This study was performed in accordance to the Justus Liebig University Giessen Animal Care Committee Guidelines. Protocols were approved by the Ethic Commission for Experimental Animal Studies of the Federal State of Hesse (Regierungspräsidium Giessen; GI 18/10 Nr. V 2/2022; JLU-No. 0002_V) and are in accordance to European Animal Welfare Legislation: ART13TFEU and currently applicable German Animal Protection Laws.

Primary bovine umbilical vein endothelial cells (BUVEC) were isolated from umbilical veins obtained from calves born by sectio caesarea at the Justus Liebig University Giessen. Therefore, umbilical cords were stored at 4°C in 0.9% HBSS–HEPES buffer (pH 7.4; Gibco, Grand Island, NY, USA) supplemented with 1% penicillin (500 U/ml; Sigma, St. Louis, MO, USA) and streptomycin (500 μg/ml; Sigma) for a maximum of 16  h before use. For endothelial cells isolation, 0.025% collagenase type II (Worthington Biochemical Corporation) suspended in Puck´s solution (Gibco) was infused into the lumen of ligated umbilical veins and incubated for 20  min at 37°C/5% CO₂ atmosphere. Cells were collected in cell culture medium supplemented with 1 ml fetal calf serum (FCS, N4637 Sigma) to inactivate collagenase. After two washes (350 × g, 12  min, 20°C), cells were re-suspended in complete endothelial cell growth medium (C-22010, ECGM, PromoCell) supplemented with 10% FCS. Then, cells were plated in 25  cm² tissue plastic culture flasks (Greiner) and cultured at 37°C/5% CO₂ atmosphere in modified ECGM medium (diluted at 30% in M199 medium) supplemented with 5% fetal bovine serum (FBS, 10270-106, Gibco) and 1% penicillin/streptomycin. Medium was replaced every 2–3 days. BUVEC cell layers were used for infection after 3 passages in vitro.

All experiments of the current study were performed with tachyzoite stages of the apicomplexan parasite B. besnoiti (Evora04 strain). Madin-Darby bovine kidney (MDBK) cells were used as host cells for tachyzoite in vitro production. Host cells were cultured in 75 cm² plastic tissue culture flasks (Greiner) at 37°C/5% CO₂ atmosphere in RPMI-1640 (R0883, Sigma-Aldrich) medium supplemented with 5% FBS and 1% penicillin/streptomycin. MDBK cell layers were infected at 80% confluency with 2.4×10⁷ tachyzoites. Parasites released from MDBK cells were scrapped and harvested from cell supernatants, filtered by a 5 μm syringe filter (Merck Millipore), washed, and pelleted (400×g, 12 min) prior to re-suspension in the working medium required. Tachyzoite numbers were determined in a Neubauer chamber, and parasite stages were placed at 37°C/5% CO₂ atmosphere for further experimental use.

Healthy adult dairy cows served as blood donors. Animals were bled by puncture of the jugular vein and peripheral blood was collected in heparinized sterile plastic tubes (Kabe Labortechnik). Heparinized blood was re-suspended at 1:1 ratio in 20 ml sterile PBS with 0.02% EDTA (CarlRoth), carefully layered on top of 12 ml Histopaque-1077 separating solution (density = 1.077 g/l; 10771, Sigma-Aldrich) and centrifuged (800×g, 45 min) without brake. After removal of plasma and peripheral blood mononuclear cells, the volume of the cell suspension was adjusted to 10 ml with Hank’s balanced salt solution (HBSS, 14065-049, Gibco). Then, 20 ml of lysis buffer (5.5 mM NaH₂PO₄, 10.8 mM KH₂PO₄, pH 7.2) were added and the sample was gently mixed for 60 s to lyse erythrocytes. Osmolarity was rapidly restored by addition of 10 ml hypertonic buffer (462 mM NaCl, 5.5 mM NaH₂PO₄, 10.8 mM KH₂PO₄, pH 7.2) and 10 ml HBSS. The lysis step was repeated twice until no erythrocytes were visible. PMN were then suspended in 5 ml HBSS, counted in a Neubauer chamber and allowed to rest on ice for 30 min prior to any experimental use.

