Animal trials were conducted following the Justus Liebig University (JLU) Giessen Animal Care Committee Guidelines. Protocols were approved by the Ethics Commission for Experimental Animal Studies of Federal State of Hesse (Regierungspräsidium Giessen; A2/2016; JLU-No. 589_AZ and G16/2017, JLU-No. 835_GP) and in accordance with European Animal Welfare Legislation: ART13TFEU and current applicable German Animal Protection Laws.

Eimeria bovis strain H was originally isolated in the field in northern Germany in 1985 and maintained by passages in calves (3). For oocyst production, two 8-week-old calves, kept in Eimeria-free housing conditions in special metabolic cages, were infected orally with 3 × 10⁴ sporulated E. bovis oocysts. Oocysts were isolated from feces beginning at 18–20 days post infectionem (p.i.) according to Jackson (44). The feces were washed with tap water through a set of sieves (pore sizes 850, 250, and 80 µm), and the resulting suspension was sedimented overnight. This sediment was mixed at a 1:1 ratio with a sucrose-saturated solution (ρ = 1.3 g/ml) and adjusted to a final density of 1.15 g/ml. For oocyst flotation, the fecal suspension was carefully transferred into plastic trays (30 × 20 × 5 cm). The trays were filled up and covered with clean glass plates, guaranteeing contact of the suspension with the surface of the glasses. Every 4 h, the glass plates were carefully removed, and adherent oocysts were collected by washings with tap water. The remaining fecal suspension was mixed, filled up, and subjected to further oocyst flotation. The process was repeated until a few oocysts were left in the suspension (microscopic control: less than 5 oocysts per power vision field of ×10 magnification). Oocyst suspension was diluted with water (1:1) and centrifuged (600 × g, 12 min). An oocyst pellet was suspended in potassium dichromate solution (2% w/v final concentration, Merck) and incubated at room temperature (RT) with constant aeration for adequate oocyst sporulation. When ≥90% of the oocysts achieved sporulation, the oocysts were pelleted (600 × g, 12 min), suspended in fresh 2% (w/v) potassium dichromate solution, and stored at 4°C until further use (5).

For sporozoite isolation, the protocol described by Silva et al. (45) was used: sporulated oocysts were incubated in 4% (v/v) sodium hypochlorite solution and stirred on ice for 20 min. After vortexing for 20 s, the oocyst solution was centrifuged (300 × g, 5 min) and the supernatant was mixed with bi-distilled water (1:1). This suspension was then filtered through sieves of 40- and 10-µm pore size (pluriStrainer, pluriSelect) to remove debris. Then, oocysts were pelleted (15 min, 600 × g), suspended in 0.02 M L-cysteine/0.2 M NaHCO₃ (Merck) solution, and incubated at 100% CO₂ atmosphere (37°C, 20 h). Thereafter, oocysts were pelleted (15 min, 600 × g) and incubated (up to 3 h, 37°C, 5% CO₂ atmosphere) in the following excystation medium: Hank’s balanced salt solution (HBSS, Gibco) containing 0.4% (w/v) trypsin (Sigma) and 8% (v/v) sterile-filtered bovine bile obtained from a local slaughterhouse. The excystation progress was microscopically controlled every hour. Freshly released sporozoites were collected, filtered through a 5-µm filter unit (pluriSelect), washed twice (600 × g, 15 min) with cell culture medium 199 [M199, Gibco, supplemented with 2% (v/v) fetal calf serum (FCS, Gibco), 1% penicillin (v/v, 500 U/ml; Sigma-Aldrich), and streptomycin (v/v, 500 μg/ml; PS; Sigma-Aldrich)], and finally suspended in RPMI 1640 medium (Sigma-Aldrich) at 2 × 10⁶ sporozoites/ml.

Healthy adult dairy cows (n = 9) served as blood donors. Animals were bled by puncture of the jugular vein, and 30 ml peripheral blood was collected in heparinized sterile plastic tubes (Kabe Labortechnik). Twenty milliliters of heparinized blood was diluted in 20 ml sterile PBS with 0.02% EDTA (Sigma-Aldrich), layered on top of 12 ml Biocoll^® separating solution (density = 1.077 g/l; Biochrom AG), and centrifuged at 800 × g for 45 min. After removal of plasma and peripheral blood mononuclear cells (PBMCs), the cell pellet was suspended in 27 ml bi-distilled water and gently mixed for 30 s to lyse erythrocytes. Osmolarity was rapidly restored by adding 3 ml of 10× Hank’s balanced salt solution (HBSS; Biochrom AG). For full erythrocyte lysis, this step was repeated twice and PMN were suspended in sterile RPMI 1640 medium (Sigma-Aldrich). PMN were counted in a Neubauer hemocytometer. Finally, freshly isolated bovine PMN were allowed to rest (37°C, 5% CO₂ atmosphere) for 30 min until further use.

