Cancers:中性粒细胞胞外陷阱在慢性粒细胞白血病中增加,并且受酪氨酸激酶抑制剂的不同影响

2022-01-16 MedSci原创 MedSci原创

这篇文章首次报告了 CML 患者中性粒细胞中 NET 形成的增加,并提供了 TKI 对 NET 形成的不同影响的证据,可能通过改变 ROS 产生和 PAD4 表达

随着使用某些酪氨酸激酶抑制剂 (TKI) 治疗慢性粒细胞白血病 (CML),这些药物也与一些短期和长期毒性有关,心血管并发症的报道越来越多。国外一研究团队描述了CML 中 NET 形成的特征和机制,并评估了 TKI 对离体和体外 NET 形成的影响。

在基线和用离子霉素 (IO) 和佛波醇 12-肉豆蔻酸酯 13-乙酸盐 (PMA) 刺激后,与匹配的对照相比,从 CML 初治患者中分离出的中性粒细胞显示 NET 形成显着增加。与对照相比,CML 样品中瓜氨酸化组蛋白 H3 (H3cit)、肽基精氨酸脱亚氨酶 4 (PAD4) 和活性氧 (ROS) 的表达显着更高。

图1:CML 患者的中性粒细胞 NETs 增加。 ( A ) 新诊断的慢性粒细胞白血病 (CML) 患者与年龄和性别匹配的健康对照组相比,在用 5 μM 离子霉素 (IO) 或二甲基亚砜 (DMSO) 刺激时,中性粒细胞胞外陷阱 (NET) 形成2小时。每组n = 8。NET 形成表示为具有 NET 形成特征的形态变化的中性粒细胞百分比(参见方法)。* p < 0.05 ** p < 0.005。(乙) 在用 4 μM IO 或 DMSO 刺激 2 小时后,来自 CML 患者和对照的中性粒细胞的代表性免疫荧光 (IF) 图像。4',6-Diamidino-2-phenylindole (DAPI) 以蓝色显示。白色箭头表示 NET 相关的形态变化。比例尺,50 μm。( C ) 用 100 μM 佛波醇 12-肉豆蔻酸酯 13-乙酸 (PMA) 或 DMSO 刺激 4 小时时,新诊断 CML 患者与健康对照组相比,NET 形成表示为 NET 结合弹性蛋白酶的量。每组n = 9。** p < 0.005。CML 患者和对照组的中性粒细胞裂解物印迹 ( D ) 肽基精氨酸脱亚氨酶 4 (PAD4) 和 ( E) 用 5µM IO 或 DMSO 刺激 2.5 小时后的瓜氨酸化组蛋白 H3 (H3cit) 表达。每组n = 3。* p < 0.05 ** p < 0.005。完整的印迹分析可在补充文件中找到(图 S3-S5)。( F ) 通过 2',7'-二氯二氢荧光素-二乙酸酯 (DCFDA) 测定新诊断 CML 患者和年龄和性别匹配的健康对照的中性粒细胞中的活性氧 (ROS) 水平。用 5 μM IO 刺激中性粒细胞 2.5 小时。每组n = 8。** p < 0.005;FU-荧光单位。

图2:Ponatinib 增强来自 CML 患者的中性粒细胞的 NET 形成。 ( A ) 来自新诊断的 CML 患者和年龄和性别匹配的对照(每组n = 9 )的中性粒细胞用各种酪氨酸激酶抑制剂 (TKI) 离体治疗。当用 100 μM PMA 或 DMSO 刺激 4 小时时,NET 形成表示为新诊断 CML 患者与健康对照相比的 NET 结合弹性蛋白酶的量。* p < 0.05 ** p < 0.005; ns——不显着。( B ) DCFDA 检测在暴露于各种 TKI 和 4 μM IO 刺激后,新诊断的 CML 患者和年龄和性别匹配的健康对照的中性粒细胞中的 ROS 水平。通过流式细胞术测量ROS水平。n= 8 用于对照,伊马替尼和普纳替尼;对于达沙替尼,n = 4。每个点代表一个患者/对照一式三份的平均值。上条涉及用 DMSO(对照)处理的 CML 细胞与用不同 TKI 处理的 CML 细胞之间的差异。下面的条形图描绘了每种条件下对照和 CML 细胞之间的差异。使用了以下 TKI 浓度:5.3 µM 伊马替尼、0.22 µM 达沙替尼和 0.17 µM 波纳替尼。* p < 0.05 ** p < 0.005; ns——不显着。

图3:BCR-ABL1 转导的 ER-HoxB8 细胞系系统概括了 CML、TKI 效应和揭示 PAD4 依赖性的 NET 表型。( A ) 绿色荧光蛋白 (GFP) 和BCR-ABL1表达以使用 MSCV-( pBabe mcs) -human p210 -BCR-ABL -IRES-GFP 系统评估HoxB8细胞的逆转录病毒转导效率。完整的印迹分析可以在补充文件中找到(图 S6)。( B ) HoxB8 - BCR-ABL1细胞和HOXB8用 IO 刺激用空载体转导的细胞,用氯脒处理或不处理,PAD4 抑制剂 (10 µM) 或二苯碘鎓 (DPI),烟酰胺腺嘌呤二核苷酸磷酸 (NADPH) 氧化酶抑制剂 (14 µM) 并用 DAPI 染色、抗 H3cit 和抗髓过氧化物酶 (MPO)。比例尺,100 μm。( C )用空载体转导的HoxB8-BCR-ABL1细胞和HoxB8细胞中的 H3cit 百分比,用 4 μM IO 刺激 2 小时并暴露于 DMSO、伊马替尼 (5.3 μM) 或普纳替尼 (0.17 μM)。* p < 0.05;ns——不显着。

用 TKI 预处理中性粒细胞与对 NET 形成的不同影响有关,BCR-ABL1逆转录病毒转导的HoxB8永生化小鼠造血祖细胞,在体外分化为中性粒细胞,显示 H3cit 和髓过氧化物酶 (MPO) 表达增加,这与过量的 NET 形成一致。这被 P​​AD4 抑制剂氯脒抑制,但不受 NADPH 抑制剂二苯碘鎓 ( DPI ) 的抑制。Ponatinib 预暴露在IO 刺激后显着增加了HoxB8-BCR-ABL1细胞中的H3cit 表达。

总之,这篇文章首次报告了 CML 患者中性粒细胞中 NET 形成的增加,并提供了 TKI 对 NET 形成的不同影响的证据,可能通过改变 ROS 产生和 PAD4 表达。研究人员提出了一种使用HoxB8细胞系统在体外评估 NET 的新方法,并概括了BCR-ABL1转导的PAD4 依赖性ER HoxB8细胞中的 NET 易感表型。这些数据进一步加深了我们对将波纳替尼与 CML 中 CVS 并发症联系起来的潜在过程的理解,并提供了潜在的破坏目标以减轻这些药物的不良反应。

 

原始出处:

Telerman, A.; Granot, G.; Leibovitch, C.; Yarchovsky-Dolberg, O.; Shacham-Abulafia, A.; Partouche, S.; Yeshurun, M.; Ellis, M.H.; Raanani, P.; Wolach, O. Neutrophil Extracellular Traps Are Increased in Chronic Myeloid Leukemia and Are Differentially Affected by Tyrosine Kinase Inhibitors. Cancers 202214, 119. https://doi.org/10.3390/cancers14010119

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    2022-11-16 anminleiryan
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    2022-01-19 jklm09
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    2022-01-17 ms6000001985805349

    学习

    0

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    2022-01-15 redcrab

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