Bovine PMN responses to extracellular vesicles released by Besnoitia besnoiti tachyzoites and B. besnoiti-infected host cells

Abstract

Bovine besnoitiosis is a re-emerging cattle disease caused by the apicomplexan parasite Besnoitia besnoiti, which severely affects individual animal welfare and profitability in cattle industry. We recently showed that B. besnoiti tachyzoite exposure to bovine polymorphonuclear neutrophils (PMN) effectively triggers neutrophil extracellular trap (NET) formation, leading to parasite immobilization hampering host cell infection. So far, the triggers of this defense mechanism remain unclear. Emerging evidence indicates that extracellular vesicles (EVs) modulate PMN effector functions, such as ROS production or NET formation. Therefore, we tested whether exposure of bovine PMN to EVs from different cellular sources affects classical PMN effector functions and cytokine/chemokine secretion. EVs were isolated from B. besnoiti-infected and non-infected host cells (bovine umbilical vein endothelial cells, BUVEC), from tachyzoite-exposed bovine PMN and from B. besnoiti tachyzoites. EV concentration and size was determined by Nano-Flow cytometry and EV nature was confirmed by both classical EV markers (CD9 and CD81) and transmission electron microscopy (TEM). Overall, PMN stimulation with both BUVEC- and tachyzoite-derived EVs significantly induced extracellular DNA release while EVs from PMN failed to affect NET formation. BUVEC and tachyzoite EV-driven NET formation was confirmed microscopically by the presence of DNA decorated with neutrophil elastase (NE) and histones in typical NET structures. Moreover, confocal microscopy revealed EVs to be internalized by bovine PMN. Referring to PMN activation, EVs from the different cellular sources all failed to affect glycolytic or oxidative responses of bovine PMN as detected by Seahorse^®-based analytics and luminol-based chemoluminescence, thereby denying any role of NADPH oxidase (NOX) activity in EV-driven NET formation. Finally, exposure to B. besnoiti-infected BUVEC-derived EVs induced IL-1β and IL-6 release, but failed to drive CXCL8 release of bovine PMN. Hence, we overall demonstrated that EVs of selected cellular origin owned the capacity to trigger NOX-independent NET formation, were incorporated by PMN and selectively fostered IL-1β and IL-6 release.

Keywords: Besnoitia besnoiti; NET formation; PMN; endothelial cells; extracellular vesicles.

Figure 1

Characterization of BUVEC-, PMN- and B. besnoiti tachyzoite-derived EVs. Extracellular vesicles were isolated from non-infected BUVEC (n.i. BUVEC), B. besnoiti-infected BUVEC (Infected BUVEC), non-exposed PMN (Plain PMN), B. besnoiti tachyzoite-exposed PMN (B. b.-exposed PMN) and from plain B. besnoiti tachyzoites (Tachyzoite). (A) Exemplary histograms on EV size distribution, (B) particle concentration and (C, D) particle release per cell as assessed by Nano-Flow cytometry. Zymosan-stimulated PMN served as positive control for PMN-derived EV production. Mean particle diameters of EVs showed values around 70 nm. (E) Western blot analysis of BUVEC-, PMN- and B. besnoiti tachyzoite-derived EV samples probed with anti-CD9, anti-CD81 and anti-vinculin antibodies. Commercially available human EV-derived proteins (EV pos) and B. besnoiti protein extract were used as controls. (F) B. besnoiti tachyzoite-derived and infected BUVEC-derived EVs were studied by TEM (black arrows), and showed a typical EV morphology (scale bars indicate 100 nm).

Figure 2

Exposure to EVs does not affect oxidative and glycolytic responses in bovine PMN. In absence of CO₂, PMN were incubated in XF RPMI media for 45 min. Four basal measurements were performed and then PMN-derived EVs (A) or BUVEC-derived EVs (B) were supplemented to bovine PMN at the time point indicated by a vertical line. OCR and ECAR values were obtained by Seahorse technology and plotted over time (n = 3 for each condition). All data are shown as mean ± SD.

Figure 3

Exposure of bovine PMN to BUVEC- and B. besnoiti tachyzoite-derived EVs induced NET formation in a ROS-independent manner. (A) Bovine PMN were stimulated with EVs derived from non-infected BUVEC (n.i. BUVEC), B. besnoiti-infected BUVEC (Infected BUVEC), unstimulated PMN (Plain PMN), B. besnoiti tachyzoite-exposed PMN (B. b.-exposed PMN) and from B. besnoiti tachyzoites (Tachyzoite) for 4 h After incubation, extracellular DNA was quantified via picogreen-derived fluorescence intensities. 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 < 0.001. (B) Exemplary immunofluorescence images showing DNA (blue), neutrophil elastase (green) and DNA-histone complexes (magenta) in EVs-exposed PMN. (C) The percentage of NET-releasing PMN was calculated via image analysis (Image J, Fiji version); bars represent mean ± SD. (D, E). Representative kinetic and total ROS production of EV-exposed PMN, evaluated by luminol-based assays after 4 h of exposure. Zymosan served as positive control. (n = 3). Scale bar = 30 µm.

Figure 4

PMN-mediated uptake of far red-labeled EVs. Bovine PMN were exposed to far red-labeled EVs for 6 h, fixed and mounted with fluoromount G (DAPI). (A, B) Representative microscopic images depicts PMN (nuclei, blue) with internalized EVs (magenta). (C) Semi-automated quantitative analysis of EV internalization showing that PMN equally internalized EVs from all cellular sources. All data are shown as mean ± SD; p-values were calculated by one-way ANOVA followed by Dunnett´s multiple comparison test. **p < 0.01; ***p < 0.001. Scale bar = 20 µm. (n = 3).

Figure 5

Selected EV exposure induced IL-1β and IL-6 release in bovine PMN. Bovine PMN or BUVEC were exposed to EVs from different cellular sources for 4 h (PMN) and 24 h (BUVEC). Thereafter, CXCL8 (A, B), IL-1β (C, D), and IL-6 (E, F) was quantified via commercially available ELISAs in co-culture-derived supernatants. Stimulation with LPS and PMA/ionomycin was used for positive controls. All data are shown as mean ± SD; p-values were calculated by one-way ANOVA followed by Dunnett´s multiple comparison test. (n = 3). **p < 0.01; *** p < 0.001; **** p < 0.0001.