For Seahorse XF-based experiments, the protocol of erythrocyte lysis was slightly modified. In brief, plasma and buffy coat containing PBMCs were aspirated following Biocoll^® gradient centrifugation. The remaining cell pellet was suspended in Hank’s balanced salt solution (HBSS; Biochrom AG). Red blood cells were removed by flash hypotonic lysis using one volume of an ice-cold phosphate-buffered water solution (5.5 mM NaH₂PO₄, 8.4 mM HK₂PO₄, pH 7.2). After 1 min of incubation, two volumes of ice-cold hypertonic phosphate-buffered solution (5.5 mM NaH₂PO₄, 8.4 mM HK₂PO₄, 0.46 M NaCl, pH 7.2) were supplemented to restore isotonicity. Then, the cells were pelleted (600 × g, 10 min) and suspended in 5 ml of HBSS. This lysis step was repeated until no erythrocytes were observed in the cell preparation as described in (46, 47). PMN were then centrifuged at 600 × g for 10 min and washed with HBSS, counted, and processed as described above. The purity of the PMN suspension was assessed in an automatic cell counter Olympus R1 (Olympus). Values of cell purity were always >92% independent of the PMN isolation protocol.

Analysis of autophagosome formation in PMN was performed according to Itakura and McCarty (49). Bovine PMN (n = 3) were deposited on poly-_L-lysine (0.01%)-pretreated coverslips (15-mm diameter, Thermo Fisher Scientific). In addition, pretreatments of PMN with rapamycin (50 nM) or wortmannin (50 nM) were performed for 30 min before exposure to E. bovis sporozoites (1:4 PMN:sporozoite ratio, 2 h). After incubation, cells were fixed with 4% paraformaldehyde (10 min; Merck), permeabilized with ice-cold methanol (Sigma-Aldrich) treatment (3 min at 4°C), and blocked with blocking buffer (5% BSA, 0.1% Triton X-100 in sterile PBS; all Sigma-Aldrich) for 60 min at RT. Thereafter, cells were incubated overnight at 4°C in anti-LC3B antibody solution (Cat# 2775 Cell Signaling Technology) diluted 1:200 in blocking buffer. After incubation, samples were washed three times with PBS and incubated for 30 min in the dark at RT in a 1:500 dilution of goat anti-rabbit IgG conjugated with Alexa Fluor 488 (Invitrogen). After three washes in sterile PBS, samples were mounted in ProLong anti-fading mounting media containing DAPI (Invitrogen) on glass slides and images were taken applying confocal microscopy (Zeiss LSM 710). To estimate LC3B-positive cells, the background fluorescence signal was determined in control conditions for FITC (green) and DAPI (blue) channels and the green fluorescence intensity was determined. An LC3B-positive cell was defined using two parameters: green fluorescence and the presence of LC3B puncta as described by Itakura and McCarthy (49). Image processing was carried out with Fiji ImageJ using Z-project and merged channel plugins and restricted to overall adjustments of brightness and contrast.

For NET analyses, 2.5 × 10⁵ PMN from three different donors (n = 3) were seeded on 12-mm glass coverslips (Thermo Fisher) coated with 0.01% poly-_L-lysin (Sigma) and left unstimulated for 30 min at 37°C and 5% CO₂. Then PMN were confronted with freshly excysted E. bovis sporozoites at 1:2 and 1:4 PMN/sporozoite ratios in HBSS for 2 h at 37°C and 5% CO₂. After incubation, the samples were fixed with paraformaldehyde (Merck) at 4% final concentration for 15 min at RT, carefully washed thrice with sterile PBS, and stored at 4°C until immunostaining was performed. Immunodetection of NETs was performed by use of anti-histone-DNA complex antibodies (Millipore Cat#MAB3864; 1:100 dilution, detected with Invitrogen’s AlexaFluor 647 Cat#A21235 1:500) and anti-neutrophil elastase (anti-NE; Abcam, Cat#Ab68672 1:200 dilution; detected with Invitrogen’s AlexaFluor 594 Cat#R37117 1:500 dilution) and counterstained for DNA using DAPI in the mounting medium (Fluoromount-G with DAPI, Dako). All antibodies were diluted in permeabilization/blocking solution consisting of PBS with 3% BSA and 0.3% Triton X-100 (all Sigma-Aldrich). The incubation period was overnight at 4°C for primary antibodies. Thereafter, samples were washed thrice with PBS. Incubation with secondary antibodies was performed for 30 min at RT, and the coverslips were washed thrice with PBS and mounted on the microscope slide. The samples were kept protected from light for 24 h at RT before analysis. Imaging was performed by a Nikon Eclipse Ti2-A inverted microscope equipped with ReScan confocal microscopic instrumentation (RCM 1.1 Visible, Confocal.nl) and a motorized Z-stage (DI1500). Image acquisition was performed using the NIS-Elements v 5.11 software (Nikon) applying a z-stack optical series with a step size of 0.1 microns. Z-series were displayed as maximum z-projections maintaining the brightness and contrast conditions within the datasets of each biological experiment using ImageJ Fiji version (50).

