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Extracellular Trap Formation by Donkey Polymorphonuclear Neutrophils Against Toxoplasma gondii
Journal of Equine Veterinary Science 73 (2019) 1e9
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Journal of Equine Veterinary Science journal homepage: www.j-evs.com
Original Research
Extracellular Trap Formation by Donkey Polymorphonuclear Neutrophils Against Toxoplasma gondii Kader Yildiz a, *, Sami Gokpinar a, Neslihan Sursal b, Cahit Babur c, Dogukan Ozen d, Ahmet Kursat Azkur e a
Department of Parasitology, Faculty of Veterinary Medicine, Kirikkale University, Kirikkale, Turkey Department of Parasitology, Ankara University Health Sciences Institute, Ankara, Turkey Ministry of Health, Public Health Institution of Turkey, Ankara, Turkey d Department of Biostatistic, Faculty of Veterinary Medicine, Ankara University, Ankara, Turkey e Department of Virology, Faculty of Veterinary Medicine, Kirikkale University, Kirikkale, Turkey b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 May 2018 Received in revised form 7 November 2018 Accepted 9 November 2018 Available online 15 November 2018
Toxoplasma (T.) gondii is an obligatory intracellular apicomplexan parasite. The donkey is one of the intermediate hosts of T. gondii. There are almost no data about T. gondii infection in donkeys, apart from a few seroprevalence reports. The aim of the study was to detect the formation of extracellular traps by donkey polymorphonuclear neutrophils (PMNs) when exposed to T. gondii tachyzoites in vitro. Tachyzoites were observed to be entrapped within extracellular traps (NET) structures. Myeloperoxidase, neutrophil elastase (NE), and histone (H3) were observed in NET structures. NE and reactive oxygen species activity increased with time and was higher in the PMN-tachyzoite cocultures. Some tachyzoites were seen close localization to the nucleus of neutrophils. Degranulation and extracellular traps were observed simultaneously in some donkey neutrophils after incubation for 1 hour. The tachyzoite invasion rate decreased in PMN-tachyzoite cocultures in comparison to the controls. NETs can negatively affect the viability of entrapped tachyzoites in addition to their action of physical entrapment. NE may play a key role in the viability of T. gondii in donkeys. The amount of extracellular DNA increased with incubation time in the PMN-tachyzoite cocultures (P < .001). This is a first report regarding the formation of donkeys NETs after exposure to T. gondii tachyzoites in vitro. Unlike sheep and cattle PMN, degranulation and extracellular traps developed in the same neutrophil. Further studies focusing on signaling pathways may shed light on what determines the behavior of donkey neutrophils after exposure to T. gondii. © 2018 Elsevier Inc. All rights reserved.
Keywords: Toxoplasma gondii Donkey Neutrophil extracellular traps Innate immunity In vitro
1. Introduction Toxoplasma (T.) gondii, an obligatory intracellular apicomplexan, is one of the most prevalent zoonotic parasites in the world [1]. Felidae members, including the domestic cat, are the definitive hosts [2]. The donkey is one of the intermediate hosts of T. gondii, alongside
Animal welfare/ethical statement: In this study, the blood samples (20 mL) were taken from vena jugularis of clinically healthy donkeys for PMN isolation. The animal owners were informed before the blood samples were taken, and the confirmation form was signed. All animal handling procedures were approved by the Kirikkale University Local Ethics Committee for Animal Experiments (Protocol no: 16/53). Conflict of interest statement: This study has received no financial support. * Corresponding author at: Kader Yildiz, Department of Parasitology, Faculty of Veterinary Medicine, Kirikkale University, Campus 71450, Kirikkale, Turkey. E-mail addresses: [email protected], [email protected] (K. Yildiz). https://doi.org/10.1016/j.jevs.2018.11.002 0737-0806/© 2018 Elsevier Inc. All rights reserved.
other well-known intermediate host species such as sheep, pigs, goats, and humans [2]. T. gondii possesses three infectious forms: sporozoite, tachyzoite, and bradyzoite [3]. After being excreted in cat feces, sporozoites develop into oocysts under suitable climatic conditions. Tachyzoites can infect all cells in an organism and proliferate intracellularly by endodyogeny [4]. During acute toxoplasmosis, tachyzoites disseminate to all organs of the body [3]. Later, cysts form in the cells of a variety of tissues including muscles, brain, and other parenchymatous organs in the intermediate hosts [2]. These cysts are filled with slow-growing bradyzoites and may remain asymptomatically in tissues for the duration of the host's life [2]. After oral infection with tissue cysts or sporulated oocysts of the parasite, bradyzoites or sporozoites are released and enter hosts' enterocytes [2]. Chemokines and cytotoxic molecules secreted by the infected cells attract immune cells to the infection area [5]. Neutrophils are the first cells to arrive in the infection area [6]. Neutrophils are
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involved in the cellular inflammatory response and possess a crucial role in innate immunity. The role of the neutrophil has previously been thought of as release of cytotoxic agents and phagocytosis during the inflammatory process [7]. Subsequently, it was discovered that neutrophils have another strategy for fighting infection [8], called NETosis [9]. During NETosis, some important nuclear and cytoplasmic changes develop in neutrophils [10]. When neutrophils encounter pathogens, reactive oxygen species (ROS) are produced [11]. The lobular form of the neutrophil nucleus disappears. Nuclear, cytoplasmic, and granular contents become mixed with one another [7]. Finally, extracellular traps (NETs), augmented with enzymes originating from granules (such as neutrophil elastase [NE] and myeloperoxidase [MPO], and histones), are excreted from neutrophils into extracellular spaces [11e13]. NET formation has been reported in many different species [14e22] including donkeys [23]. Extracellular traps form not only from neutrophils but also from other granulocytic cells including eosinophils [24] and monocytes [11,25,26]. Extracellular trap structures are known to develop in response to some parasitic protozoon species [18,27e30] including T. gondii [17,26,31]. T. gondii tachyzoites trigger the formation of extracellular trap structures from neutrophils in murine, humans [17], sheep, and cattle [31] and from polymorphonuclear neutrophils (PMNs) and monocytes in harbor seals [26]. NETs also have negative effects on the viability of entrapped tachyzoites in addition to the physical entrapment, which prevents invasion of host cells by tachyzoites [17,31]. Infection rates reduced significantly when T. gondii tachyzoites were exposed to PMNs and monocytes from harbor seals [26]. The donkey has become more popular with the increasing importance of its milk for children with allergies to cow's milk [32]. Some researchers have focused on the potential risk of jenny milk for foodborne toxoplasmosis [33]. As a member of the Equidae family, donkeys are generally accepted to be biologically similar to horses [34]. Although clinical toxoplasmosis has not been confirmed in horses, specific antibodies develop between 6 and 12 days after experimental infection with T. gondii [35]. There are almost no data about T. gondii infection in donkeys, apart from a few seroprevalence reports [36e38]. Toxoplasma gondii has the ability to infect a broad range of animal species [2]. Some hosts, such as the donkey, can be infected with the parasite but do not play an important role in parasite epidemiology. One of the reasons for this may be innate immunity. Following infection with T. gondii, the parasites are first exposed to innate immune cells, especially neutrophils. The main aim of the study was to detect the formation of extracellular traps in donkey PMNs when exposed to T. gondii tachyzoites in vitro. Activities of MPO and NE were measured during the incubation period in the donkey PMN-tachyzoite coculture. The effects of different parasite concentrations and incubation periods on NET development in donkey PMNs and the effect of NET structures on host cell invasion by T. gondii were studied. 