Bovine besnoitiosis is a re-emerging cattle disease caused by the cyst-forming apicomplexan parasite Besnoitia besnoiti. Neutrophil extracellular trap (NET) formation represents an efficient innate immune mechanism of polymorphonuclear neutrophils (PMN) against apicomplexan parasites, including B. besnoiti. PMN purinergic signaling was proposed as a critical factor for NET formation. One important purinergic ligand is ATP, which is recognized as a danger signal and released into the extracellular space acting as an autocrine/paracrine signaling molecule. ATP-driven effects on PMN via the nucleotide P2 receptor family include chemotaxis, reactive oxygen species (ROS) production, and NET formation. So far, data on both PMN ATP concentrations and the role of ATP as a key modulator of purinergic signaling in B. besnoiti tachyzoite-triggered bovine NETosis is scarce. Current data showed that B. besnoiti tachyzoite exposure to bovine PMN neither changed total PMN ATP nor extracellular ATP quantities even though it significantly triggered NET formation. Moreover, B. besnoiti tachyzoite-exposed PMN revealed enhanced oxygen consumption rates (OCR) as quantified by the Seahorse metabolic analyzer. Exogenous supplementation of ATP or non-hydrolizable ATP (ATPγS) led to increased extracellular acidification rates (ECAR) but failed to alter tachyzoite-induced oxidative responses (OCR) in exposed PMN. In addition, exogenous supplementation of ATPγS, but not of ATP, boosted B. besnoiti tachyzoite-induced anchored NET formation. Referring to purinergic signaling, B. besnoiti tachyzoite-triggered anchored NET formation revealed P2X1 purinergic as receptor-dependent since it was blocked by the P2X1 inhibitor NF449 at an IC₅₀ of 1.27 µM. In contrast, antagonists of P2Y2, P2Y6, P2X4, and P2X7 purinergic receptors all failed to affect parasite-driven NETosis. As an interesting finding, we additionally observed that B. besnoiti tachyzoite exposure induced PMN clustering in a P2X1-dependent manner. Thus, we identified P2X1 purinergic receptor as a pivotal molecule for both B. besnoiti tachyzoite-induced PMN clustering and anchored NET formation.

Bovine besnoitiosis is a re-emerging cattle disease caused by the apicomplexan parasite Besnoitia besnoiti, which severely affects individual animal welfare and profitability in cattle industry. We recently showed that B. besnoiti tachyzoite exposure to bovine polymorphonuclear neutrophils (PMN) effectively triggers neutrophil extracellular trap (NET) formation, leading to parasite immobilization hampering host cell infection. So far, the triggers of this defense mechanism remain unclear. Emerging evidence indicates that extracellular vesicles (EVs) modulate PMN effector functions, such as ROS production or NET formation. Therefore, we tested whether exposure of bovine PMN to EVs from different cellular sources affects classical PMN effector functions and cytokine/chemokine secretion. EVs were isolated from B. besnoiti-infected and non-infected host cells (bovine umbilical vein endothelial cells, BUVEC), from tachyzoite-exposed bovine PMN and from B. besnoiti tachyzoites. EV concentration and size was determined by Nano-Flow cytometry and EV nature was confirmed by both classical EV markers (CD9 and CD81) and transmission electron microscopy (TEM). Overall, PMN stimulation with both BUVEC- and tachyzoite-derived EVs significantly induced extracellular DNA release while EVs from PMN failed to affect NET formation. BUVEC and tachyzoite EV-driven NET formation was confirmed microscopically by the presence of DNA decorated with neutrophil elastase (NE) and histones in typical NET structures. Moreover, confocal microscopy revealed EVs to be internalized by bovine PMN. Referring to PMN activation, EVs from the different cellular sources all failed to affect glycolytic or oxidative responses of bovine PMN as detected by Seahorse^®-based analytics and luminol-based chemoluminescence, thereby denying any role of NADPH oxidase (NOX) activity in EV-driven NET formation. Finally, exposure to B. besnoiti-infected BUVEC-derived EVs induced IL-1β and IL-6 release, but failed to drive CXCL8 release of bovine PMN. Hence, we overall demonstrated that EVs of selected cellular origin owned the capacity to trigger NOX-independent NET formation, were incorporated by PMN and selectively fostered IL-1β and IL-6 release.

Besnoitia besnoiti is an obligate intracellular apicomplexan parasite and the causal agent of bovine besnoitiosis. Bovine besnoitiosis has a considerable economic impact in Africa and Asia due to reduced milk production, abortions, and bull infertility. In Europe, bovine besnoitiosis is classified as an emerging disease. Polymorphonuclear neutrophils (PMN) are one of the most abundant leukocytes in cattle blood and amongst the first immunological responders toward invading pathogens. In the case of B. besnoiti, bovine PMN produce reactive oxygen species (ROS), release neutrophil extracellular traps (NETs), and show increased autophagic activities upon exposure to tachyzoite stages. In that context, the general processes of NETosis and autophagy were previously reported as associated with AMP-activated protein kinase (AMPK) activation. Here, we study the role of AMPK in B. besnoiti tachyzoite-induced NET formation, thereby expanding the analysis to both upstream proteins, such as the calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK), and downstream signaling and effector molecules, such as the autophagy-related proteins ULK-1 and Beclin-1. Current data revealed early AMPK activation (<30 min) in both B. besnoiti-exposed and AMPK activator (AICAR)-treated bovine PMN. This finding correlated with upstream responses on the level of CAMKK activation. Moreover, these reactions were accompanied by an augmented autophagic activity, as represented by enhanced expression of ULK-1 but not of Beclin-1. Referring to neutrophil effector functions, AICAR treatments induced both AMPK phosphorylation and NET formation, without affecting cell viability. In B. besnoiti tachyzoite-exposed PMN, AICAR treatments failed to affect oxidative responses, but led to enhanced NET formation, thereby indicating that AMPK and autophagic activation synergize with B. besnoiti-driven NETosis.