To isolate EVs from B. besnoiti-infected BUVEC, 8 x 10⁶ BUVEC (n = 3) in 75 cm² plastic tissue culture flasks were infected with tachyzoites (ratio: 1:6) in modified ECGM medium (C-22210, Promocell) for 4 h (37°C, 5% CO₂ atmosphere). After washing with sterile PBS, cells were resuspended in vesicle-depleted modified ECGM medium (EV medium) and incubated for 24 h. Equal numbers of plain tachyzoites and non-infected BUVEC were equally treated for controls. For isolation of EVs from tachyzoite-exposed PMN (n = 3), 5 x 10⁷ PMN were incubated with tachyzoites (ratio 1:6) in EV medium for 4 h. Equal numbers of plain PMN and of zymosan-stimulated PMN (0.1 mg/ml, 4 h) were used as controls. After incubation, EV-enriched supernatants were collected and pooled by experimental condition in conical tubes and differentially centrifuged (300×g for 5 min, 2,000×g for 10 min and 10,000×g for 30 min) to eliminate cell debris. Supernatants were concentrated using Amicon Ultra-15 100 kDa MWCO filter devices (Millipore, Billerica, MA, USA) to a final volume of 500 µl. Then, EV isolation was performed by size-exclusion chromatography (SEC) with an Automatic Fraction Collector V2 (Izon) using a qEV sepharose column (ICO-70, qEVoriginal/70 nm, Izon) according to IZON´s protocol. The columns were equilibrated with filtered (0.22 µm) PBS, pH 7.4 before loading EV samples (500 µl). After discarding 2.9 ml eluate, 0.5 ml fractions were collected into 2 ml Eppendorf tubes. EV fractions were concentrated using Amicon Ultra-15 100 kDa MWCO filter devices to a final volume of 100-150 µl. EV samples were stored at −20°C until further use.

For EV characterization, Nano-flow cytometry was conducted using a Flow NanoAnalyzer (NanoFCM Co., Ltd, Nottingham, UK) equipped with a 488 nm and a 638 nm laser. The instrument was calibrated using 200 nm polystyrene beads (NanoFCM Co. Ltd.) at a defined concentration of 2.08 x 10⁸ particles/ml, serving as reference for particle concentration. Monodispersed silica beads (NanoFCM Co., Ltd) of four different diameters (68 nm, 91 nm, 113 nm and 155 nm) were utilized as size reference standards. Measurements of freshly filtered (0.1 μm), plain 1x TE buffer pH 7.4 (Lonza, Basel, Switzerland) were defined as background signals; consequently, respective values were subtracted from all other measurements. Particle concentration and size distribution of EV samples (diluted in 0.1 μm pore size-filtered 1x TE buffer) were calculated using NanoFCM software (NF Profession V2.0), based on data collected for one minute under a sample pressure of 1.0 kPa.

Bovine PMN (n = 3) were co-cultured with BUVEC- and B. besnoiti tachyzoite-derived EVs (1 x 10⁸) in RPMI-1640 medium (without phenol red, R7509, Sigma-Aldrich) for 4 h (37°C, 5% CO₂ atmosphere) on poly-_L-lysine (0.01%) -pretreated coverslips (15 mm diameter, Thermo Fisher Scientific), fixed in 4% paraformaldehyde (Merck) and stored at 4°C until further use. For NET visualization, DAPI (Fluoromount G, ThermoFisher, 495952) was applied to stain DNA; anti-histone (clone H11-4, 1:100, Merck Millipore MAB3422, Darmstadt, Germany) and anti-NE (ab68672, 1:200, Abcam, Cambridge, UK) primary antibodies were used to detect respective proteins on NET structures. Therefore, fixed samples were washed three times with PBS, blocked with 1% bovine serum albumin (BSA, Sigma-Aldrich, Steinheim, Germany, 30 min, RT), and incubated in corresponding primary antibody solutions (1 h, RT). After three washings in PBS, samples were reacted with secondary antibody solutions (Alexa Fluor 488 goat anti-rabbit IgG and Alexa Fluor 594 goat anti-mouse IgG, Life Technologies, Eugene, USA; 60 min, 1:500, RT). Finally, samples were washed three times in PBS and mounted in DAPI-containing mounting media (Fluoromount G, ThermoFisher, 495952). Image acquisition was achieved by a BZ-X800 microscope (Keyence), thereby applying identical brightness and contrast conditions within the datasets of each biological experiment. Percentages of NET-forming PMN were calculated semi-automatically by dividing the events counted in the histone channel (multiplied by 100) by the events counted in the DAPI channel (44).

