Canine Neutrophil Extracellular Traps Release Induced by the Apicomplexan Parasite Neospora caninum In Vitro

Neosporosis is considered as one of the main causes of abortion and severe economic losses in dairy industry. The Canis genus serving as one of the confirmed definitive hosts of the apicomplexan parasite Neospora caninum (N. caninum) plays a critical role in its life cycle. However, the effects of N. caninum on its definitive hosts of neutrophils extracellular traps (NETs) formation remain unclear. In the present study, N. caninum tachyzoite-induced canine NETs formation was observed by scanning electron microscopy (SEM). Visualization of DNA decorated with H3, neutrophil elastase (NE), and myeloperoxidase (MPO) within N. caninum tachyzoite-induced NETs were examined using fluorescence confocal microscopy analyses. Furthermore, the formation of canine NETs was quantified using Sytox Green staining, and the LDH levels in supernatants were examined by an LDH Cytotoxicity Assay® kit. The results clearly showed that NETs-like structures were induced by N. caninum tachyzoites, and the major components within these structures induced by N. caninum tachyzoite were further confirmed by fluorescence confocal microscopy visualization. These results suggest that N. caninum tachyzoites strongly induced NETs formation in canine polymorphonuclear neutrophils (PMN). In functional inhibition assays, the blockings of NADPH oxidase, NE, MPO, SOCE, ERK 1/2, and p38 MAPK signaling pathways significantly inhibited N. caninum tachyzoite-induced NETs formation. To our knowledge, this study is the first to report the formation of NETs in canine PMN against N. caninum infection.

Neosporosis is considered as one of the main causes of abortion and severe economic losses in dairy industry. The Canis genus serving as one of the confirmed definitive hosts of the apicomplexan parasite Neospora caninum (N. caninum) plays a critical role in its life cycle. However, the effects of N. caninum on its definitive hosts of neutrophils extracellular traps (NETs) formation remain unclear. In the present study, N. caninum tachyzoite-induced canine NETs formation was observed by scanning electron microscopy (SEM). Visualization of DNA decorated with H3, neutrophil elastase (NE), and myeloperoxidase (MPO) within N. caninum tachyzoite-induced NETs were examined using fluorescence confocal microscopy analyses. Furthermore, the formation of canine NETs was quantified using Sytox Green staining, and the LDH levels in supernatants were examined by an LDH Cytotoxicity Assay ® kit. The results clearly showed that NETslike structures were induced by N. caninum tachyzoites, and the major components within these structures induced by N. caninum tachyzoite were further confirmed by fluorescence confocal microscopy visualization. These results suggest that N. caninum tachyzoites strongly induced NETs formation in canine polymorphonuclear neutrophils (PMN). In functional inhibition assays, the blockings of NADPH oxidase, NE, MPO, SOCE, ERK 1/2, and p38 MAPK signaling pathways significantly inhibited N. caninum tachyzoite-induced NETs formation. To our knowledge, this study is the first to report the formation of NETs in canine PMN against N. caninum infection.
Keywords: neTs, canine, neutrophils, Neospora caninum inTrODUcTiOn Neosporosis is caused by the apicomplexan protozoa Neospora caninum and is considered as one of the main diseases causing abortion, reproduction disorders, and thus severe economic losses in dairy industry worldwide (1,2). N. caninum is an apicomplexan parasite closely related to Toxoplasma gondii, which infects a wide intermediate host range (3,4). As an important veterinary pathogen, several researches have been focused on N. caninum in the past two decades. To date, its confirmed definitive hosts of N. caninum is the genus Canis, including domestic dogs (Canis familiaris) (5), dingoes (Canis lupus dingo) (6), and gray wolves (Canis lupus) (7). Neosporosis in canines principally results in a neuromuscular disease, i.e., polyradiculoneuritis-myositis, but a variety of less common lesions due to focal necrosis can also occurs in other organs (8,9). Although several studies on cattle N. caninum infections are well reported and analyzed by serologic and/or molecular diagnostic assays, the definitive host-parasite interactions remain not well clarified. Polymorphonuclear neutrophils (PMN) are one of the most abundant and important leukocyte population in blood, thereby considered as the first-line of defense in the host innate immune system against invasive microorganisms. Neutrophils extracellular traps (NETs) have been recognized as a novel effector mechanism of PMN in many immune processes. NETs are mainly compost of DNA, antibacterial proteins/peptides, and granule proteins, such as histones, neutrophil elastase (NE), myeloperoxidase (MPO), lactoferrin, gelatinase, pentraxin, and cathelicidin, among other molecules (10,11). During N. caninum infection of bovine endothelial cells, PMN adhesion was increased and the expression of adhesion molecules, such as E-selectin, VCAM-1, and ICAM-1, was significantly upregulated, which revealed the critical role of PMN in the host innate immune system (12). However, whether the novel effector mechanism of PMN-NETs involved in the interactions between N. caninum and its definitive hosts has not been investigated during the acute infection of N. caninum. In this study, the influence of N. caninum on canine NETs formation was examined, and the key molecular signaling pathways were further elucidated.

isolation of canine PMn
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.

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

statistical analysis
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. resUlTs

Neospora caninum Tachyzoites induced neTs Formation in canine PMn
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.
Visualization of Dna Decorated with histones (h3), neutrophil elastase, and Myeloperoxidase in N. caninum

Tachyzoite-induced neTs structures
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.

