Parasitology Unit, Department of Animal Pathology, Faculty of Veterinary Medicine, University of Las Palmas de Gran Canaria, Las Palmas, Spain.
Escuela de Medicina Veterinaria, Facultad de Recursos Naturales y Medicina Veterinaria, Universidad Santo Tomas, Chile.
Exp Parasitol. 2021 Jan;220:108034. doi: 10.1016/j.exppara.2020.108034. Epub 2020 Nov 12.
Eimeria ninakohlyakimovae represents a highly pathogenic coccidian parasite causing severe haemorrhagic typhlocolitis in goat kids worldwide. NETosis was recently described as an efficient defense mechanism of polymorphonuclear neutrophils (PMN) acting against different parasites in vitro and in vivo. In vitro interactions of caprine PMN with parasitic stages of E. ninakohlyakimovae (i. e. oocysts and sporozoites) as well as soluble oocyst antigens (SOA) were analyzed at different ratios, concentrations and time spans. Extracellular DNA staining was used to illustrate classical molecules induced during caprine NETosis [i. e. histones (H3) and neutrophil elastase (NE)] via antibody-based immunofluorescence analyses. Functional inhibitor treatments with DPI and DNase I were applied to unveil role of NADPH oxidase (NOX) and characterize DNA-backbone composition of E. ninakohlyakimovae-triggered caprine NETosis. Scanning electron microscopy (SEM)- and immunofluorescence-analyses demonstrated that caprine PMN underwent NETosis upon contact with sporozoites and oocysts of E. ninakohlyakimovae, ensnaring filaments which firmly entrapped parasites. Detailed co-localization studies of E. ninakohlyakimovae-induced caprine NETosis revealed presence of PMN-derived DNA being adorned with nuclear H3 and NE corroborating molecular characteristics of NETosis. E. ninakohlyakoimovae-induced caprine NETosis was found to be NOX-independent since DPI inhibition led to a slight decrease of NETosis. Exposure of caprine PMN to vital E. ninakohlyakimovae sporozoites as well as SOA resulted in up-regulation of IL-12, TNF-α, IL-6, CCL2 and iNOS gene transcription in stimulated PMN. Since vital E. ninakohlyakimovae-sporozoites induced caprine NETosis, this effective entrapment mechanism might reduce initial sporozoite epithelial host cell invasion during goat coccidiosis ultimately resulting in less macromeront formation and reduced merozoites I production.
柔嫩艾美耳球虫代表一种高致病性的球虫寄生虫，可导致全球山羊幼崽发生严重的出血性回肠炎。NETosis 最近被描述为多形核中性粒细胞（PMN）对抗不同寄生虫的有效防御机制，无论是在体外还是体内。在不同比例、浓度和时间跨度下，分析了山羊 PMN 与柔嫩艾美耳球虫的寄生虫阶段（即卵囊和孢子）以及可溶性卵囊抗原（SOA）的体外相互作用。通过基于抗体的免疫荧光分析，使用细胞外 DNA 染色来阐明山羊 NETosis 期间诱导的经典分子[即组蛋白（H3）和中性粒细胞弹性蛋白酶（NE）]。应用 DPI 和 DNase I 功能抑制剂处理来揭示 NADPH 氧化酶（NOX）的作用，并阐明柔嫩艾美耳球虫触发的山羊 NETosis 中 DNA 骨架的组成。扫描电子显微镜（SEM）和免疫荧光分析表明，山羊 PMN 在与柔嫩艾美耳球虫的孢子和卵囊接触后发生 NETosis，缠绕的丝紧紧困住寄生虫。详细的柔嫩艾美耳球虫诱导的山羊 NETosis 共定位研究表明，存在被 PMN 衍生的 DNA 装饰的核 H3 和 NE，这证实了 NETosis 的分子特征。发现柔嫩艾美耳球虫诱导的山羊 NETosis 不依赖于 NOX，因为 DPI 抑制导致 NETosis 略有下降。暴露于活的柔嫩艾美耳球虫孢子和 SOA 会导致刺激的 PMN 中 IL-12、TNF-α、IL-6、CCL2 和 iNOS 基因转录的上调。由于活的柔嫩艾美耳球虫孢子诱导了山羊 NETosis，这种有效的捕获机制可能会减少山羊球虫病期间初始孢子对上皮宿主细胞的入侵，最终导致较少的大配子体形成和减少裂殖子 I 的产生。