2. Materials and Methods 2.1. Toxoplasma gondii Tachyzoites Toxoplasma gondii RH strain tachyzoites were passaged intraperitoneally in Swiss albino mice. The tachyzoites were obtained from the peritoneal fluid 48 hours after the experimental infection of mice. The fluid was centrifuged at room temperature (200 g for 10 minutes). The supernatant was centrifuged (1,000 g for 10 minutes), and the pellet was washed with a sterile saline solution (pH 7.2). After staining with Diff-Quick stain (Bio Optica, Italy), murine inflammatory cells were evaluated by light microscopy (Leica DM750), the pellet did not include any murine inflammatory
cells. Tachyzoites were counted using a Neubauer chamber with a light microscope and then resuspended in RPMI 1640. 2.2. PMN Isolation From Donkey Blood All animal handling procedures were approved by the Kirikkale University Local Ethics Committee for Animal Experiments (Protocol no: 16/53). This study was carried out according to Munoz Caro et al. [18], with some modifications. The blood samples (20 mL) were collected from clinically healthy donkeys (n ¼ 3) into heparinized tubes. The samples were layered into sterile tubes with Biocoll Separating Solution (Biochrom) after mixing with equal volumes of sterile 0.02% PBS-EDTA. The plasma, lymphocytes, and monocytes were removed after centrifugation (800 g, with no brake, 45 minutes, 22 C; Thermo Scientific 16S). Erythrocytes were lysed by addition of ultrapure sterile water (25 mL); 30 seconds later, osmolarity was adjusted by addition of 3 mL sterile HBSS (10; Sigma). Polymorphonuclear neutrophils were centrifuged with RPMI 1640 (without phenol red; Sigma) (400 g, 10 minutes, 4 C). After staining with Diff-Quick stain (Bio Optica, Italy), neutrophil purity was evaluated by light microscopy (Leica DM750) and was higher than 90%. Polymorphonuclear neutrophil viability was determined using the trypan blue dye test and was higher than 95%. Polymorphonuclear neutrophils were counted using a Neubauer chamber and then reconstituted as 1 105/100 mL in RPMI 1640. 2.3. Fluorescence Microscope Examination of NET Structures Against T. gondii Just before starting the experiment, to facilitate their observation within NETs, tachyzoites were stained with CellTrace CFSE Cell Proliferation Kit (5 mM; Thermo Fisher Scientific) according to the manufacturer's instructions. Briefly, CFSE and tachyzoites were incubated (37 C, 10 minutes). Then, ice-cold PBS (containing 10% FCS) was added. CFSE-stained tachyzoites were protected from light until the start of the experiment after centrifugation for three times (10 minutes, 400 g). Donkey PMNs and CFSE-stained tachyzoites (1:3) were placed on poly-L-lysine-coated coverslips (Corning, BioCoat) and incubated for 1 hour (37 C, 5% CO2). After fixing with paraformaldehyde (4%) and treatment with Triton X-100 (0.1%), the cells were blocked with 1% BSA. To detect MPO, NE, and histone (H3) in NET structures, the samples were incubated with anti-MPO (1:1000, sc-52707, Santa Cruz), anti-NE (1:1000, sc-55548, Santa Cruz), and antihistone (H3) monoclonal antibodies (1:1000, sc-374669; Santa Cruz; 1 hour, 37 C, 5% CO2). After three washes, secondary antibodies (IgG1-FITC, 1:100, sc-358546; Santa Cruz for MPO and NE, and IgG2b-FITC, 1:100, ABIN 637988; antibodies.online for histone) were added for 1 hour (37 C, 5% CO2). Then, the cocultures were stained with SYTOX orange dye (Invitrogen) at room temperature (1:30,000, 5 minutes, in dark). The coverslips were placed gently onto microscope slides using a drop of anti-fading buffer (Mowiol). The slides were examined with the fluorescence microscope (Leica DM IL Led, 470 nm excitation/515 nm emission). Composite images were generated using Image J (version 2.0.0-rc-43/1.50 g). The isotype controls, mouse IgG1 (1:1000, sc-3877; Santa Cruz), were used to confirm the MPO and NE signals, and mouse IgG2b (1:1000, sc-3879; Santa Cruz) for histone (H3) signals. 2.4. Detection of NE, ROS, and MPO Activities in PMN-Tachyzoite Cocultures Tachyzoite and donkey PMN (1:1) cocultures were placed into the 96 wells of a flat bottom immunoplate (Nunc, Sigma-Aldrich). To detect NE and ROS activities, respectively, MeOSuc-Ala-Ala-
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Pro-Val-chloromethyl-ketone (the chromogenic substrate, 3 mg/ mL; Sigma-Aldrich) and 20 ,70 -dichlorofluorescin diacetate (DCFHDA, 10 mg/mL; Sigma Aldrich) were added into the wells. To detect MPO activity, Amplex red hydrogen peroxide/peroxidase assay kit (Thermo Fisher) was used according to manufacturer's protocol. The immunoplate was incubated in the fluorometer, and NE, MPO, and ROS activities were measured every 10 minutes during the incubation period (37 C, 150 minutes) (485 nm excitation/538 nm emission). In the experiments, phorbol 12-myristate 13-acetate (PMA)einduced PMNs and the samples containing untreated PMNs were used as the positive and negative controls, respectively.
2.5. Host Cell Invasion Assay During host-cell invasion assays, three groups were classified as follows: Donkey PMNs and tachyzoite cocultures (1:2) were incubated for 3 hours (37 C, 5% CO2). The cocultures were treated with deoxyribonuclease I (DNase I, 90 U; Sigma) added 15 minutes before the end of the incubation period. The cocultures were seeded onto a confluent
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monolayer of Vero cells in a 6-well plate (group 1). Donkey PMNs and tachyzoite cocultures (1:2) were incubated for 3 hours (37 C, 5% CO2). The cocultures were seeded onto a confluent monolayer of Vero cells in a 6-well plate (without treatment with DNase I) (group 2). As a control, equal numbers of tachyzoites were seeded onto a confluent monolayer of the Vero cells in a 6-well plate (group 3). After 2 hours of incubation, the plates were washed twice with prewarmed Dulbecco's Modified Eagle Medium to remove extracellular tachyzoites and PMNs. The plates were incubated for 22 hours under the same conditions (37 C, 5% CO2), the percentage of infected cells was determined quantitatively in 10 randomly selected microscopic areas (Olympus CKX41 inverted microscope). The percentage of the infected cells was accepted as 100% in the control group (group 3). The assay was performed at least three independent times. The arithmetic means of the results were calculated.