Quantitation of neTs
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 timedependent process.
naDPh Oxidase, ne, and MPO are involved in N. caninum Tachyzoiteinduced neTs Formation 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 tachyzoiteinduced 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.

Neospora caninum Tachyzoite-induced neTs Formation is an erK 1/2-, p38 MaPK-, and sOce-Dependent Process
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 Ca 2+ 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 pathwayand SOCE-dependent process.

N. caninum Tachyzoite-induced neTs Formation is independent of lDh activity
To account for the novel form of cell death program-NETosis, we tested LDH activity in the progress of N. caninum tachyzoiteinduced 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.

DiscUssiOn
As the first-line of defense within the host innate immune system, PMN are endowed with powerful effector mechanisms, such as phagocytosis and degranulation, to resist and kill microbes (13). In 2004, another novel effector mechanism of PMN was first described and named "NETs" (10). This novel effector mechanism has been considered as a physical barrier to trap microbes and to   avoid their dissemination, and even kill these entrapped microbes. It has been demonstrated that several stimuli, including bacteria, fungi, viruses, and crystal salts can induce the activation of this novel mechanism-NETs in activated PMN (14)(15)(16). While most researches concentrated on the effects of NETs on bacteria, fungi, and viral pathogens, increasing evidence on NETs formation triggered by parasites was reported recently, such as T. gondii, Eimeria bovis, and Leishmania donovani (17)(18)(19). However, the effects of N. caninum on NETs formation have not been investigated.   (17)(18)(19). However, we do not know the effects of NETs on N. caninum infection in bovine, and whether neutrophils would also be able to recognize and attack N. caninum by NETs formation in vivo need to be further investigated.
In response to pathogens infection, the process of NETs formation has been accompanied by the concentration of several antibacterial proteins and granule proteins, including histones, MPO, NE, and cathelicidin at the site of infection (20)(21)(22). In this study, the decoration of DNA with H3, MPO, and NE in N. caninum-triggered NETs were demonstrated. These colocalization results clearly proved the nature of these NET structures after the exposure of canine PMN to vital tachyzoites. Furthermore, inhibitors of NE and MPO significantly inhibited N. caninum tachyzoite-induced NETs formation, which suggest the critical role of NE and MPO in N. caninum tachyzoite-induced NETs. These proteins were also previously observed as key molecules in T. gondii-, Besnoitia besnoiti-, and E. bovis-triggered ETs (17,23,24). Previous studies showed that NETs formation was a NADPH oxidase-dependent process, which resulted in the production of ROS (25). Thus, DPI was used to investigate the role of NADPH oxidase in N. caninum tachyzoite-induced NETs. As a result, pretreatment with the NADPH oxidase inhibitor significantly decreased N. caninum tachyzoite-induced NETs formation, and N. caninum tachyzoite significantly increased ROS production. Moreover, ROS production has been proved to be dependent on SOCE (26), so we next explored the role of SOCE in N. caninum tachyzoite-induced NETs formation. The results showed that N. caninum tachyzoite-induced NETs formation was significantly decreased by the SOCE inhibitor 2-APB. There is another report showing that SOCE is regulated via ERK 1/2 phosphorylation and via a ROS production-dependent activation of the Raf-MEK-ERK pathway, which has also been proved to be required for NETs formation induced by parasites (23,27,28). Then, we analyzed the critical role of Ca 2+ influx and the role of ERK 1/2 and p38 signaling pathway in N. caninum tachyzoite-induced NETs. Our results show that 2-APB, UO126, and SB202190 significantly inhibited N. caninum tachyzoite-induced NETs formation thereby proving the potential role of these molecules in N. caninum-mediated NETosis. As reported for several pathogens, N. caninum tachyzoite-induced NETs formation may be also a NADPH oxidase-, NE-, MPO-, SOCE-, ERK 1/2-, and p38 MAPK-dependent process (23,28). In addition, we detected LDH activity in the process of N. caninum tachyzoite-induced NET formation. These results showed that no significant necrosis occurred during N. caninum tachyzoite-induced NETs formation when compared to positive controls, which was in accordance with the characteristics of typical NETosis induction as previously demonstrated (10,15,29).
Taken together, N. caninum tachyzoites are strong inducers of canine NETs, which suggested a critical role of NETs in the early host innate immune response against tachyzoites. Whether other N. caninum stages, such as sporozoites or bradyzoites, might be capable to induce NETosis needs further investigations. Furthermore, several molecular mechanisms have been proved to Canine PMN were stimulated with N. caninum tachyzoite (ratio 1:1) for 30, 60, 90, and 120 min. The LDH levels in supernatant were examined by an LDH Cytotoxicity Assay kit according to the manufacturer's protocols. Values are presented as mean ± SD (n = 5). P values of <0.05 were considered significant (***P < 0.001).
FigUre 7 | effects of sOce inhibitor, erK, and P38 signaling pathway inhibitors on N. caninum tachyzoite-induced neTs in canine PMn. Prior to stimulation with N. caninum tachyzoite, the PMN were pretreated with the following inhibitors of SOCE, ERK, and P38 signaling pathway. Zymosan was used as positive controls. The formation of canine NETs was quantified using Sytox Green (Invitrogen). The samples were examined with a fluorometric reader using an excitation wavelength of 488 nm and detecting at 523 nm. Values are presented as mean ± SD (n = 5). P values of <0.05 were considered significant (**P < 0.01 and ***P < 0.001).