Metabolic requirements of Besnoitia besnoiti tachyzoite-triggered NETosis
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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, α-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 P2X1 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.
KeywordsBesnoitia besnoiti PMN NETosis Metabolic signatures Glycolysis ATP
The authors would like to acknowledge Anika Seipp (Institute of Anatomy and Cell Biology, Justus Liebig University Giessen, Germany) for her excellent assistance in processing samples for scanning electron microscopy analysis. Many thanks to Prof. Mazurek’s assistants Sandra Rühl and Bianca Kulik for their help in measuring metabolites and creating the metabolic scheme. We also want to thank to Hannah Salecker and Dr. Christin Ritter for the technical support in B. besnoiti cell culture. We further thank all staff members of JLU Gießen teaching and research station Oberer Hardthof.
This research was funded by the German Research Foundation (Deutsche Forschungsgemeinsachaft, DFG, grant no. TA291/4-2). EZ is a recipient of a PhD fellowship from China Scholarship Council (file number: 201506170042).
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Conflict of interest
The authors declare that they have no conflict of interest.
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