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

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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.