Bovine PMN (n = 3) suspended in RPMI-1640 were confronted with 1 x 10⁸ EVs from all cellular sources (see 2.5) and incubated for 4 h (37°C, 5% CO₂). After incubation, picogreen (Invitrogen, Eugene, USA, 1:200 dilution in 10 mM Tris base buffered with 1 mM EDTA, 100 μl/well) was added to each sample. Extracellular DNA was quantified by picogreen-derived fluorescence intensities using an automated multiplate reader (Varioskan, Thermo Scientific) at 484 nm excitation/520 nm emission as described elsewhere (24, 28, 45).

Oxidative and glycolytic responses of bovine PMN were monitored using a Seahorse XFp analyzer (Agilent). Briefly, 1 x 10⁶ PMN from three blood donors were pelleted (500 × g, 10 min) and re-suspended in 0.25 ml of XF assay medium (Agilent) supplemented with 2 mM of _L-glutamine, 1 mM pyruvate and 10 mM glucose. 2 x 10⁵ cells were gently placed in each well of an eight-well XF analyzer plate (Agilent) pre-coated for 30 min with 0.001% poly-_L-lysine (Sigma-Aldrich). Then, XF assay medium (Agilent) was adjusted to 180 μl total volume per well and cells were incubated at 37°C without CO₂ supplementation for 45 min before Seahorse measurements. 1 x 10⁸ EVs from different cellular sources (see 2.5) were supplemented to the cells via instrument-own injection ports following 4 baseline measurements. The total assay duration was 160 min. Background subtraction and determination of OCR/ECAR registries were performed by using Seahorse Agilent analytics platform (https://seahorseanalytics.agilent.com).

The protein concentration of each EV isolate was estimated by the absorbance at 562 nm using the micro BCA protein assay kit (ref 23235, Thermo Scientific) following manufacturer’s protocol. EV-derived protein samples were supplemented with Laemmli-β-mercaptoethanol loading buffer (1x final concentration, #1610747, BioRad). Commercially available human EV-derived proteins (EV pos, EXOAB-POS-1, System Biosciences) and B. besnoiti tachyzoite protein extracts were used as positive controls. After boiling (95°C) for 5 min, proteins (20 µg/slot) were separated in 4-20% polyacrylamide gels (#4561095, BioRad) via electrophoresis (120 V constant for 1 h, tetra system BioRad) and then transferred to 0.2 µm PVDF membranes (trans-blot turbo #1704156, 2.5 A constant, up to 25 V, 7 min, BioRad). Samples were blocked in 3% BSA in TBS [50 mM Tris-Cl, pH 7.6; 150 mM NaCl containing 0.1% Tween (blocking solution); Sigma-Aldrich] for 1 h at RT, and then reacted with primary antibodies diluted in blocking solution (overnight, 4°C). Primary antibodies were anti-CD9 (ThermoFisher, Cat #MA1-19301, Mouse, 1:500), anti-CD81 (ThermoFisher, Cat #MA5-28419, Rabbit, 1:500), and anti-vinculin (Santa Cruz, Cat #sc-73614, Mouse, 1:500). Vinculin was detected as sample loading control. Following three washes in TBS-Tween 0.1% buffer, blots were incubated with secondary antibody solutions (dilution in blocking solution, 30 min, RT). Secondary antibodies were anti-Mouse (Pierce, Cat #31430, 1:40000) and anti-Rabbit (Pierce, Cat #31466, 1:40000). After three further washes in TBS-Tween (0.1%) buffer, signal detection was accomplished by an enhanced chemiluminescence detection system (Clarity Max Western ECL substrate, #1705062, BioRad) and recorded using a ChemiDOC Imager (BioRad). Protein masses were controlled by a protein ladder (PageRuler Plus Prestained Protein Ladder ~10-180 kDa, #26616, Thermo Fisher Scientific).

