ICAAM-Instituto Ciências Agrárias e Ambientais Mediterrânicas, IIFA/Universidade de Évora, Núcleo da Mitra, Apart 94, 7002-542, Évora, Portugal,
Parasitol Res. 2014 Aug;113(8):2797-807. doi: 10.1007/s00436-014-3939-0. Epub 2014 May 22.
As a novel effector mechanism polymorphonuclear neutrophils (PMN) release neutrophil extracellular traps (NETs), which represent protein-labeled DNA matrices capable of extracellular trapping and killing of invasive pathogens. Here, we demonstrate for the first time NET formation performed by caprine PMN exposed to different stages (sporozoites and oocysts) of the goat apicomplexan protozoan parasite Eimeria arloingi. Scanning electron microscopy as well as fluorescence microscopy of sporozoites- and oocysts-PMN co-cultures revealed a fine network of DNA fibrils partially covering the parasites. Immunofluorescence analyses confirmed the co-localization of histones (H3), neutrophil elastase (NE), and myeloperoxidase (MPO) in extracellular traps released from caprine PMN. In addition, the enzymatic activity of NE was found significantly enhanced in sporozoite-exposed caprine PMN. The treatment of caprine NET structures with deoxyribonuclease (DNase) and the NADPH oxidase inhibitor diphenylene iodondium (DPI) significantly reduced NETosis confirming the classical characteristics of NETs. Caprine NETs efficiently trapped vital sporozoites of E. arloingi since 72% of these stages were immobilized-but not killed-in NET structures. As a consequence, early infection rates were significantly reduced when PMN-pre-exposed sporozoites were allowed to infect adequate host cells. These findings suggest that NETs may play an important role in the early innate host response to E. arloingi infection in goats.
作为一种新型效应机制,多形核粒细胞(PMN)会释放中性粒细胞胞外陷阱(NETs),这是一种能够捕获和杀伤入侵病原体的蛋白标记 DNA 基质。在这里,我们首次证明了暴露于山羊顶复门原虫 Eimeria arloingi 不同阶段(孢子和卵囊)的山羊 PMN 可以形成 NET。扫描电子显微镜和孢子和卵囊-PMN 共培养物的荧光显微镜观察显示,DNA 纤维的细网部分覆盖了寄生虫。免疫荧光分析证实了组蛋白(H3)、中性粒细胞弹性蛋白酶(NE)和髓过氧化物酶(MPO)在山羊 PMN 释放的细胞外陷阱中的共定位。此外,在暴露于孢子的山羊 PMN 中,NE 的酶活性显著增强。用脱氧核糖核酸酶(DNase)和 NADPH 氧化酶抑制剂二苯基碘(DPI)处理山羊 NET 结构,显著降低了 NETosis,证实了 NETs 的经典特征。山羊 NET 有效地捕获了 E. arloingi 的有活力的孢子,因为这些阶段中有 72%被固定在 NET 结构中,但没有被杀死。因此,当 PMN 预先暴露的孢子被允许感染足够的宿主细胞时,早期感染率显著降低。这些发现表明,NETs 可能在山羊对 E. arloingi 感染的早期固有宿主反应中发挥重要作用。