Abstract
Given that B. besnoiti tachyzoites infect host endothelial cells of vessels in vivo, 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 B. besnoiti-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 B. besnoiti 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 B. besnoiti tachyzoites. Nuclear area expansion (NAE) analysis and cell-free and anchored NETs quantification were performed in B. besnoiti-induced NET formation. Interestingly, tachyzoites of B. besnoiti additionally induced LC3B-related autophagosome formation in parallel to NET formation in bovine PMN. Notably, both rapamycin- and wortmannin-treatments failed to influence B. besnoiti-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 B. besnoiti-triggered NET formation indeed occurs in parallel to autophagy.
Highlights
Besnoitia besnoiti is a cyst-forming apicomplexan protozoan parasite that causes bovine besnoitiosis which is traditionally endemic in Africa and Asia
Suicidal neutrophil extracellular traps (NETs) formation was described as a NADPH oxidase (NOX)-dependent cellular mechanism which induces the extrusion of DNA and nuclear and cytoplasmic granule enzymes leading to the formation of DNArich networks being decorated with histones and various potent antimicrobial granular effector molecules, such as neutrophil elastase (NE), myeloperoxidase (MPO), lactoferrin, pentraxin, peptidoglycan recognition proteins, or calprotectin [19,20,21,22]
Scanning Electron Microscopy (SEM) analysis showed that bovine polymorphonuclear neutrophils (PMN) (Figure 1) exposed to vital B. besnoiti tachyzoites released NET-like structures and many B. besnoiti tachyzoites were firmly trapped by these filaments (Figures 1A–C)
Summary
Very little data is available on early host innate immune reactions during primary acute B. besnoiti infections in vivo [11] and in vitro [12, 13] despite the fact that early host innate defense reactions should be critical for the outcome of the disease. In this sense, PMN play a pivotal role since they are the most abundant leukocyte population in blood and the first ones to be recruited to sites of infection. A variety of invasive pathogens such as bacteria, virus, fungi, protozoan, and metazoan parasites, might either be immobilized within released sticky NET structures or be killed via local high concentration of antimicrobial histones, peptides, and proteases [16, 20, 23,24,25]
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