The percentage of cells releasing NETs was assessed as described by Brinkmann et al. (51) with minor modifications. Three random images per animal (n = 3) were obtained at ×10 magnification using a confocal microscope. A manual threshold was applied to each channel using the clustering algorithm of Otsu, and the total number of particles was counted.

To illustrate early interactions of PMN with vital and motile E. bovis sporozoites, live-cell 3D holotomographic videos were recorded. In total, 5 × 10⁵ PMN were seeded into a 35-mm tissue culture µ-dish (ibidi) in imaging medium [RPMI 1640 lacking phenol red and serum, supplemented with 0.5 µM SYTOX Green (Life Technologies) and 2 µM DRAQ 5 (Thermo Scientific)]. After 30 min of incubation in the ibidi^® Stage Top Incubation Systems, E. bovis sporozoites were added (1:1, 5 × 10⁵). Then, holotomographic videos were obtained by using a 3D Cell Explorer microscope (Nanolive 3D) equipped with a ×60 magnification (λ = 520 nm, sample exposure 0.2 mW/mm²) and a depth of field of 30 µm. The FITC channel was used to visualize extracellular DNA (present in NETs and death cells, = green fluorescence), and the TRITC channel (red fluorescence) was used for nuclear DNA visualization. The video was post-processed and analyzed using STEVE software (Nanolive).

Bovine PMN from three donors were suspended in RPMI 1640 medium lacking phenol red (Sigma) and serum. Then the PMN were confronted with viable E. bovis sporozoites at a final PMN: sporozoite ratio of 1:4 (2 h, 37°C, 5% CO₂, 96-well plates). For negative controls, PMN or sporozoites in plain medium were used.

For pH-related experiments, RPMI 1640 medium was adjusted to different pH values (pH 6.6, 7.0, 7.4, and 7.8) by HCl or NaOH (both Merck, Darmstadt, Germany) supplementation as previously described (52). Bovine PMN were suspended in pH-adjusted RPMI 1640 medium and then exposed to sporozoites. Experiments were performed as follows: 2 × 10⁵ PMN were seeded in duplicates into 96-well plates and cocultured with 8 × 10⁵ E. bovis sporozoites or incubated in plain pH-adjusted medium (controls) for 2 h at 37°C and 5% CO₂.

For inhibition assays, bovine PMN (n = 3) were pretreated with inhibitors for 30 min and then cocultured with E. bovis sporozoites (1:4 PMN: sporozoite ratio, 2 h, 37°C, 5% CO₂). The following chemical blockers were used: 2-fluor-2-deoxy-D-glucose (FDG, 2 mM, Sigma-Aldrich; glucose analogue, inhibitor of glycolysis), sodium dichloroacetate (DCA, 8 mM, Sigma-Aldrich; inhibitor of pyruvate dehydrogenase kinase), oxythiamine (OT, 50 µM, Sigma-Aldrich; inhibitor of pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, and transketolase), sodium oxamate (OXA, 50 mM, Sigma-Aldrich; structural analogue of pyruvate, inhibitor of lactate dehydrogenase), 6-diazo-5-oxo-L-norleucin (DON, 4 µM, inhibitor of glutaminolysis), oligomycin A (5 µM, Sigma-Aldrich; inhibitor of ATP-synthase in mitochondrial respiration), theobromine (100 µM, Sigma-Aldrich; inhibitor of P1A1-mediated purinergic signaling), NF449 (100 µM, Tocris; purinergic receptor antagonist with high specificity for P2X₁), AR-C141990 (1 µM, Tocris; MCT1 inhibitor), and AR-C155858 (1 µM, Tocris; inhibitor of MCT1 and MCT2). Inhibitor concentrations were chosen according to previous studies (26, 42, 53–56). Stimulation of PMN with zymosan (1 mg/ml; Sigma-Aldrich) served as positive control.