2.6. Quantitative Analysis of NETs Against T. gondii Donkey PMN-tachyzoite cultures (1:1 and 1:3) were incubated for four different incubation times (30, 60, 120, and 180 minutes;
Fig. 1. Fluorescence micrographs of extracellular trap (NET) structures triggered by Toxoplasma gondii tachyzoites. Cocultures of donkey polymorphonuclear neutrophils (PMNs) and tachyzoites stained with CellTrace CFSE Cell Proliferation Kit (5 mM; Thermo Fisher Scientific) (1:3) were stained with SYTOX orange dye for extracellular DNA backbone (A, D, G) and probed for histones (B), myeloperoxidase (MPO) (E), and neutrophil elastase (NE) (H) using anti-histone, anti-MPO, and anti-NE antibody, respectively. Composite images (C, F, I) were generated using Image J (version 2.0.0-rc-43/1.50 g). Tachyzoites to be entrapped in NET structures (arrows in the composite images) (Bar: 20 mm).
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37 C, 5% CO2). Micrococcal nuclease (5 U, NEB) was added, and then samples were incubated for 15 minutes under same conditions. The supernatants were transferred into 96-well flat bottom immunoplates after centrifugation (300 g, 5 minutes), each sample was tested in duplicate. SYTOX green dye (5 mM, Invitrogen) was added into the wells and incubated in the dark (15 minutes). Fluorescence levels were measured with a fluorometer (Fluoroskan Ascent; Thermo Scientific, 485 nm excitation/538 nm emission). Zymosan(1 mg/mL; Sigma Aldrich) and PMA- (50 nM; Sigma Aldrich) induced PMNs were used as positive controls and untreated PMNs as negative controls. Diphenyleneiodonium (DPI), a potent NADPH oxidase inhibitor, was used as an inhibitor in the experiment (10 mM; Sigma Aldrich). The assay was performed at least three independent times.
extracellular traps were observed simultaneously in some donkey neutrophils after 1-hour incubation. Some tachyzoites were also observed in PMNs that developed extracellular traps (Fig. 2). Light microscopic observation of donkey PMNs after being stained with Diff-Quick stain is submitted as Supplemental Fig. 1. Fluorescence micrographs of isotype controls for MPO, NE, and histone (H3) immunostainings are submitted as Supplemental File 2. 3.2. Detection of NE, ROS, and MPO Activities in PMN-Tachyzoite Cocultures
The data were analyzed using SPSS 14.01 (SPSS Inc, Chicago, IL, USA). Dose-dependent analyses were evaluated with one-way variance analysis and a Tukey post hoc test. Normality of data and homogeneity of variance were analyzed by the Shapiro Wilk test and Levene test, respectively. Time-dependent analyses of different parasite concentrations were evaluated with the General Linear Model for Repeated Measure. The terms of “dose (betweensubject factor),” “time (within-subject factor),” and “dose*time interaction” were used in the model. The Huynh-Feldt correction was used for sphericity. The Bonferroni test was used for simple effect analysis. Differences were considered significant at P < .05.
Neutrophil elastase activity was higher in the PMN-tachyzoite cocultures than in the positive controls in the experiments. This activity increased rapidly up to the 100th minute of incubation in all groups, after this time the activity slowed down and even began to decrease in the control groups (P < .001) (Fig. 3). Quantitative analysis revealed that ROS activity increased in a time-dependent fashion and was higher in tachyzoite-PMN cocultures than in control groups during the incubation periods (P < .001) (Fig. 4). Myeloperoxidase activity increased over the standard curve in PMN-tachyzoite cocultures during all incubation periods (Fig. 5). Surprisingly, MPO activity was higher in all groups in the experiments. In PMN-tachyzoite cocultures, MPO activity increases and peaked at 80 minutes, and then it decreased slowly until the end of the incubation. Similar results were recorded in the positive and negative controls (P < .04).
3. Results
3.3. Host Cell Invasion Assay
3.1. Fluorescence Microscopic Examination of Effect of NET Structures Against T. gondii
In this experiment, the invasion rate by tachyzoites in host cells not incubated with PMNs was set as 100% (the control group). The tachyzoite invasion rate decreased by 75% in the untreated groups when compared with the controls. Although the invasion rate increased in the DNase I treatment groups (53%), it was nearly half that of the control groups (Fig. 6).
2.7. Statistical Analysis
Extracellular trap structures were visualized in tachyzoitesPMN cocultures (1:3) after incubation for 1 hour. Toxoplasma gondii tachyzoites stained with CFSE were seen to be entrapped within cloud-like extracellular trap structures (Fig. 1). The nuclei of some PMNs had almost disappeared. Extracellular DNA, the backbone of the NET structures, was observed after staining with SYTOX orange dye (Figs. 1A, 1D, and 1G). Histone (H3), MPO, and NE were observed in NETs after staining with primary and secondary antibodies (Figs. 1B, 1E, and 1H). NETs developed in donkey PMNs after 1-hour incubation with tachyzoites. Some tachyzoites were seen to be located near the nucleus of neutrophils. Degranulation and
3.4. Quantitative Analysis of NETs Efficacy Against T. gondii According to dose-dependent studies, the fluorescence intensities of extracellular DNA contents were increased in parallel with tachyzoite concentrations (P < .001) (Fig. 7). Quantities of T. gondiieinduced extracellular DNA were significantly increased in donkey PMNs compared with the negative controls (P < .001). The amount of
Fig. 2. Various functions of donkey neutrophils against Toxoplasma gondii tachyzoites. Cocultures of donkey polymorphonuclear neutrophils (PMNs) and tachyzoites stained with CellTrace CFSE Cell Proliferation Kit (5 mM; Thermo Fisher Scientific) (1:3) were probed for neutrophil elastase (NE) (A), myeloperoxidase (MPO) (B), and histones (C) using antihistone, anti-MPO, and anti-NE antibody, respectively. CFSE-stained tachyzoites were in crescent shape seen to be located near the nucleus of neutrophils (stars) (A). Some tachyzoites were seen to be located close to the nucleus of neutrophils that developed extracellular traps (arrow) (B). Degranulation and extracellular traps were observed simultaneously in some donkey neutrophils after 1-hour incubation (arrowhead) (C). The structure of the nucleus disappeared in these neutrophils during degranulation and NETosis. Granules were in spherical forms around neutrophils (Bar: 15 mm).
