Besnoitia besnoiti-induced neutrophil clustering and neutrophil extracellular trap formation depend on P2X1 purinergic receptor signaling.

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.

Cryptosporidium parvum-induced neutrophil extracellular traps in neonatal calves is a stage-independent process.

Introduction: Infections with the apicomplexan obligate intracellular parasite Cryptosporidium parvum lead to cryptosporidiosis-a worldwide zoonotic infection. C. parvum is one of the most common diarrheal pathogens in young calves, which are the main reservoir of the pathogen. Cryptosporidiosis leads to severe economic losses in the calf industry and being a major contributor to diarrhea morbidity and mortality in children. Polymorphonuclear neutrophils (PMN) are part of the innate immune system. Their effector mechanisms directed against invasive parasites include phagocytosis, production of antimicrobial molecules as well as the formation of so-called neutrophil extracellular traps (NETs). Like other leukocytes of the innate immune system, PMN are thus able to release chromatin fibers enriched with antimicrobial granular molecules extracellularly thereby immobilizing and partially killing invasive bacteria, viruses, fungi and parasites. Methods: In vitro interactions of neonatal bovine PMN and C. parvum-oocysts and sporozoites were illustrated microscopically via scanning electron microscopy- and live cell imaging 3D holotomographic microscopy analyses. C. parvum-triggered NETosis was quantified via extracellular DNA measurements as well as verified via detection of NET-typical molecules [histones, neutrophil elastase (NE)] through immunofluorescence microscopy analysis. To verify the role of ATP in neonatal-derived NETosis, inhibition experiments were performed with NF449 (purinergic receptor antagonist with high specificity to P2X1 receptor). Results and discussion: Using immunofluorescence- and SEM-based analyses, we demonstrate here for the first time that neonate bovine PMN are capable of forming NETs against C. parvum-sporozoites and oocysts, thus as a stage-independent cell death process. Our data further showed that C. parvum strongly induces suicidal neonatal NETosis in a P2X1-dependent manner, suggesting anti-cryptosporidial effects not only through firm sporozoite ensnarement and hampered sporozoite excystation, but also via direct exposure to NETs-associated toxic components.