Metabolic requirements of Besnoitia besnoiti tachyzoite-triggered NETosis

Besnoitia besnoiti is the causative agent of bovine besnoitiosis, a disease affecting both, animal welfare and cattle productivity. NETosis represents an important and early host innate effector mechanism of polymorphonuclear neutrophils (PMN) that also acts against B. besnoiti tachyzoites. So far, no data are available on metabolic requirements of B. besnoiti tachyzoite-triggered NETosis. Therefore, here we analyzed metabolic signatures of tachyzoite-exposed PMN and determined the relevance of distinct PMN-derived metabolic pathways via pharmacological inhibition experiments. Overall, tachyzoite exposure induced a significant increase in glucose and serine consumption as well as glutamate production in PMN. Moreover, tachyzoite-induced cell-free NETs were significantly diminished via PMN pre-treatments with oxamate and dichloroacetate which both induce an inhibition of lactate release as well as oxythiamine, which inhibits pyruvate dehydrogenase, alpha-ketoglutarate dehydrogenase, and transketolase, thereby indicating a key role of pyruvate- and lactate-mediated metabolic pathways for proper tachyzoite-mediated NETosis. Furthermore, NETosis was increased by enhanced pH conditions; however, inhibitors of MCT-lactate transporters (AR-C141900, AR-C151858) failed to influence NET formation. Moreover, a significant reduction of tachyzoite-induced NET formation was also achieved by treatments with oligomycin A (inhibitor of ATP synthase) and NF449 (purinergic receptor P2X(1) antagonist) thereby suggesting a pivotal role of ATP availability for tachyzoite-mediated NETosis. In summary, the current data provide first evidence on carbohydrate-related metabolic pathways and energy supply to be involved in B. besnoiti tachyzoite-induced NETosis.