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

Abstract

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.

Keywords: ATP; Besnoitia besnoiti; NET formation; PMN; immunometabolism; purinergic receptors.

Figure 1

B. besnoiti tachyzoites are trapped in released NETs. (A, B) Unstimulated PMN. NETs defined as extracellular chromatin filaments containing proteins like neutrophil elastase and histone H1 formed a network in contact with B. besnoiti tachyzoites, as visualized via SEM (C, D) and immunostaining (E, F). White arrows indicate entrapped B. besnoiti tachyzoites. Co-localization of DNA (DAPI, blue); B. besnoiti tachyzoites (red); Histone H1 clone TNT-1 staining (pink) and neutrophil elastase (NE, green).

Figure 2

Exposure to B. besnoiti tachyzoites induces oxygen consumption in bovine PMN. In the absence of CO₂, 2x10⁵ PMN were incubated in XF RPMI media for 45 min. Four basal measurements were made and then either B. besnoiti tachyzoites (A, B) or ATP (0.05-50 µM; (C, D) was supplemented at the time point indicated by a vertical line. OCR (A, C) and ECAR (B, D) values were obtained by Seahorse technology and plotted over time (n = 3, for each condition). All data are shown as mean ± SD; p-values were calculated by unpaired two-tailed t-test analysis (n = 3).

Figure 3

Exposure to B. besnoiti tachyzoites induces a metabolic shift toward aerobic carbohydrate catabolism in bovine PMN. After basal measurements, PMN were treated with ATP, ATPγS (50 µM), or vehicle, followed by supplementation of B. besnoiti tachyzoites as indicated by vertical lines. Differences in OCR and ECAR from means in the same curve and sharing boxes of the same color were calculated (A, B, D, E). Energetic maps (C, F) were generated by presenting OCR (Y-axis) and ECAR (X-axis) as means of the different measurements over time. All data are shown as mean ± SD; p-values were calculated by unpaired t-test (A, B, n = 6; D, E n = 3).

Figure 4

Total PMN ATP and extracellular ATP concentration in B. besnoiti tachyzoite-PMN co-cultures. (A) For positive control of ATP release, PMN were treated for 15 s with a hypertonic buffer (HBSS supplemented with 1 M sodium chloride). ***p < 0,001. (B) For positive control for PMN ATP consumption, PMN were stimulated with PMA/ionomycin (100 nM/5 µM) for 15 and 60 min. **p < 0,01; ****p < 0,0001. (C–E) To assess parasite-driven effects on PMN ATP release and consumption, bovine PMN were confronted with B. besnoiti tachyzoites (1:6) for 15 s and 15 min. ATP in PMN (total ATP) or supernatants (extracellular ATP) was measured with a commercial kit. All data are shown as mean ± SD; p-values were calculated by unpaired two-way t-test (A), or one-way ANOVA followed by Tukey´s multiple comparison (B-E) analysis (A, B, n = 3; D, n = 4; C, n = 6).

Figure 5

ATPγS supplementation induces NET release and boosts Besnoitia besnoiti tachyzoite-driven anchored NET formation. Bovine PMN were pre-treated with increasing concentrations of non-modified ATP or non-hydrolyzable ATPγS (0.05-50 µM) for 10 min. Then, PMN were incubated for 4 h in plain medium (A, B, E, F) or exposed to B. besnoiti tachyzoites (C, D, G, H). After incubation, extracellular DNA was detected and quantified via picogreen-derived fluorescence intensities using a multi-plate reader at 480 nm excitation/520 nm emission wavelengths. All data are shown as mean ± SD; p-values were calculated by one-way ANOVA followed by Dunnett´s multiple comparison test. *p < 0,05; **p < 0,01; ****p < 0,0001.

Figure 6

Effects of purinergic receptor antagonist pre-exposure treatments on Besnoitia besnoiti triggered NET formation. Bovine PMN were pre-treated for 10 min with increasing concentrations of MRS2179, MRS2578, NF449, 5-BDBD, and AZ10606120 (0.1-100 µM) targeting P2Y1, P2Y6, P2X1, P2X4, and P2X7 receptors, respectively, and then exposed to B. besnoiti tachyzoites for 4 (h) Thereafter, cell-free (A, C, E, G, I) and anchored (B, D, F, H, J) NETs were analyzed. For both NET types, extracellular DNA was detected and quantified via picogreen-derived fluorescence intensities using a multi-plate reader at 480 nm excitation/520 nm emission wavelengths. All data are shown as mean ± SD; p-values were calculated by Kruskal-Wallis test followed by Dunn´s multiple comparison test (n =6). *p < 0,05; **p < 0,01.

Figure 7

Effects of purinergic receptor antagonist post-exposure treatments on Besnoitia besnoiti triggered NET formation. Bovine PMN were first exposed to B. besnoiti tachyzoites for 40 min and then treated with 100 µM of MRS2179, MRS2578, NF449, and 5-BDBD targeting P2Y1, P2Y6, P2X1, P2X4, and P2X7 receptors, respectively. After 4 h of incubation, cell-free (A, C) and anchored (B, D) NETs were analyzed. For both NET types, extracellular DNA was detected and quantified via picogreen-derived fluorescence intensities using a multi-plate reader at 480 nm excitation/520 nm emission wavelengths. All data are shown as mean ± SD; p-values were calculated by an ordinary one-way ANOVA with Dunnett´s multiple comparison analysis. (n =3). *p < 0,05; **p < 0,01; ***p < 0,001.

Figure 8

NF449 does not affect PMN’ viability and inhibits Besnoitia besnoiti triggered NET formation in an ATP-independent but dose-dependent manner. (A, B) Bovine PMN were pre-treated with NF449 (100 µM) in the presence or absence of ATP/ATPγS and then confronted with B. besnoiti tachyzoites. After 4 h of incubation, extracellular DNA was detected and quantified via picogreen-derived fluorescence intensities. (C) Annexin V-FITC and propidium iodide staining of PMN treated with NF449 for 4 h. (D) NF449-based dose-response-inhibition of B. besnoiti tachyzoite-induced NET formation. The IC50 was calculated by a nonlinear regression analysis. All data are shown as mean ± SD; p-values were calculated by one-way ANOVA with Dunnett´s multiple comparison analysis. (n = 3). **p < 0,01; ***p < 0,001; ****p < 0,0001.

Figure 9

B. besnoiti tachyzoite-induced clustering of bovine PMN depends on P2X1-based purinergic signaling. PMN were co-cultured with tachyzoites for 4 h in the presence or absence of NF449 (100 µM). (A) Exemplary illustrations of tachyzoite-PMN co-cultures stained for DNA (DAPI, blue) and parasite stages (red). (B) DANA-based quantification of cluster formation. All values are presented as mean ± SD, and p-values were calculated using one-way ANOVA followed by Tukey´s multiple comparisons. (n = 4). *p < 0,05; **p < 0,01.