Total ROS measurement was performed by a chemiluminescence-based assay using luminol (A4685, Sigma-Aldrich). Therefore, 1 x 10⁷ PMN were suspended in 1 ml of HBSS; 100 µl (1 × 10⁶ PMN) was transferred per well to a white 96 well plate. Then, 90 µl luminol (80 µM final concentration) were added per well. For negative controls, non-stimulated PMN were used. After 30 readings accounting for 12 min, 10 µl of 1 x 10⁸ EVs isolated from BUVEC controls, B. besnoiti-infected BUVEC, B. besnoiti tachyzoites or Zymosan (0.1 mg/ml, Z4250, Sigma) were added to PMN. Chemiluminescence was measured for 3 h in a luminometer (Luminoskan, Thermo Scientific).

TEM analysis was performed on 1 x 10¹⁰ EVs derived from infected BUVEC and B. besnoiti tachyzoites. EV samples (10 μl) were fixed in a drop of 0.1 M cacodylate buffer containing 4% formaldehyde and 1.5% glutaraldehyde. Specimen suspensions were absorbed immediately after fixation on formvar-coated grids and stained with 1% ammonium molybdate. Negatively stained samples were inspected in a transmission electron microscope (EM 902N, Zeiss, Oberkochen, Germany) equipped with a slow-scan 2K CCD camera (TRS, Tröndle, Moorenweis, Germany).

1 x 10⁹ EVs isolated from non-infected BUVEC, B. besnoiti-infected BUVEC and B. besnoiti tachyzoites were stained by far red staining (CellTrace™ far red, C34564, ThermoFisher) as described before (46). Briefly, EVs (15 µl) in PBS were mixed with 15 μl far red dye solution (40 μM) and incubated for 2 h at 37°C. To remove unbound dye, EV samples were loaded on a qEV sepharose column (qEVoriginal/70 nm, Izon) and processed as described before (see 2.5). Cell Trace far red dye alone was used as dye control.

1 x 10⁶ PMN were incubated with 1 x 10⁹ far red-labeled EVs derived from non-infected BUVEC, B. besnoiti-infected BUVEC and B. besnoiti tachyzoites in RPMI-1640 medium (without phenol red, R7509, Sigma-Aldrich) at 37°C for 6 h. Far red dye alone was used as dye control and unstimulated/unstained PMN were used as negative controls. Samples were mixed by gently pipetting every hour. Cells were washed twice with PBS before analysis. Confocal microscopy was performed with 0.5 x 10⁶ EV-stimulated PMN seeded in 24-well plates containing poly-_L-lysine (0.01%, 20 min, RT and subsequent washing with PBS) -pretreated 15 mm coverslip. After 30 min of incubation, cells were fixed in 4% paraformaldehyde (Merck) and mounted in anti-fading buffer with DAPI (Fluoromount G, ThermoFisher, 495952). Images were acquired 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). Two channels were recorded for signal detection: DAPI/405 nm and far red dye/640 nm. Images were acquired by a sCMOS camera (PCO edge) using a CFI Plan Apochromat X20 and x60 lambda-immersion oil objective (NA 1.4/0.13; Nikon) controlled by NIS-Elements v 5.11 (Nikon, Tokyo, Japan) software. Identical brightness and contrast conditions were applied for each data set within one experiment using Fiji software.

