Simultaneous and Positively Correlated NET Formation and Autophagy in Besnoitia besnoiti Tachyzoite-Exposed Bovine Polymorphonuclear Neutrophils

Abstract

Given that <i>B. besnoiti</i> tachyzoites infect host endothelial cells of vessels <i>in vivo</i>, they become potential targets for professional phagocytes [e.g., polymorphonuclear neutrophils (PMN)] when in search for adequate host cells or in case of host cell lysis. It was recently reported that <i>B. besnoiti</i>-tachyzoites can efficiently be trapped by neutrophil extracellular traps (NETs) released by bovine PMN. So far, the potential role of autophagy in parasite-triggered NET formation is unclear. Thus, we here analyzed autophagosome formation and activation of AMP-activated protein kinase α (AMPKα) in potentially NET-forming innate leukocytes being exposed to <i>B. besnoiti</i> tachyzoites. Blood was collected from healthy adult dairy cows, and bovine PMN were isolated via density gradient centrifugation. Scanning electron microscopy confirmed PMN to undergo NET formation upon contact with <i>B. besnoiti</i> tachyzoites. Nuclear area expansion (NAE) analysis and cell-free and anchored NETs quantification were performed in <i>B. besnoiti-induced</i> NET formation. Interestingly, tachyzoites of <i>B. besnoiti</i> additionally induced LC3B-related autophagosome formation in parallel to NET formation in bovine PMN. Notably, both rapamycin- and wortmannin-treatments failed to influence <i>B. besnoiti</i>-triggered NET formation and autophagosome formation. Also, isolated NETs fail to induce autophagy suggesting independence between both cellular processes. Finally, enhanced phosphorylation of AMP activated kinase α (AMPKα), a key regulator molecule of autophagy, was observed within the first minutes of interaction in tachyzoite-exposed PMN thereby emphasizing that <i>B. besnoiti</i>-triggered NET formation indeed occurs in parallel to autophagy.