For pH-related and inhibitor assay, ‘anchored’ and ‘cell-free’ NETs were distinguished according to Tanaka et al. (57). Therefore, the samples were directly centrifuged in the cell culture plate (300 × g, 5 min) after incubation. Supernatants were transferred to a new 96-well plate to measure ‘cell-free’ NETs, and the remaining pellets were used for ‘anchored’-NET estimation. For both sample types, 50 µl PicoGreen (Invitrogen, diluted 1:200 in 10 mM Tris base buffered with 1 mM EDTA) was added to each well. Extracellular DNA was quantified based on PicoGreen-derived fluorescence intensities using an automated multiplate reader (Varioskan, Thermo Scientific) at 484 nm excitation/520 nm emission (42, 58).

Unless otherwise stated, comparisons between two groups were performed using a paired t-test analysis. When more than two datasets were analyzed, a paired one-way analysis of variance (ANOVA) followed by Dunnett’s multiple-comparison test was applied. Statistical significance was defined by a p-value < 0.05. All graphs (mean ± SEM) and statistical analyses were generated by GraphPad software (v. 7.03).

As a direct indicator of PMN activation, we here measured metabolic key functions in bovine PMN being exposed to E. bovis sporozoites. Thus, OCR were estimated via Seahorse-based technology over a time period of 4 h. After detecting basal respiration in non-exposed PMN for 30 min, sporozoites were added to resting PMN in the presence of the mitochondrial activity inhibitor rotenone/antimycin A. An immediate and ongoing increase in OCR was here detected in response to sporozoite exposure ( Figure 1A ). When analyzing the area under the curve (AUC), a significant overall induction of OCR was found (sporozoite-exposed PMN vs. non-exposed PMN: p = 0.004, Figure 1B ), thereby reflecting PMN activation. Additionally, proton efflux rates (PER), reflecting ECAR, were measured in sporozoite-exposed PMN via Seahorse-based analyses. In contrast to OCR, no significant differences were here detected (p = 0.768) even though PER seemed moderately but constantly induced by sporozoite supplementation ( Figures 1C, D ).

We furthermore analyzed whether sporozoite exposure would influence the glycolytic activity of bovine PMN. In the current Seahorse-based experimental setting (glycolysis stress test, without mitochondrial inhibitors), a short period (18 min) of basal ECAR estimation in non-exposed PMN was followed by sporozoite supplementation to bovine PMN ( Figure 2A , indicated by an arrow + Eb). PMN confrontation with sporozoites led to an immediate and significant increase of ECAR, thereby reflecting an enhancement of the acute glycolytic response in exposed cells ( Figure 2A ). Additional glucose supplementation revealed that also the general glycolysis was significantly enhanced in PMN:sporozoite cocultures (sporozoite-exposed PMN vs. non-exposed PMN: p = 0.03) ( Figure 2B ). Also, ECAR measurements after the injection of oligomycin (blocks mitochondrial ATP production) revealed that glycolytic capacity was increased in PMN:sporozoite cocultures when compared to non-stimulated PMN. This increase did not reach statistical significance (sporozoite-exposed PMN vs. non-exposed PMN: p = 0.07) ( Figure 2C ). The contribution of E. bovis-sporozoites alone to these results is not significant given the low OCR and ECAR values measured in E. bovis-sporozoites using the same experimental settings ( Supplementary Figure 1 ). Additionally, we performed glycolysis stress tests in the presence of 2 mM FDG, an analogue of glucose and inhibitor of glycolysis that cannot be metabolized. Results showed that FDG treatments indeed inhibited glycolysis, but this result was not statistically significant (p = 0.16). The same was observed when the glycolytic capacity of E. bovis-confronted PMN was evaluated ( Figures 2D–F ).