1 x 10⁶ PMN or BUVEC plated in 12 well plates (at 80% confluence) were exposed to 1 x 10⁸ EVs derived from non-infected BUVEC, B. besnoiti-infected BUVEC and B. besnoiti tachyzoites for 4 h (PMN) and 24 h (BUVEC) at 37°C, 5% CO₂. LPS (1 µg/ml) and PMA/ionomycin (100 nM/5 µM) for PMN and LPS (0.01 µg/ml) for BUVEC were use as positive controls. After incubation, aliquots (100 µl) of cell supernatants (each n = 3) were analyzed for the presence of interleukin (IL)-1β (bovine IL-1β ELISA Kit, #ESS0027, Thermo Fisher Scientific, MA, USA), IL-6 (#ESS0029, Thermo Fisher Scientific, MA, USA) and CXCL8 (IL-8, bovine IL-8 ELISA kit, ABIN6957183, Antibodies Online, Germany), following manufacturer’s instructions. In the case of IL-1β and IL-6 ELISA kit, a high binding 96 well plate (#655061, Greiner bio one) were used. The samples were analyzed at 450 nm and 550 nm in an automatic Varioskan Flash Reader (Thermo Fisher Scientific, MA, USA). Standard curves and sample concentrations of IL-1β, IL-6 and CXCL8 were calculated using GraphPad PRISM^® V10.3.0 software package (GraphPad software, USA).

All experiments were repeated at least three times. Statistical significance was defined by a p value ≤ 0.05. The p values were determined by one-way ANOVA followed by a Dunnett´s multiple comparison test, with single pooled variance. Bars graphs represent the mean ± SD, and statistical analyses were generated using GraphPad PRISM^® V10.3.0.

EVs were isolated from all cell sources using size exclusion chromatography (SEC) with qEV/70 nm Original (IZON) columns, which are optimized for high EV recovery and minimal lipoprotein contamination. EV numbers and sizes from pooled fractions were assessed by Nano-Flow cytometry, EV-specific marker detection was performed by western blotting and EV morphology was visualized by TEM ( Figure 1 ). In all cases, EVs peaked in size around 60-80 nm ( Figure 1A ). Overall, mean EV sizes from control BUVEC, B. besnoiti-infected BUVEC, plain PMN, B. besnoiti-confronted PMN, zymosan-stimulated PMN and B. besnoiti tachyzoites were detected, consistent with literature data describing a general size of 30-120 nm for small EVs ( Figure 1B ) (47). The mean concentration of particles per cell after 24 h incubation showed comparable EV secretion from BUVEC regardless of infection ( Figure 1C ). In contrast, EV secretion experienced a 3-fold increment when PMN were exposed for 4 h to B. besnoiti tachyzoites in comparison to plain PMN or zymosan-stimulated PMN ( Figure 1D ). Western blot analyses confirmed the EV nature of the particles since the samples proved positive for CD9 and showed weak signals for CD81 (besides vinculin as loading control), both representing typical EV markers ( Figure 1E ). TEM analyses revealed for the first time B. besnoiti tachyzoite-derived and B. besnoiti-infected BUVEC-derived EVs by confirming the typical EV morphology ( Figure 1F ).

To explore if exposure of unprimed PMN to EVs of different cellular sources changed the energetic status and oxidative responses, we analyzed the PMN metabolic parameters of oxygen consumption (OCR) and extracellular acidification rates (ECAR) via Seahorse analytics ( Figure 2 ). Overall, encounter with EVs neither affected oxidative nor glycolytic responses of bovine PMN, irrespective of the EV source ( Figure 2 ).