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Fig. 3. Neutrophil elastase (NE) activity in polymorphonuclear neutrophils (PMNs)-tachyzoite cocultures. Tachyzoite and donkey PMN (1:1) cocultures were placed into the wells. MeOSuc-Ala-Ala-Pro-Val-chloromethyl-ketone (3 mg/mL; Sigma-Aldrich) was added into the wells for detection of NE activity. Neutrophil elastase activities were measured every 10 minutes during the incubation period (37 C, 150 minutes) (485 nm excitation/538 nm emission). Phorbol 12-myristate 13-acetate (PMA)einduced PMNs and PMNs alone were used as the positive and negative controls, respectively. The assay was performed at least three independent times with the same results (P < .001) (arbitrary unit [AU]).
extracellular DNA in PMN-tachyzoite cocultures significantly increased with incubation time (P < .001). Phorbol 12-myristate 13acetate (50 nM) and Zymosan (1 mg/mL) were good inducers of extracellular traps for donkey PMNs. NET reactions were inhibited in the groups where DPI (10 mM; Sigma Aldrich) was added.
4. Discussion Toxoplasma gondii has a wide intermediate host range, and it can infect nearly all cell types of its hosts [2]. Following oral infection, infectious forms of T. gondii disseminate through the bloodstream
Fig. 4. Reactive oxygen species (ROS) activity in polymorphonuclear neutrophil (PMN)-tachyzoite cocultures. Tachyzoite and donkey PMN (1:1) cocultures were placed into the wells. 20 ,70 -Dichlorofluorescin diacetate (DCFH-DA) (10 mg/mL; Sigma Aldrich) was added into the wells for detection of ROS activity. ROS activities were measured every 10 minutes during the incubation period (37 C, 150 minutes) (485 nm excitation/538 nm emission). Phorbol 12-myristate 13-acetate (PMA)einduced PMNs and PMNs alone were used as the positive and negative controls, respectively. The assay was performed at least three independent times with the same results (P < .001) (arbitrary unit [AU]).
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Fig. 5. Detection of myeloperoxidase (MPO) activity in polymorphonuclear neutrophils (PMNs)-tachyzoite cocultures. Tachyzoite and donkey PMN (1:1) cocultures were placed into the wells. Amplex red hydrogen peroxide/peroxidase assay kit (Thermo Fisher) was used according to manufacturer's instructions for detection of MPO activity. A standard curve was prepared with horseradish peroxidase (HRP) and 1 reaction buffer (1xRB) supplied by the kit. MPO activities were measured every 10 minutes during the incubation period (37 C, 150 minutes) (485 nm excitation/538 nm emission). Phorbol 12-myristate 13-acetate (PMA)einduced PMNs and PMNs alone were used as the positive and negative controls, respectively. The assay was performed at least three independent times with the same results (P < .04) (arbitrary unit [AU]).
and cause inflammation [39]. The inflammatory reaction is important for innate immunity. As one of the most important components of innate immunity, neutrophils are the first to reach affected areas [40] and fight pathogens by a variety of mechanisms including NETosis [41], where DNA augmented with granular
Fig. 6. Host cell invasion assay. Donkey polymorphonuclear neutrophils (PMNs) and tachyzoite cocultures (1:2) were incubated 3 hours. The cocultures were seeded in Vero cells after treated with deoxyribonuclease I (DNase I) (90 U; Sigma) 15 minutes before the end of the incubation period (group 1). Donkey PMN and tachyzoite cocultures (1:2) were incubated for 3 hours (37 C, 5% CO2). The cocultures were seeded onto a confluent monolayer of Vero cells in a 6-well plate (without treatment with DNase I) (group 2). As a control, equal numbers of tachyzoites were seeded onto a confluent monolayer of the Vero cells in a 6-well plate (group 3). After 2 hours of incubation, the plates were washed twice to remove extracellular tachyzoites and PMNs. The plates were incubated for 22 hours under the same conditions, and the percentage of infected cells was determined quantitatively in 10 randomly selected microscopic areas. The percentage of the infected cells was accepted as 100% in the control group. The assay was performed at least three independent times. The graphic shows the arithmetic means of three experiments.
contents is released extracellularly from neutrophils after exposure to a pathogen [7]. Studies [14e22] suggested that extracellular trap structures are from PMNs of many different animal species. There is only one report on NETosis from donkey neutrophils, in which extracellular trap structures developed against donkey spermatozoa in vitro [23]. Neutrophils possess three defense strategies: phagocytosis, degranulation, and NETosis [6]. There is no clear information on which defense strategy is used when they are exposed to pathogens [11]. Some authors suggested that neutrophils used defense strategies in a consecutive order after they are exposed to a pathogen [11]. Neutrophils first try to internalize pathogens by phagocytosis for a few minutes. After about 10 minutes, secretory vesicles secrete their substances into the extracellular space, followed by secretions from specific and azurophilic granules. Eventually, extracellular traps develop against pathogens [11]. This hypothesis has not been widely accepted because pathogens internalized in the phagosome will be released again into the extracellular space if the same neutrophil develops extracellular trap structures after phagocytosis of pathogens. Others suggest that microbial size is a critical factor in regulation of NETosis and NETs are released in response to larger pathogens [42]. Others have claimed that there are subsets of neutrophils, which possess different fighting capabilities, some of them being responsible for phagocytosis and others for NETosis [41]. This view is supported by some observations that suggest that extracellular traps develop from only some neutrophils in vitro [41]. In the present study, the extracellular traps developed in donkey PMN-tachyzoite cocultures at the end of the 1-hour incubation period. Moreover, some tachyzoites were observed on close localization to the nucleus of neutrophils. It was thought that these tachyzoites had entered donkey neutrophils during the incubation period. However, this observation has not previously been reported in sheep and cattle PMNeT. gondii tachyzoite cocultures [31].
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Fig. 7. Quantitative dose and time studies. Donkey polymorphonuclear neutrophils (PMNs)-tachyzoite cultures (1:1 and 1:3) were incubated for 30, 60, 120, and 180 minutes (37 C, 5% CO2). Then, SYTOX green dye (5 mM, Invitrogen) was added, and fluorescence levels were measured with a fluorometer (485 nm excitation/538 nm emission). Phorbol 12myristate 13-acetate (PMA)einduced and Zymosan-induced PMNs were used as positive controls, whereas PMNs alone were used as negative controls. Diphenyleneiodonium (DPI) was used as an inhibitor in the experiments. The assay was performed at least three independent times with the same results (dose P < .001, time P < .001, dose*time P < .001) (arbitrary unit [AU]).