LC3B was detected in immunofluorescence analyses to evaluate the activation of autophagy in bovine PMN being confronted with E. bovis sporozoites. Here, non-stimulated PMN showed a non-punctuated, homogeneous distribution of the LC3B (green) signal ( Figure 3A , first row). This pattern changed to a typically punctuated phenotype by the formation of autophagic vesicles upon sporozoite confrontation ( Figure 3A , second row). Furthermore, we performed co-incubation assays on PMN + E. bovis sporozoites in the presence of wortmannin (PI3K and autophagy inhibitor) and rapamycin (mTOR inhibitor, an activator of autophagy). Representative images are shown in Figure 3A (third and fourth rows, respectively). When the percentage of LC3B-positive cells for each experimental condition was determined, a significant increase (p = 0.01) of autophagy-positive cells was only observed in conditions of plain sporozoite supplementation ( Figure 3B ). Treatments with wortmannin partially decreased this activation but on a non-significant level. Unexpectedly, also rapamycin showed insignificant inhibitory effects on the proportion of LC3B-positive cells in sporozoite-exposed PMN. Moreover, these results were also analyzed in terms of punctuated and diffuse LC3B fluorescent signals. In this context, the punctuated pattern increased in the presence of E. bovis (p = 0.03; Figure 3C ) but not the diffuse pattern (p = 0.29; Figure 3D ). Interestingly, the diffuse pattern was now increased significantly in the presence of the mTOR activator rapamycin (p < 0.001; Figure 3D ).

Activation of bovine PMN after confrontation with E. bovis sporozoites was evaluated further by determining the NET formation induced by E. bovis at PMN:sporozoite ratios of 1:2 and 1:4 after 2 h of co-incubation. The percentage of NET-forming PMN was determined according to Brinkmann et al. (51) using an anti-DNA–histone complex and anti-NE antibodies ( Figure 4 ). Chromatin was counterstained by DAPI. NET formation was observed as chromatin structures being released from PMN and positive for NE and histone–DNA complex. Unstimulated PMN were used as negative controls ( Figure 4 ). Current data showed that E. bovis sporozoites in a 1:2 PMN:parasite ratio induced NETs in 12.55 ± 0.48% of PMN versus 1.56 ± 0.72% of unstimulated PMN ( Figure 4 ; p < 0.001). When a 1:4 PMN:parasite ratio was analyzed, 19.81 ± 1.87% of PMN were NET-positive indicating that E. bovis-induced NET formation is dependent on the PMN:E. bovis sporozoite ratio ( Figure 4 ; p < 0.001).

A holotomographic analysis with 3D Cell Explorer Fluo (Nanolive) on early interactions between bovine PMN and viable and motile E. bovis sporozoites was performed (please refer to Supplementary video 1 ). Activation of bovine PMN after addition of motile parasites to imaging media was evident by the observation by rapid pseudopod formation and increased kinetic activities. These changes in morphology and motility were documented for 100 min. Figure 5 shows representative images of interactions between PMN and motile E. bovis sporozoites. The imaging media contained DRAQ5 (red) to stain PMN nuclei and SYTOX Orange to stain extracellular DNA, as a component of NETs. Here, six PMN nuclei were stained red and one dead E. bovis sporocyst was SYTOX Green-positive ( Figure 5A ) at time point 0. After 13 min of interaction, extrusion of a DNA diffuse web-like structure was observed in one of the analyzed PMN (white arrow, green staining in Figure 5A and zoomed in Figure 5B ), which was in close contact to motile sporozoites. This spread NET (sprNET) became larger and was located in close proximity to sporozoites during the total incubation period. The PMN which released sprNET (yellow arrow) were alive during the whole process and died at minute 45 (i.e., 32 min after sprNET extrusion), thereby suggesting the cell process of vital NETosis. Of note, some sporozoites which were exposed to sprNET or in direct contact this NET structure died after 20 min (blue arrow).

Worthwhile to mention is that at minute 9 of coculture, one E. bovis sporozoite actively invaded a bovine PMN (please refer to Video 1, Figure 5 , red arrow), and the whole invasion process took 36 s. The parasite moved inside the cytoplasm—as illustrated by the deformation of the invaded PMN cell—for less than a minute and even tried to escape. Then, a slow retrograde movement of the sporozoite back into the cell was seen, a process that took in total of 7.5 min. Thereafter, the invaded PMN began its nuclear expansion and finally died at minute 35, with the sporozoite still inside the cytoplasm ( Figure 5A red arrow).