To address if EV exposure has an impact on PMN effector mechanisms, we first focused on NET formation. Bovine PMN were exposed to EVs derived from non-infected BUVEC, B. besnoiti-infected BUVEC, non-stimulated PMN, tachyzoite-exposed PMN and B. besnoiti tachyzoites ( Figure 3 ). Extracellular DNA quantification based on picogreen-derived fluorescence intensities was performed at 4 h of incubation, thereby rather reflecting a late phase of NET formation. Relative DNA level analysis showed a significant increase of extracellular DNA release only for PMN stimulated with EVs derived from BUVEC (p < 0.001) and from B. besnoiti tachyzoites (p < 0.05) when compared to medium controls ( Figure 3A ). In the former case, EV-driven NET formation revealed independent of the infection status of BUVEC since EVs from non-infected and B. besnoiti-infected BUVEC equally induced NET formation. In contrast, PMN-derived EVs failed to induce extracellular DNA release, irrespective of PMN stimulation ( Figure 3A ). Therefore, we focused further experimentation on BUVEC- and B. besnoiti tachyzoite-derived EVs. To confirm typical characteristics of NET formation, classical NET markers (NE and histone-DNA) were visualized by immunostaining ( Figure 3B ), applying a semi-automatic image analysis for NET quantification ( Figure 3C ). Here, the presence of extracellular DNA concomitant with histone and NE was confirmed for NET structures from PMN stimulated with BUVEC- and tachyzoite-derived EVs at 4 h ( Figure 3B ). Further analysis revealed a tendency to increase in the percentage of PMN extruding NETs in case of tachyzoite-derived EVs, B. besnoiti-infected BUVEC-derived EVs, non-infected BUVEC-derived EVs, and B. besnoiti tachyzoite-derived EVs in comparison with control ( Figure 3C ).

To study, if extracellular DNA release coincided with PMN ROS production, total ROS production was measured in PMN stimulated with BUVEC- and tachyzoite-derived EVs ( Figures 3D, E ). However, current data revealed that EVs from all tested sources failed to affect PMN-derived total ROS production ( Figure 3E ). In contrast, stimulation of PMN with zymosan, serving as positive control for ROS synthesis, indeed triggered significant ROS production.

To study PMN-EV-interactions on the level of EV internalization, bovine PMN (n = 3) were co-cultured for 6 h with far red-labeled EVs derived from non-infected BUVEC, B. besnoiti-infected BUVEC and B. besnoiti tachyzoite ( Figure 4 ). PMN-mediated EV uptake was assessed by confocal microscopy ( Figure 4A ) visualizing a rather globular staining within the PMN cytoplasm, most likely reflecting endosomal localization of internalized EVs, as described in the literature (48) ( Figure 4B ). Semi-automated microscopic quantification revealed a significant increase in PMN-derived far red signals upon EV encounter. Thus, almost equal fractions of PMN with far red signals were detected in case of EVs from tachyzoites, B. besnoiti-infected BUVEC and non-infected BUVEC ( Figure 4C ).

Since EVs are well-documented for their role in intercellular communication, we explored their capacity to induce inflammatory responses in PMN and BUVEC by assessing the release of IL-1β, IL-6 and CXCL8. These inflammatory mediators were quantified via commercially available ELISAs in supernatants from both PMN and BUVEC being exposed to EVs from BUVEC and B. besnoiti tachyzoites ( Figure 5 ). At 4 and 24 h of exposure for PMN and BUVEC, respectively, only trace amounts of IL-1β, IL-6 and CXCL8 were detected in supernatants of PMN ( Figures 5A, C, D ) and BUVEC ( Figures 5B, D, F ). Nevertheless, PMN-derived IL-1β and IL-6 release was significantly increased after PMN exposure to EVs derived from B. besnoiti-infected BUVEC (p = <0.0001 and p = <0.0001, respectively, Figures 5C, E ). In contrast, EVs failed to induce CXCL8 secretion in PMN. BUVEC stimulation with EVs of different origin all failed to affect endothelial IL-1β, IL-6 and CXCL8 release. Interestingly, PMN showed differential cytokine secretion depending on the stimuli initially used for positive controls. Thus, LPS induced an increase in IL-1β secretion while stimulation with PMA/ionomycin enhanced IL-6 release. Furthermore, LPS worked as a positive stimulus for BUVEC by inducing an enhanced secretion of both IL-1β and IL-6.