Neospora caninum tachyzoites (strain Nc-1) were maintained in VERO cells monolayer at 37°C/5% CO₂. VERO cells were cultured in RPMI 1640 medium (Hyclone, USA) supplemented with 2% fetal bovine serum (FBS, Biological Industries, Israel) and 1% penicillin/streptomycin (Hyclone, USA). N. caninum tachyzoites were collected and harvested from VERO cells (3000 r/min, 10 min, room temperature), then passed through 20, 5, 1-ml syringe and a 27-gage needle in turn, purified by 40% Percoll reagent through centrifugation (3000 r/min, 30 min, room temperature), collected precipitates and washed them twice with RPMI 1640 medium (3000 r/min, 10 min, room temperature).

Adult healthy canines (n = 3) were bleeding by puncture of the femoral vein, and blood was collected in blood collection tubes containing heparin (Jun Nuo, Shandong Chengwu County Medical Products Co., China). The PMN were isolated by using the commercially available Canine PMN isolation kit^® (TianJin HaoYang Biological Manufacture Co., China) according to the manufacturer’s instructions. All animal experiments were approved by the NIH Guide for the Care and Use of Laboratory Animals of the Jilin University and in accordance to the current Animal Protection Laws of China.

Canine PMN were stimulated with N. caninum tachyzoites (ratio 1:1, 90 min, 37°C) on cover glass slides. These cover glass slides were pre-coated with poly-l-lysine (0.1 mg/ml, Sigma-Aldrich) for 12 h and washed three times with distilled water. After incubation, the cells were fixed with 4.0% glutaraldehyde (Merck) for 24 h, washed with PBS and post-fixed in 1.0% osmium tetroxide (Merck). Then, the samples were dehydrated in ascending ethanol concentrations (30, 50, 70, 80, 90, 100%), frozen in tertiary butyl alcohol at −20°C and sputtered with gold. Finally, specimens were examined using a scanning electron microscope (Hitachi S-3400N, Japan).

Canine PMN were stimulated with N. caninum tachyzoite (ratio 1:1, 90 min, 37°C) on poly-l-lysine (0.1 mg/ml, Sigma-Aldrich) pre-coated cover glass slides. After incubation, the cells were fixed with 4% (w/v) paraformaldehyde for 20 min at room temperature, washed thrice with PBS, permeabilized with 0.1% Triton X-100 for 15 min and blocked in 3% goat serum/PBS, followed by incubation with antibodies to H3, MPO, and NE at 4°C overnight. Anti-histone antibody (LS-C353149; Life Span BioSciences, Inc, 1:200, dissolved in 3% goat serum), anti-MPO antibody (Orb16003; Biorbyt, 1:200, dissolved in 3% goat serum), and anti-NE antibody (AB68672; Abcam, 1:200, dissolved in 3% goat serum) were used for the detection of H3, MPO, and NE in N. caninum tachyzoite-triggered NETs-like extracellular structures. Then, the samples were incubated with the second conjugated antibody (goat anti-rabbit IgG-FITC conjugated, Bioworld Technology Inc) and washed two times with PBS, then stained with 5-μM Sytox Orange (dissolved in PBS, Invitrogen) for 10 min at room temperature. Finally, the specimens were washed two times with PBS, mounted in anti-fading reagents (Beyotime Biotechnology, China), and examined using scanning confocal microscope (Olympus FluoView FV1000).

Canine PMN were stimulated with viable N. caninum tachyzoites (ratio 1:1, 1:2, 1:3, 1:6, or 1:12, 37°C) for 30, 60, and 90 min, respectively. In parallel settings, prior to stimulation with N. caninum tachyzoite (1:6), the canine PMN were pretreated with the following specific inhibitors for 30 min including the NE inhibitor (CMK, 1 mM, Sigma-Aldrich), the NADPH oxidase inhibitor diphenylene iodonium (DPI, 10 μM, Sigma-Aldrich), the MPO inhibitor (ABAH, 100 μM, Calbiochem), the inhibitors of ERK 1/2 (UO126, 50 μM, Sigma-Aldrich) and p38 MAKP (SB202190, 10 μM, Sigma-Aldrich) signaling pathway, and with the store-operated calcium entry (SOCE) inhibitor (2-amindethoxydiphery borate, 100 μM, Sigma-Aldrich) for 15 min. The formation of canine NETs was quantified using 5-μM Sytox Green (Invitrogen). The samples were examined with a fluorometric reader Infiniti M200^® (TECAN, Austria) using an excitation wavelength of 488 nm and detecting at 523 nm the emission wavelength.

