor-bearing mice produced more NETs than those from tumor-free counterparts following stimulation with PMA. The increase in NET production correlated with significantly higher histone H3 citrullination levels and increased measurements of CitH3DNA. Thus, our data indicate that bone marrow neutrophils from myeloma-bearing hosts are prone to NET formation.
中性粒细胞胞外诱捕网（NETs）的形成与包括癌症在内的多种病理状况相关。虽然通过显微镜观察NETs是一项常规技术，但其定量分析存在诸多挑战。通常，由于NET形成需要组蛋白H3的瓜氨酸化，这种修饰的组蛋白与DNA的共存被认为是NETs的一个标志。在此，我们描述并验证了一种基于检测与DNA结合的瓜氨酸化组蛋白H3（瓜氨酸化组蛋白H3-DNA结合测定法）对NETs进行定量分析的新方法。使用该方法，我们研究了佛波酯12-肉豆蔻酸酯13-乙酸酯（PMA）对从对照小鼠和荷瘤小鼠骨髓中分离出的中性粒细胞NET形成的影响。我们发现，PMA诱导了组蛋白H3的瓜氨酸化、瓜氨酸化组蛋白H3-DNA水平的增加以及无瘤小鼠和荷瘤小鼠中性粒细胞中NET的形成。通过我们的测定法测得的NET组分中瓜氨酸化组蛋白H3-DNA的水平，与通过蛋白质免疫印迹法检测到的中性粒细胞中组蛋白H3的瓜氨酸化直接相关，并且与未刺激的中性粒细胞相比，PMA刺激的中性粒细胞中该水平显著更高。用PMA刺激后，荷瘤小鼠的中性粒细胞产生的NETs比无瘤小鼠的更多。NET产生的增加与组蛋白H3瓜氨酸化水平显著更高以及瓜氨酸化组蛋白H3-DNA的测量值增加相关。因此，我们的数据表明荷瘤宿主的骨髓中性粒细胞易于形成NETs。