Since lactate or other proton-based efflux revealed crucial for NET formation and considering the observed ECAR-related effects in parasite-stimulated PMN, we here additionally studied the effects of different extracellular pH conditions (i.e., pH of 6.6, 7.0, 7.4, and 7.8) on sporozoite-triggered cell-free and ‘anchored’ NETosis phenotypes. Overall, only moderate effects were detected at different pH conditions, evidencing that the most consistent response is observed at pH 7.4 (p = 0.009 and p = 0.02 for anchored and cell-free NETs, respectively) ( Figure 6 ). E. bovis sporozoite-induced NETosis depended on ATP synthase and lactate dehydrogenase activities.

We next investigated the relevance of single metabolic pathways during NETosis via the use of selected metabolic inhibitors interfering with distinct steps of glycolysis, glutaminolysis, and ATP generation. In detail, we studied sporozoite-induced NET formation and the related impact of PMN pretreatments with chemical compounds acting on the glucose lactate and citric acid cycle axis (FDG: blocks glycolysis at the hexokinase level; oxamate: inhibits lactate formation by blocking lactate dehydrogenase; oxythiamine: blocks pyruvate conversion to acetyl-CoA via pyruvate dehydrogenase inhibition, succinyl-CoA production by inhibition of α-ketoglutarate dehydrogenase as well as the non-oxidative pentose phosphate pathway by inhibiting transketolase; DCA: activates the conversion of pyruvate to acetyl-CoA by inhibiting pyruvate dehydrogenase kinase) and on mitochondrial ATP regeneration by inhibiting ATP synthase within the mitochondrial respiration chain (oligomycin). A scheme illustrating the inhibitors and their corresponding targets is shown in Figure 8 .

Quantification of ‘anchored’ and ‘cell-free’ NET phenotypes confirmed that sporozoites indeed triggered exposed bovine PMN to release both forms of NETs ( Figures 7A–D ). Functional inhibition experiments showed that this process seemed independent of glycolysis since FDG treatments failed to block NET formation ( Figures 7A, B ). Overall, the formation of both parasite-triggered ‘anchored’ and ‘cell-free’ NETs was significantly reduced by oxamate treatments (OXA; treated PMN + sporozoites vs. non-treated PMN + sporozoites, oxamate: ‘anchored’ p = 0.002, ‘cell-free’ p = 0.01) which indicated a key role of lactate generation during the NETosis process ( Figures 7A, B ). In addition, a significant decrease of ‘cell-free’ and ‘anchored’ NET formation was observed in the case of oligomycin A treatments (treated PMN + sporozoites vs. non-treated PMN + sporozoites, oligomycin A: ‘cell-free’ p = 0.002, ‘anchored’ p = 0.003), suggesting that efficient E. bovis sporozoite-induced NET formation was also dependent on ATP synthase activities. In contrast, treatments with DON, DCA, and oxythiamine failed to influence sporozoite-induced NETosis ( Figures 7A, B ).

Given that lactate synthesis seemed of major importance during parasite-triggered NETosis, we additionally studied the relevance of lactate efflux in sporozoite-exposed PMN by applying chemical blockers of MCT which control the proton-linked transport of monocarboxylates, such as L-lactate, pyruvate, and ketone bodies, across the plasma membrane (30). Indeed, pretreatments of PMN with AR-C 141990 (blocks MCT1) and AR-C 155858 (blocks MCT1 and MCT2) both significantly reduced sporozoite-induced ‘cell-free’ and ‘anchored’ NET formation (treated PMN + sporozoites vs. non-treated PMN + sporozoites for both inhibitors: ‘anchored’ p ≤ 0.0001, ‘cell-free’ p ≤ 0.0001) ( Figure 7C, D ).

To further elucidate the relevance of purinergic signaling pathways in E. bovis sporozoite-induced NETosis, PMN were pretreated with theobromine (inhibits P1A1 receptor-mediated purinergic signaling) and NF449 (blocks P2X1 receptor-mediated purinergic signaling). As an interesting finding, we here showed that NF449 pretreatments entirely abolished E. bovis sporozoite-induced ‘cell-free’ and ‘anchored’ NET formation (levels were even below those of plain PMN) when compared to non-treated controls (treated PMN + sporozoites vs. non-treated PMN + sporozoites: both p < 0.001) ( Figures 7C, D ). In line, theobromine treatments also significantly reduced the formation of both types of NETs (treated PMN + sporozoites vs. non-treated PMN + sporozoites: ‘anchored’ p < 0.001 and ‘cell-free’ p ≤ 0.001) but to a lesser degree than NF449 ( Figures 7C, D ). These results strongly suggest that sporozoite-induced NETosis depends on both P2X1-mediated ATP binding and P1A1-mediated purinergic signaling.