The reactive oxygen species (ROS) production of PMN induced by N. caninum tachyzoites (ratio 1:1, 90 min) was determined with 2,7 dichlorofluorescein diacetate (DCFH-DA, Sigma-Aldrich). PMN stimulated with zymosan (1 mg/ml, Sigma) were used as positive controls. The samples were examined with a fluorometric reader using an excitation wavelength of 485 nm and detecting at 530 nm the emission wavelength.

The LDH levels in supernatant were examined by the LDH Cytotoxicity Assay kit^® (Beyotime Biotechnology, China) according to the manufacturer’s protocols.

Values were expressed as the means ± SD. Data were analyzed by the GraphPad 5.0 software. Differences among the groups were performed with one-way analysis of variance (ANOVA) with Tukey multiple comparison test. P values of <0.05 were considered as significant.

Neospora caninum tachyzoites strongly induced NETs formation in canine PMN and was confirmed by scanning electron microscopy (SEM) analyses (Figure 1). NETs released from canine PMN were observed in Figure 1, and N. caninum tachyzoites were captured in these thicker and thinner network extracellular structures. Furthermore, N. caninum tachyzoite-induced NETs in canine PMN were demonstrated by fluorescence confocal microscopy analyses (Figures 2B,E,H). These results confirmed that N. caninum tachyzoite surely induced NETs in canine PMN as an additional anti-parasitic effector mechanism.

Fluorescence confocal microscopy analyses revealed that NETs formation can be induced by N. caninum tachyzoites, and the nature of these structures was mainly composed by DNA (Figures 2B,E,H). Besides DNA backbone structure of N. caninum-induced NETs, H3 (Figure 2A), MPO (Figure 2D), and NE (Figure 2G) were also observed corroborating the colocalization of these molecules and DNA of these NETs. Merge image of DNA decorated with H3, MPO and NE was showed in Figures 2C,F and I. Taken together, these results confirm the main classical characteristics of NETs induced by N. caninum tachyzoites in canine PMN.

Canine NETs formation was quantified using Sytox Green. As shown in Figures 3 and 4, N. caninum tachyzoites significantly induced increasing quantitation of NETs when compared to negative controls but lower levels than zymosan (positive controls). These results revealed that the formation of NETs induced by N. caninum tachyzoite was a dose- and time-dependent process.

To investigate the role of NADPH oxidase, NE, and MPO of whether to be involved in N. caninum tachyzoite-induced NETs, inhibitors of NADPH oxidase, NE, and MPO were here used. As shown in Figure 5, DPI, ABAH, CMK, and DNase I treatments significantly inhibited N. caninum tachyzoite-induced NETs. In addition, N. caninum tachyzoites significantly increased ROS production when compared to negative controls (Figure 6). These results suggest that NADPH oxidase, NE, and MPO are involved in N. caninum tachyzoite-induced NETs formation. Moreover, the DNase I treatment resulted in significant reduction of N. caninum-induced NET formation, thereby confirming that the primary nature of these NETs was a DNA backbone.

In order to investigate in more detail, molecular signaling pathways of N. caninum-triggered NET formation, inhibition assays were performed. For this purpose, inhibitors of the SOCE, ERK1/2, and p38 MAPK signaling pathways were used to analyze the critical role of Ca²⁺ and these two kinases-dependent signaling pathways. As shown in Figure 7, 2-APB, UO126, and SB202190 significantly inhibited N. caninum tachyzoite-induced NETs. These results indicated that N. caninum tachyzoite-induced NETs formation was an ERK 1/2 and p38 MAPK signaling pathway- and SOCE-dependent process.

To account for the novel form of cell death program-NETosis, we tested LDH activity in the progress of N. caninum tachyzoite-induced NETs formation. As shown in Figure 8, LDH activities in the supernatant were markedly induced by lysis buffer, but there were no significant changes in the progress of N. caninum tachyzoite-induced NETs formation.