Fasciola hepatica is the causative agent of fasciolosis, a worldwide distributed zoonotic disease, leading to hepatitis in humans and livestock. Newly excysted juveniles (NEJ) of F. hepatica are the first invasive stages to encounter leukocytes of host innate immune system in vivo. Among leukocytes, polymorphonuclear neutrophils (PMN) are the most abundant granulocytes of blood system and first ones to migrate into infection sites. PMN are able to cast neutrophil extracellular traps (NETs), also known as NETosis, consisting of nuclear DNA, decorated with histones, enzymes and antimicrobial peptides, which can entrap and eventually kill invasive parasites. Given that only few large parasitic helminths have been identified as potent NETosis inducers, here we studied for first time whether different F. hepatica stages can also trigger NETosis. Therefore, isolated bovine PMN were co-cultured with viable F. hepatica-NEJ, -metacercariae, -eggs and soluble antigen (FhAg). Interestingly, all stages failed to induce considerable levels of NETosis as detected by immunofluorescence- and scanning electron microscopy (SEM) analyses. NEJ remained motile until the end of incubation period. In line, NETosis quantification via nuclear area expansion (NAE) analysis revealed NEJ as weak NETosis inducers. However, bovine PMN frequently displaced towards motile NEJ and were found attached to NEJ surfaces. Functional PMN chemotaxis assays using vital F. hepatica-NEJ revealed a slight increase of PMN migration when compared to non-exposed controls. Additional experiments on intra- and extracellular reactive oxygen species (ROS) production revealed that soluble FhAg failed to induce ROS production of exposed PMN. Finally, mitochondrial oxygen consumption rates (OCR), extracellular acidification rates (ERAC) and proton production rates (PPR) were not significantly increased in FhAg-stimulated PMN. In summary, data suggest that F. hepatica might effectively evade PMN activation and NETosis by secreting parasite-specific molecules to either resolve NETs or to impair NETosis signaling pathways. We call for future molecular analysis not only on F. hepatica-derived NETosis modulation but also on its possible role in fasciolosis-associated pathology in vivo.

肝片形吸虫病是一种全球分布的人畜共患病，导致人类和牲畜肝炎的病原体。F.肝的新脱囊幼体（NEJ）是在体内遇到宿主先天免疫系统白细胞的第一个侵入阶段. 在白细胞中，多形核中性粒细胞（PMN）是血液系统中最丰富的粒细胞，也是最先迁移到感染部位的粒细胞。PMN 能够投射中性粒细胞胞外陷阱 (NETs)，也称为 NETosis，由核 DNA 组成，装饰有组蛋白、酶和抗菌肽，可以捕获并最终杀死侵入性寄生虫。鉴于只有少数大型寄生蠕虫已被确定为有效的 NETosis 诱导剂，我们在这里首次研究了不同的 F.livera 阶段是否也可以触发 NETosis。因此，分离的牛 PMN 与活的F.livera -NEJ、-metacrcariae、-eggs 和可溶性抗原 ( Fh银）。有趣的是，通过免疫荧光和扫描电子显微镜 (SEM) 分析检测到，所有阶段都未能诱导相当程度的 NETosis。NEJ 一直活动到潜伏期结束。与此一致，通过核面积扩展 (NAE) 分析进行的 NETosis 量化显示 NEJ 是弱 NETosis 诱导剂。然而，牛 PMN 经常向运动的 NEJ 移动，并被发现附着在 NEJ 表面。与未暴露的对照相比，使用重要的肝菌-NEJ进行的功能性 PMN 趋化性测定显示 PMN 迁移略有增加。关于细胞内和细胞外活性氧 (ROS) 产生的额外实验表明，可溶性FhAg 未能诱导暴露的 PMN 产生 ROS。最后， Fh Ag 刺激的 PMN中线粒体耗氧率 (OCR)、细胞外酸化率 (ERAC) 和质子产生率 (PPR) 没有显着增加。总而言之，数据表明，肝镰刀菌可能通过分泌寄生虫特异性分子来解决 NETs 或损害 NETosis 信号通路，从而有效地逃避 PMN 激活和 NETosis。我们呼吁未来的分子分析不仅要对肝梭菌衍生的 NETosis 调制进行，而且还要对其在体内与片形吸虫病相关的病理学中的可能作用进行分析。