Besnoitia besnoiti is the causative agent of bovine besnoitiosis, a disease that affects both, animal welfare and cattle productivity. Neutrophil extracellular trap (NET) formation represents an important innate effector mechanism of polymorphonuclear neutrophils (PMN) acting against various pathogens. Recently, NETs release was reported in response to B. besnoiti tachyzoites. However, limited knowledge is available on the role of metabolic pathways during parasite-triggered NET formation, nowadays known as NETosis. By analysing metabolic signatures of tachyzoite-exposed PMN and applying functional inhibition experiments, we here aimed to investigate the importance of distinct PMN-derived metabolic pathways for effective B. besnoiti tachyzoite-induced NETosis. Overall, exposure to heat-inactivated tachyzoites induced an increase in glucose and serine consumption, a drop in glutamine consumption and an enhancement of glutamate and alanine production in bovine PMN. Moreover, tachyzoite-induced formation of cell free NETs was significantly diminished via PMN pretreatments with dichloroacetate (pyruvate dehydrogenase inhibitor), oxythiamine (inhibitor of pyruvate dehydrogenase kinase) and oxamate (inhibitor of lactate dehydrogenase), thereby indicating a key role of pyruvate- and lactate-mediated metabolic pathways for proper tachyzoite-mediated NETosis. In line, NET formation was also induced by enhanced pH, however, blockers (AR-C141900, AR-C151858) of lactate transporters failed to influence NETosis. Moreover, a significant reduction of tachyzoite-induced NET formation was also achieved by treatments with oligomycin (inhibitor of ATP synthase) and NF449 (purinergic receptor P2X1 antagonist) which suggested a pivotal role of ATP availability in this effector mechanism. In contrast, treatments with inhibitors of early steps of glycolysis or glutaminolysis did not affect parasite-triggered NETosis. These current data will provide a better understanding of metabolic pathways involved in B. besnoiti tachyzoite-induced NETosis.Moreover, tachyzoites of B. besnoiti induced LC3B-related autophagosome formation in parallel to NETosis in bovine PMN. Notably, both rapamycin- and wortmannin-treatments failed to influence B. besnoiti-triggered NETosis and autophagosome formation. Also, isolated NETosis failed to induce autophagy suggesting independence between both cellular processes. Interestingly, enhanced phosphorylation of AMPKα, a key regulator molecule of autophagy, was observed within the first minutes of interaction in tachyzoite-exposed bovine PMN thereby emphasizing that B. besnoiti-triggered NETosis indeed occurred in parallel to autophagy.In addition, we investigated early effector mechanisms of bovine PMN being exposed to vital B. besnoiti bradyzoite stages, which were isolated from skin tissue cysts of a naturally infected animal presenting characteristic symptoms of bovine besnoitiosis. Histopathological examinations confirmed the presence of typical roundish, large-sized cysts in subdermal biopsies. After PMN:B. besnoiti bradyzoites co-cultures, scanning electron microscopy (SEM)- and epifluorescence microscopy-analyses demonstrated a fine network of NET-like structures being released by bovine PMN and efficiently ensnaring bradyzoites. Classical NETosis-associated components were confirmed in these extracellular structures via immunofluorescence analyses on extracellular DNA, histone (H1-H4) and neutrophil elastase (NE) colocalization. Besides NETosis, an increased formation of autophagosomes (visualized by specific-LC3B staining) was observed in bradyzoite-stimulated PMN. Statistical analyses revealed a significant positive correlation (p = 0.042) between the occurrence of NETosis and autophagy in these immunocompetent cells. These findings suggest NETosis plays a pivotal role in early innate host responses against bradyzoite stages and furthermore deliver first evidence on autophagy being associated with B. besnoiti bradyzoite-induced NETosis.
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