College of Life Sciences and Engineering, Foshan University, Foshan 528225, Guangdong Province, PR China.
College of Life Sciences and Engineering, Foshan University, Foshan 528225, Guangdong Province, PR China.
Poult Sci. 2023 Oct;102(10):102946. doi: 10.1016/j.psj.2023.102946. Epub 2023 Jul 18.
Zearalenone (ZEA) is produced mainly by fungi belonging to genus Fusarium in foods and feeds. Heterophil extracellular traps (HETs) are a novel defense mechanism of chicken innate immunity involving activated heterophils. However, the conditions and requirements for ZEA-triggered HET release remain unknown. In this study, immunostaining analysis demonstrated that ZEA-triggered extracellular fibers were composed of histone and elastase assembled on DNA skeleton, showing that ZEA can induce the formation of HETs. Further experiments indicated that ZEA-induced HET release was concentration-dependent (ranging from 20 to 80 μM ZEA) and time-dependent (ranging from 30 to 180 min). Moreover, in 80 μM ZEA-exposed chicken heterophils, reactive oxygen species (ROS) level, catalase (CAT), superoxide dismutase (SOD) activity, malondialdehyde (MDA) content, and glutathione (GSH) content were increased. Simultaneously, ZEA at 80 μM activated ERK and p38 MAPK signaling pathways by increasing the phosphorylation level of ERK and p38 proteins. Pharmacological inhibition assays revealed that blocking nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, ERK, and p38 mitogen-activated protein kinase (MAPK) reduced ZEA-induced ROS levels but had no impact on HET formation. Furthermore, immunostaining analysis indicated that the heterophil underwent the formation of autophagosome based on being stained with LC3B. The pharmacological inhibition assays demonstrated that rapamycin-, wortmannin-, and 3-methyladenine (3-MA)-treatments modulated ZEA-triggered HET formation, indicating that heterophil autophagy played a key role in ZEA-induced HET formation. Further studies on energy metabolism showed that inhibition of lactate/glucose transport, hexokinase-2 (HK-2), fructose-2,6-biphosphatase 3 (PFKFB3) in glycolysis abated ZEA-induced HETs, implying that glycolysis was one of the factors influencing the ZEA-induced HET formation. Besides, inhibition of the peptidylarginine deiminase (PAD) enzyme and P2X significantly reduced the ZEA-induced HET formation. In conclusion, we demonstrated that ZEA-triggered HET formation, which was associated with glycolysis, autophagy, PAD enzyme, and P2X receptor activation, providing valuable insight into the negative effect of ZEA on chicken innate immunity.
玉米赤霉烯酮（ZEA）主要由镰刀菌属真菌在食品和饲料中产生。嗜中性粒细胞细胞外陷阱（HET）是一种新的鸡先天免疫防御机制，涉及激活的嗜中性粒细胞。然而，ZEA 触发 HET 释放的条件和要求尚不清楚。在这项研究中，免疫染色分析表明，ZEA 触发的细胞外纤维由组蛋白和弹性蛋白酶组装在 DNA 骨架上组成，表明 ZEA 可以诱导 HET 的形成。进一步的实验表明，ZEA 诱导的 HET 释放是浓度依赖性的（范围从 20 到 80 μM ZEA）和时间依赖性的（范围从 30 到 180 分钟）。此外，在 80 μM ZEA 暴露的鸡嗜中性粒细胞中，活性氧（ROS）水平、过氧化氢酶（CAT）、超氧化物歧化酶（SOD）活性、丙二醛（MDA）含量和谷胱甘肽（GSH）含量增加。同时，ZEA 在 80 μM 时通过增加 ERK 和 p38 蛋白的磷酸化水平激活 ERK 和 p38 MAPK 信号通路。药理学抑制试验表明，阻断烟酰胺腺嘌呤二核苷酸磷酸（NADPH）氧化酶、ERK 和 p38 丝裂原活化蛋白激酶（MAPK）可降低 ZEA 诱导的 ROS 水平，但对 HET 形成没有影响。此外，免疫染色分析表明，嗜中性粒细胞基于 LC3B 的染色经历自噬体的形成。药理学抑制试验表明，雷帕霉素、wortmannin 和 3-甲基腺嘌呤（3-MA）处理调节了 ZEA 触发的 HET 形成，表明嗜中性粒细胞自噬在 ZEA 诱导的 HET 形成中起关键作用。进一步的能量代谢研究表明，抑制乳酸/葡萄糖转运、己糖激酶-2（HK-2）和果糖-2,6-二磷酸酶 3（PFKFB3）可减弱 ZEA 诱导的 HET，表明糖酵解是影响 ZEA 诱导的 HET 形成的因素之一。此外，抑制肽基精氨酸脱氨酶（PAD）酶和 P2X 显著降低了 ZEA 诱导的 HET 形成。总之，我们证明了 ZEA 触发的 HET 形成与糖酵解、自噬、PAD 酶和 P2X 受体激活有关，为玉米赤霉烯酮对鸡先天免疫的负面影响提供了有价值的见解。
