P2X1 receptor antagonist NF449 inhibits Besnoitia besnoiti-induced neutrophil clustering and anchored extracellular trap (NET) formation

Abstract text

Introduction

Bovine besnoitiosis is a re-emerging cattle disease caused by the apicomplexan parasite Besnoitia besnoiti. Neutrophil extracellular traps (NETs) formation represents an efficient innate immune mechanism of polymorphonuclear neutrophils (PMN) against parasites, including B. besnoiti. PMN-derived purinergic signaling was proposed as critical factor for NET formation. ATP is an important purinergic ligand, which regulates several PMN actions, such as chemotaxis, ROS production and NET formation, via the P2 nucleotide receptor family. However, little data are available on the role of ATP as key modulator of purinergic signaling in B. besnoiti-triggered NETosis.

Material and methods

Bovine PMN were isolated from the blood of healthy cows by density gradient and exposed to B. besnoiti tachyzoites in the presence of ATP and purinergic receptor antagonists. Total and extracellular ATP levels were quantified by luminometry. NET formation was estimated as extracellular DNA by picogreen-derived fluorescence intensities. PMN oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) were quantified by Seahorse instrumentation. NET and cluster formation of PMN exposed to B. besnoiti was evaluated by epifluorescence microscopy.

Results

Current data showed that B. besnoiti tachyzoite exposure neither changed total ATP levels in PMN nor in the extracellular environment even though significantly triggering NET formation. Interestingly, exogenous supplementation of ATP failed to affect B. besnoiti-induced OCR but led to an increase of ECAR in PMN. Likewise, parasite exposure enhanced ECAR in PMN. However, neither the latter effect nor tachyzoite-triggered total NET formation was influenced by exogenous ATP levels. B. besnoiti-induced anchored NET formation was selectively blocked by the inhibition of the purinergic receptor P2X1 whilst antagonists of P2Y2, P2Y6, P2X4 and P2X7 receptors all failed to affect parasite-driven NETosis. [AT1] As an interesting finding, we additionally observed that tachyzoites exposure increased the percentage of PMN forming clusters and that this effect was P2X1-dependent.

Conclusion

B. besnoiti induces an increase in clustering, oxidative activity and NET formation in bovine PMN. We identified the P2X1 as a pivotal purinergic receptor to be involved in B. besnoiti-induced anchored NET formation therefore suggesting this receptor as an interesting target for future treatments of NET-mediated tissue damage.