Equine neutrophils possess different chemotactic and secretory stimuli responses from other animal species [43]. Donkey PMNs can possess varied subtypes, which have different fighting strategies against pathogens. It is well known that neutrophils are shortlived immune system cells with a life span of only 6e8 hours if there is no inflammation [44]. When neutrophils are exposed to pathogens, there is insufficient time for subtypes to develop; for this reason, they may be released into the blood stream as separate subtypes in the donkey. However, degranulation and extracellular traps were observed simultaneously in some donkey neutrophils after incubation for 1 hour in the present study. Toxoplasma gondii can invade and replicate in all nucleated cells, including immune system cells [3]. Neutrophils may contain T. gondii as a result of phagocytosis in addition to active invasion by the parasites [45]. Active invasion develops rapidly, and T. gondii penetrates into the host cell by the formation of a parasitophorous vacuole (PV) [46]. The identification of PVs is important in the discrimination of the route of entrance of tachyzoites into host cells, especially in phagocytic cells to discriminate active entry from phagocytosis [45]. The parasites survive in the PV, which avoids lysosome fusion and acidification in the host cell after invasion [46]. Neutrophils may therefore play a role in parasite dissemination within the host and being involved in fighting infection [45]. Neutrophils are actively invaded and harbor live tachyzoites in the small intestine of mice after oral infection [45]. In the present study, tachyzoites were located near the nucleus of some donkey neutrophils. The entry route for tachyzoites into neutrophils could not be established. Tachyzoites used in our experiment were part of the T. gondii RH strain known as a virulent strain. Hypervirulent T. gondii strains are known to enter cells using active penetration [47]. Neutrophils possess many enzymes and proteins in their granules [10,44]. These granular substances have antimicrobial effects on pathogens [10]. Neutrophil elastase is abundant in the azurophilic granules and is involved in oxygen-independent
microbicidal mechanism [48]. Neutrophil elastase may have a crucial role in the destruction of microorganisms via different routes such as microbicidal peptide activation and outer wall protein proteolysis [49,50]. In this study, NE concentrations increased rapidly up to the 100th minute of incubation, and then the activity slowed in all groups. Neutrophil elastase concentrations are higher in PMN-tachyzoite cocultures than those of positive controls. Neutrophil elastase can have some lethal effects on T. gondii tachyzoites in vitro experiments after 3 hours incubation in the present study. Myeloperoxidase originating from azurophilic granules is one of the most potent in vivo oxidant agents [51]. Myeloperoxidase catalyzes halide oxidation by hydrogen peroxide [52]. Myeloperoxidase participates actively in the destruction of microorganism within the phagocytic vacuole of neutrophils. Also, during NETosis, MPO is released from neutrophils to fight against microorganisms [51]. Neutrophil activation is generally associated with an inflammatory response in equine medicine [53]. In the horse, increased MPO concentrations are considered a marker of inflammatory pathologies such as laminitis and recurrent airway obstruction [54], which are common problems in horses [55]. Myeloperoxidase from stimulated neutrophils can lead to tissue damage and plays an important role in inflammation in the horses [53,54]. Increased MPO concentrations due to neutrophil degranulation have also been shown in plasma of horses, even following exercise [54,56]. Unlike horses, no data were found on MPO activity in donkeys. In our study, MPO activity increased over the standard curve during all incubation periods in PMN-tachyzoite cocultures. Surprisingly, MPO activity was lower in the PMN-tachyzoite cocultures in all experiments. In the host cell invasion assay, the tachyzoite invasion rate decreased by 75% in the untreated groups in comparison to the control. Tachyzoites entrapped by extracellular traps can be one of the factors contributing to this low invasion rate. NETs can be
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destroyed by DNase activity [7]. In the present study, the cell invasion rate increased after destroying extracellular traps with DNase I, but it was still almost half that of the invasion rate in the control group. Granular enzymes and proteins released from donkey neutrophils may have a lethal effect on tachyzoites in vitro. Notably, NE concentration was higher in PMN-tachyzoite cocultures in this study (P < .001). Besides granular enzymes, extracellular histones released from neutrophils also play an important role in destruction of pathogens [57]. Histones, basic proteins, located in the neutrophil nucleus, are toxic to pathogens in the extracellular spaces [57]. In the present study, it is possible that histones had detrimental effects on tachyzoites. Histones are known to have harmful effects on promastigotes of Leishmania spp. [58]. Also, some tachyzoites were observed inside donkey neutrophils in addition to those entrapped within extracellular traps. These tachyzoites might have been removed from the cell culture together with neutrophils during washing procedures in the invasion assay. This may help explain the low invasion rate of both the untreated and the DNase I treatment groups. Extracellular trap structures are triggered by some parasitic protozoa [18,27e30]. Toxoplasma gondii tachyzoites are extracellular trap inducers for mouse and human PMNs [17], harbor seal PMNs and monocytes [26], and sheep and cattle PMNs [31]. Extracellular traps are formed a time-dependent manner in bovine and caprine PMNs against parasites such as Eimeria bovis, Cryptosporidium parvum, and Eimeria arloingi sporozoites ([19,28,59], respectively). However, no such time-dependent development was seen in cattle PMNeBesnoitia besnoiti tachyzoite cocultures [18]. Formation of extracellular traps increases in a time-dependent manner in relation to incubation time in mouse [17], sheep, and cattle PMNs [31] against T. gondii tachyzoites. In the present