Studies on triggers and modulators of coccidia-induced NETosis: parasite glycans, ATP, AMPK, O2 and extracellular vesicles

感染球虫，如Besnoitia benoiti或Eimeria bovis，在很大程度上损害了动物的福利，并给养牛业造成了巨大的经济损失。总体而言，适当的免疫反应在控制这些寄生虫感染方面发挥着关键作用。在我们最近的工作中，我们发现了几种球虫和球虫阶段是中性粒细胞胞外陷阱(Net)形成的有效触发因素，这意味着天然免疫细胞的一个重要效应机制。上述研究数据表明，寄生虫激活的中性粒细胞(PMN)同时发生网络分裂和自噬过程，且两者呈正相关。作为一个有趣的发现，我们在这里检测到在寄生虫暴露的PMN中AMPK作为关键调控分子的早期和选择性激活。此外，我们还发现，贝氏杆菌引发的网状结构损害了内皮细胞的完整性，但不能显著改变这种寄生虫在内皮细胞内的发育。此外，NETotic PMN的代谢特征表明糖酵解和葡萄糖-丙酮酸-乳酸-轴的关键作用。功能抑制研究一方面揭示了单羧酸转运体与NETsis的物种相关性，但另一方面强调了嘌呤能信号和线粒体ATP合成对这一效应机制的正常功能所起的共同关键作用。此外，我们在这里表明，通过对寄生阶段进行化学处理来去除表面碳水化合物残基，可以减少蚊虫侵染，这表明多糖可能是寄生虫衍生的蚊虫侵染的触发物。凝集素印迹显示在寄生虫阶段有相当多的多糖存在，荧光分析表明这些多糖存在于速殖子和子孢子的内室和表面。在当前的提案中，我们想要集中在以下主题上以更好地理解球虫驱动的蚊虫感染的分子基础：i)寄生虫表面的多糖是否真的是球虫诱导的蚊虫感染的触发因素？ii)ATP是寄生虫驱动的蚊帐形成的代谢主调节因子吗？iii)AMPK是否作为寄生虫介导的早期蚊虫感染的调节因子？iv)在寄生虫诱导的蚊虫感染中，寄生虫、PMN或受感染的宿主细胞衍生的胞外小泡起什么作用？以及v)生理氧气条件对球虫触发的网络形成有哪些影响？通过所有这些努力，我们希望揭示寄生虫诱导的蚊虫感染高度复杂的诱导和调控的新方面。

Infections with coccidian parasites, such as Besnoitia besnoiti or Eimeria bovis, considerably impair animal welfare and cause high economic losses in cattle industry. Overall, proper immune reactions play a key role in control of these parasitic infections. In our recent work, we identified several coccidian species and stages as potent triggers of “Neutrophil extracellular Trap” (NET) formation, which signifies an important effector mechanism of innate immune cells. The data of the preceding proposal show that the processes of NETosis and autophagy occur simultaneously in parasite-activated polymorphonuclear neutrophils (PMN) and are positively correlated. As an interesting finding, we here detected an early and selective activation of AMPK as key regulatory molecule in parasite-exposed PMN. Furthermore, we document that B. besnoiti-triggered NET structures impair endothelial integrity but fail to significantly alter intracellular development of this parasite in endothelial cells. In addition, metabolic signatures of NETotic PMN indicated a key role of glycolysis and the glucose-pyruvate-lactate-axis. Functional inhibitor studies revealed a species-dependent relevance of monocarboxylate transporters for NETosis on one hand, but emphasized the common pivotal role of purinergic signaling and mitochondrial ATP synthesis for proper functionality of this effector mechanism on the other hand. Moreover, we here showed that chemical treatments of parasitic stages to remove surface carbohydrate residues led to diminished NETosis indicating that glycans may serve as parasite-derived triggers of NETosis. Respective lectin blots indicated a considerable variety of glycans in parasite stages, fluorescence analyses showed that these were present in both, inner compartments and on the surface of tachyzoites and sporozoites.In the current proposal, we want to focus on the following topics to better understand the molecular basis of coccidia-driven NETosis: i) Are parasite surface glycans indeed triggers of coccidian-induced NETosis?, ii) Is ATP a metabolic masterregulator of parasite-driven NET formation?, iii) Does AMPK function as an regulator of early parasite-mediated NETosis?, iv) Which role play parasite-, PMN- or infected host cell-derived extracellular vesicles in parasite-induced NET induction?, and v) Which influence do physiological oxygen conditions have on coccidian-triggered NET formation? With all this effort, we expect to reveal new aspects of the highly complex induction and regulation of parasite-induced NETosis.