study, NET production from donkey PMNs was seen after exposure to T. gondii tachyzoites in vitro. The amount of extracellular DNA increased significantly in a time-dependent fashion in the donkey PMNs (P < .001). Parasite concentrations are an important factor for the induction of extracellular traps in vitro [18,28,30,60]. NETs are induced by Leishmania promastigotes and Entamoeba histolytica in dosedependent manner [30,60]. NETs released from bovine PMNs are dependent on the number of B. besnoiti tachyzoites and E. bovis sporozoites [18,28]. Toxoplasma gondiieinduced NETs are also dose dependent in harbor seal PMNs and monocytes [26] and sheep and cattle PMNs [31]. In the present study, a parasite concentrationdependent relationship was detected in donkey PMNs after encounters with T. gondii tachyzoite in vitro (P < .001). Phorbol 12-myristate 13-acetate is used for in vitro studies as a potential inducer of neutrophils from mice and humans, sheep, and cattle [17,31]. Zymosan are used as a potential inducer of neutrophils from different animals and human in vitro experiments [18,19,61]. In the present study, PMA and Zymosan were used as positive controls, and they were better extracellular trap inducers for donkey PMNs than T. gondii tachyzoites. In previous studies, PMA has been shown to be a worse NETosis inducer in human, sheep, and cattle PMNs than T. gondii tachyzoites [17,31]. 5. Conclusion To the authors' knowledge, this is the first report regarding the formation of extracellular traps in donkey PMNs when exposed to T. gondii tachyzoites in vitro. Some tachyzoites were seen to be located close to the nucleus of neutrophils. Degranulation and extracellular traps were observed simultaneously in some donkey neutrophils after incubation for 1 hour. Toxoplasma gondiieinduced extracellular DNA contents increased with increasing tachyzoite concentrations and incubation time. The host cell invasion rate
decreased in tachyzoites after exposure to donkey PMNs. NETs can negatively affect the viability of entrapped tachyzoites in addition to their role in physical entrapment. Our results suggest that NE may play a role in the viability of T. gondii in donkeys. Further studies focusing on signaling pathways may shed light on what determines the behavior of donkey neutrophils after exposure to T. gondii. Acknowledgments Portion of data was presented at the fifth European Immunology & Innate Immunity, Berlin, Germany, July 21e23, 2016. Supplementary Data Supplementary data related to this article can be found at https://doi.org/10.1016/j.jevs.2018.11.002. References [1] Tenter AM, Heckeroth AR, Weiss LM. Toxoplasma gondii: from animals to humans. Int J Parasitol 2000;30:1217e58. [2] Dubey JP. Toxoplasmosis of animals and humans. Second Edition. Boca Raton: CRC Press; 2010. [3] Weiss LM, Kim K. Toxoplasma gondii. The model apicomplexan: Perspective and methods. San Diego: Academic Press; 2007. [4] Hill DE, Chirukandoth S, Dubey JP. Biology and epidemiology of Toxoplasma gondii in man and animals. Anim Health Res Rev 2005;6:41e61. [5] Mennechet FJ, Kasper LH, Rachinel N, Li W, Vandewalle A, Buzoni-Gatel D. Lamina propria CD4þ T lymphocytes synergize with murine intestinal epithelial cells to enhance proinflammatory response against an intracellular pathogen. J Immunol 2002;168:2988e96. [6] Kumar V, Sharma A. Neutrophils: cindirella of innate immune system. Int Immunopharmacol 2010;10:1325e34. [7] Papayannopoulos V, Zychlinsky A. NETs: a new strategy for using old weapons. Trends Immunol 2009;30:513e21. [8] Brinkmann V, Reichard U, Goosmann C, Fauler B, Uhlemann Y, Weiss DS, et al. Neutrophil extracellular traps kill bacteria. Science 2004;303:1532e5. [9] Steinberg BE, Grinstein S. Unconventional roles of the NADPH oxidase: signaling, ion homeostasis, and cell death. Sci STKE 2007;379:pe11. [10] Guimaraes-Costa AB, Nascimento MT, Wardini AB, Pinto-da-Silva LH, Saraiva EM. ETosis: A microbicidal mechanism beyond cell death. J Parasitol Res 2012;2012:929743. [11] Kaplan MJ, Radic M. Neutrophil extracellular traps: double-edged swords of innate immunity. J Immunol 2012;189:2689e95. [12] Fuchs TA, Abed U, Goosmann C, Hurwitz R, Schulze I, Wahn V, et al. Novel cell death program leads to neutrophil extracellular traps. J Cell Biol 2007;176: 231e41. [13] Papayannopoulos V, Metzler KD, Hakkim A, Zychlinsky A. Neutrophil elastase and myeloperoxidase regulate the formation of neutrophil extracellular traps. J Cell Biol 2010;191:677e91. [14] Palic D, Ostojic J, Andreasen CB, Roth JA. Fish cast NETs: neutrophil extracellular traps are released from fish neutrophils. Dev Comp Immunol 2007;31: 805e16. [15] Chuammitri P, Ostojic J, Andreasen CB, Redmond SB, Lamont SJ, Palic D. Chicken heterophil extracellular traps (HETs): novel defense mechanism of chicken heterophils. Vet Immunol Immunopathol 2009;129:126e31. [16] Wardini AB, Guimaraes-Costa AB, Nascimento MT, Nadaes NR, Danelli MG, Mazur C, et al. Characterization of neutrophil extracellular traps in cats naturally infected with feline leukemia virus. J Gen Virol 2010;91:259e64. [17] Abi Abdallah DS, Lin C, Ball CJ, King MR, Duhamel GE, Denkers EY. Toxoplasma gondii triggers release of human and mouse neutrophil extracellular traps. Infect Immun 2011;80:768e77. [18] Munoz Caro T, Hermosilla C, Silva LMR, Cortes H, Taubert A. Neutrophil extracellular traps as innate immune reaction against the emerging apicomplexan parasite Besnoitia besnoiti. PLoS One 2014;9:e91415. [19] Silva LMR, Munoz Caro T, Gerstberger R, Vila-Vicosa MJM, Cortes HCE, Hermosilla C, et al. The apicomplexan parasite Eimeria arloingi induces caprine neutrophil extracellular traps. Parasitol Res 2014;113:2797e807. [20] Jeffery U, Kimura K, Gray R, Lueth P, Bellaire B, LeVine D. Dogs cast NETs too: Canine neutrophil extracellular traps in health and immune-mediated hemolytic anemia. Vet Immunol Immunopathol 2015;168:262e8. [21] Wei Z, Hermosilla C, Taubert A, He X, Wang X, Gong P, et al. Canine neutrophil extracellular traps release induced by the apicomplexan parasite Neospora caninum in vitro. Front Immunol 2016;7:436. [22] Koiwai K, Alenton RR, Kondo H, Hirono I. Extracellular trap formation in kuruma shrimp (Marsupenaeus japonicus) hemocytes is coupled with c-type lysozyme. Fish Shellfish Immunol 2016;52:206e9.
K. Yildiz et al. / Journal of Equine Veterinary Science 73 (2019) 1e9 J, Vile s K, García W, Jordana J, Yeste M. Effect of donkey seminal plasma [23] Miro on sperm movement and sperm-polymorphonuclear neutrophils attachment in vitro. Anim Reprod Sci 2013;140:164e72. [24] Yousefi S, Simon D, Simon HU. Eosinophil extracellular DNA traps: molecular mechanisms and potential roles in disease. Curr Opin Immunol 2012;24: 736e9. [25] Munoz-Caro T, Silva LM, Ritter C, Taubert A, Hermosilla C. Besnoitia besnoiti tachyzoites induce monocyte extracellular trap formation. Parasitol Res 2014;113:4189e97. [26] Reichel M, Munoz-Caro T, Sanchez Contreras G, Rubio Garcia A, Magdowski G, G€ artner U, et al. Harbour seal (Phoca vitulina) PMN and monocytes release extracellular traps to capture the apicomplexan parasite Toxoplasma gondii. Dev Comp Immunol 2015;50:106e15. [27] Baker VS, Imade GE, Molta NB, Tawde P, Pam SD, Obadofin MO, et al. Cytokine-associated neutrophil extracellular traps and antinuclear antibodies in Plasmodium falciparum infected children under six years of age. Malar J 2008;7:41e64. [28] Behrendt JH, Ruiz A, Zahner H, Taubert A, Hermosilla C. Neutrophil extracellular trap formation as innate immune reactions against the apicomplexan parasite Eimeria bovis. Vet Immunol Immunopathol 2010;133:1e8. [29] Gabriel C, McMaster WR, Girard D, Descoteaux A. Leishmania donovani promastigotes evade the antimicrobial activity of neutrophil extracellular traps. J Immunol 2010;185:4319e27. [30] Avila EE, Salaiza N, Pulido J, Rodríguez MC, Díaz-Godínez C, Laclette JP, et al. Entamoeba histolytica trophozoites and lipopeptidophosphoglycan trigger human neutrophil extracellular traps. PLoS One 2016;11:e0158979. [31] Yildiz K, Gokpinar S, Gazyagci AN, Babur C, Sursal N, Azkur AK. Role of NETs in the difference in host susceptibility to Toxoplasma gondii between sheep and cattle. Vet Immunol Immunopathol 2017;189:1e10. [32] Polidori P, Vincenzetti S. Use of donkey milk in children with cow's milk protein allergy. Foods 2013;2:151e9. [33] Mancianti F, Nardoni S, Papini R, Mugnaini L, Martini M, Altomonte I, et al. Detection and genotyping of Toxoplasma gondii DNA in the blood and milk of naturally infected donkeys (Equus asinus). Parasit Vectors 2014;7:165. [34] Dubey JP, Ness SL, Kwok OCH, Choudhary S, Mittel LD, Divers TJ. Seropositivity of Toxoplasma gondii in domestic donkeys (Equus asinus) and isolation of T. gondii from farm cats. Vet Parasitol 2014;199:18e23. [35] Dubey JP, Desmonts G. Serological responses of equids fed Toxoplasma gondii oocysts. Equine Vet J 1987;19:337e9. [36] Balkaya I, Babur C, Celebi B, Utuk AE. Seroprevalence of toxoplasmosis in donkeys in Eastern Turkey. Isr J Vet Med 2011;66:39e42. [37] Yang N, Mu MY, Yuan GM, Zhang GX, Li HK, He JB. Seroprevalence of Toxoplasma gondii in slaughtered horses and donkeys in Liaoning province, northeastern China. Parasit Vectors 2013;6:140. [38] Alvarado-Esquivel C, Alvarado-Esquivel D, Dubey JP. Prevalence of Toxoplasma gondii antibodies in domestic donkeys (Equus asinus) in Durango, Mexico slaughtered for human consumption. BMC Vet Res 2015;11:6. [39] Barragan A, Sibley LD. Migration of Toxoplasma gondii across biological barriers. Trends Microbiol 2003;11:426e30. [40] Branzk N, Papayannopoulos V. Molecular mechanisms regulating NETosis in infection and disease. Semin Immunopathol 2013;35:513e30. [41] Manda A, Pruchniak MP, Ara zna M, Demkow UA. Neutrophil extracellular traps in physiology and pathology. Cent Eur J Immunol 2014;39:116e21. [42] Branzk N, Lubojemska A, Hardison SE, Wang Q, Gutierrez MG, Brown GD, et al. Neutrophils sense microbe size and selectively release neutrophil extracellular traps in response to large pathogens. Nat Immunol 2014;11:1017e25.
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[43] Dagleish MP, Brazil TJ, Scudamore CL. Potentiation of the extracellular release of equine neutrophil elastase and alpha-1-proteinase inhibitor by a combination of two bacterial cell wall components: fMLP and LPS. Equine Vet J 2003;35:35e9. [44] Mesa MA, Vasquez G. NETosis. Autoimmune Dis 2013;2013:651497. [45] Coombes JL, Charsar BA, Han SJ, Halkias J, Chan SW, Koshy AA, et al. Motile invaded neutrophils in the small intestine of Toxoplasma gondii-infected mice reveal a potential mechanism for parasite spread. Proc Natl Acad Sci U S A 2013;110:E1913e22. [46] Morisaki JH, Heuser JE, Sibley LD. Invasion of Toxoplasma gondii occurs by active penetration of the host cell. J Cell Sci 1995;108:2457e64. [47] Zhao Y, Marple AH, Ferguson DJP, Bzik DJ, Yapa GS. Avirulent strains of Toxoplasma gondii infect macrophages by active invasion from the phagosome. Proc Natl Acad Sci U S A 2014;111:6437e42. [48] Shapiro SD. Neutrophil elastase: path clearer, pathogen killer, or just pathologic? Am J Respir Cell Mol Biol 2002;26:266e8. [49] Belaaouaj A, McCarthy R, Baumann M, Gao Z, Ley TJ, Abraham SN, et al. Mice lacking neutrophil elastase reveal impaired host defense against gram negative bacterial sepsis. Nat Med 1998;4:615e8. [50] Weinrauch Y, Drujan D, Shapiro SD, Weiss J, Zychlinsky A. Neutrophil elastase targets virulence factors of enterobacteria. Nature 2002;417:91e4. [51] Weiss DJ, Wardrop KJ. Schalm's veterinary hematology. Iowa, USA: WileyBlackwell; 2010. [52] Tobler A, Koeffler HP. Myeloperoxidase: Localization, structure and function. In: Robin HJ, editor. Blood cell biochemistry, Volume 3 Lymphocytes and granulocytes. NewYork: Springer ScienceþBusiness Media; 1991. p. 255e88. [53] Franck T, Grulke S, Deby-Dupont G, Deby C, Duvivier H, Peters F, et al. Development of an enzyme-linked immunosorbent assay for specific equine neutrophil myeloperoxidase measurement in blood. J Vet Diagn Invest 2005;17:412e9. [54] Art T, Franck T, Gangl M, Votion D, Kohnen S, Deby-Dupont G, et al. Plasma concentrations of myeloperoxidase in endurance and 3-day event horses after a competition. Equine Vet J Suppl 2006;36:298e302. [55] Orsini JA, Divers TJ. Equine emergencies: treatment and procedures. Fourth Edition. St. Louis: Saunders Elsevier; 2014. [56] Fonseca RG, Kenny DA, McGivney BA, Murphy BA, Hill EW, Katz LM. Effect of training on plasma myeloperoxidase concentrations measured before and following intense exercise in thoroughbred racehorses. Comp Exerc Physiol 2016;12:17e25. [57] Kaplan MJ, Radic M, Herrmann M. NETosis 2: the Excitement Continues. Front Immunol 2017;8:1318. [58] Wang Y, Chen Y, Xin L, Beverley SM, Carlsen ED, Popov V, et al. Differential microbicidal effects of human histone proteins H2A and H2B on Leishmania promastigotes and amastigotes. Infect Immunol 2011;79:1124e33. [59] Munoz-Caro T, Lendner M, Daugschies A, Hermosilla C, Taubert A. NADPH oxidase, MPO, NE, ERK1/2, p38 MAPK and Ca2þ influx are essential for Cryptosporidium parvum-induced NET formation. Dev Comp Immunol 2015;52:245e54. [60] Guimaraes-Costa AB, Nascimento MT, Froment GS, Soares RP, Morgado FN, Conceicao-Silva F, et al. Leishmania amazonensis promastigotes induce and are killed by neutrophil extracellular traps. Proc Natl Acad Sci U S A 2009;106:6748e53. [61] Makni-Maalej K, Chiandotto M, Hurtado-Nedelec M, Bedouhene S, GougerotPocidalo MA, Dang PM, et al. Zymosan induces NADPH oxidase activation in human neutrophils by inducing the phosphorylation of p47phox and the activation of Rac2: Involvement of protein tyrosine kinases, PI3Kinase, PKC, ERK1/2 and p38MAPkinas. Biochem Pharmacol 2013;85:92e100.
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Original Research
Extracellular Trap Formation by Donkey Polymorphonuclear Neutrophils Against Toxoplasma gondii Kader Yildiz a, *, Sami Gokpinar a, Neslihan Sursal b, Cahit Babur c, Dogukan Ozen d, Ahmet Kursat Azkur e a
Department of Parasitology, Faculty of Veterinary Medicine, Kirikkale University, Kirikkale, Turkey Department of Parasitology, Ankara University Health Sciences Institute, Ankara, Turkey Ministry of Health, Public Health Institution of Turkey, Ankara, Turkey d Department of Biostatistic, Faculty of Veterinary Medicine, Ankara University, Ankara, Turkey e Department of Virology, Faculty of Veterinary Medicine, Kirikkale University, Kirikkale, Turkey b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 May 2018 Received in revised form 7 November 2018 Accepted 9 November 2018 Available online 15 November 2018
Toxoplasma (T.) gondii is an obligatory intracellular apicomplexan parasite. The donkey is one of the intermediate hosts of T. gondii. There are almost no data about T. gondii infection in donkeys, apart from a few seroprevalence reports. The aim of the study was to detect the formation of extracellular traps by donkey polymorphonuclear neutrophils (PMNs) when exposed to T. gondii tachyzoites in vitro. Tachyzoites were observed to be entrapped within extracellular traps (NET) structures. Myeloperoxidase, neutrophil elastase (NE), and histone (H3) were observed in NET structures. NE and reactive oxygen species activity increased with time and was higher in the PMN-tachyzoite cocultures. Some tachyzoites were seen close localization to the nucleus of neutrophils. Degranulation and extracellular traps were observed simultaneously in some donkey neutrophils after incubation for 1 hour. The tachyzoite invasion rate decreased in PMN-tachyzoite cocultures in comparison to the controls. NETs can negatively affect the viability of entrapped tachyzoites in addition to their action of physical entrapment. NE may play a key role in the viability of T. gondii in donkeys. The amount of extracellular DNA increased with incubation time in the PMN-tachyzoite cocultures (P < .001). This is a first report regarding the formation of donkeys NETs after exposure to T. gondii tachyzoites in vitro. Unlike sheep and cattle PMN, degranulation and extracellular traps developed in the same neutrophil. Further studies focusing on signaling pathways may shed light on what determines the behavior of donkey neutrophils after exposure to T. gondii. © 2018 Elsevier Inc. All rights reserved.
Keywords: Toxoplasma gondii Donkey Neutrophil extracellular traps Innate immunity In vitro
1. Introduction Toxoplasma (T.) gondii, an obligatory intracellular apicomplexan, is one of the most prevalent zoonotic parasites in the world [1]. Felidae members, including the domestic cat, are the definitive hosts [2]. The donkey is one of the intermediate hosts of T. gondii, alongside
Animal welfare/ethical statement: In this study, the blood samples (20 mL) were taken from vena jugularis of clinically healthy donkeys for PMN isolation. The animal owners were informed before the blood samples were taken, and the confirmation form was signed. All animal handling procedures were approved by the Kirikkale University Local Ethics Committee for Animal Experiments (Protocol no: 16/53). Conflict of interest statement: This study has received no financial support. * Corresponding author at: Kader Yildiz, Department of Parasitology, Faculty of Veterinary Medicine, Kirikkale University, Campus 71450, Kirikkale, Turkey. E-mail addresses: [email protected], [email protected] (K. Yildiz). https://doi.org/10.1016/j.jevs.2018.11.002 0737-0806/© 2018 Elsevier Inc. All rights reserved.
other well-known intermediate host species such as sheep, pigs, goats, and humans [2]. T. gondii possesses three infectious forms: sporozoite, tachyzoite, and bradyzoite [3]. After being excreted in cat feces, sporozoites develop into oocysts under suitable climatic conditions. Tachyzoites can infect all cells in an organism and proliferate intracellularly by endodyogeny [4]. During acute toxoplasmosis, tachyzoites disseminate to all organs of the body [3]. Later, cysts form in the cells of a variety of tissues including muscles, brain, and other parenchymatous organs in the intermediate hosts [2]. These cysts are filled with slow-growing bradyzoites and may remain asymptomatically in tissues for the duration of the host's life [2]. After oral infection with tissue cysts or sporulated oocysts of the parasite, bradyzoites or sporozoites are released and enter hosts' enterocytes [2]. Chemokines and cytotoxic molecules secreted by the infected cells attract immune cells to the infection area [5]. Neutrophils are the first cells to arrive in the infection area [6]. Neutrophils are
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involved in the cellular inflammatory response and possess a crucial role in innate immunity. The role of the neutrophil has previously been thought of as release of cytotoxic agents and phagocytosis during the inflammatory process [7]. Subsequently, it was discovered that neutrophils have another strategy for fighting infection [8], called NETosis [9]. During NETosis, some important nuclear and cytoplasmic changes develop in neutrophils [10]. When neutrophils encounter pathogens, reactive oxygen species (ROS) are produced [11]. The lobular form of the neutrophil nucleus disappears. Nuclear, cytoplasmic, and granular contents become mixed with one another [7]. Finally, extracellular traps (NETs), augmented with enzymes originating from granules (such as neutrophil elastase [NE] and myeloperoxidase [MPO], and histones), are excreted from neutrophils into extracellular spaces [11e13]. NET formation has been reported in many different species [14e22] including donkeys [23]. Extracellular traps form not only from neutrophils but also from other granulocytic cells including eosinophils [24] and monocytes [11,25,26]. Extracellular trap structures are known to develop in response to some parasitic protozoon species [18,27e30] including T. gondii [17,26,31]. T. gondii tachyzoites trigger the formation of extracellular trap structures from neutrophils in murine, humans [17], sheep, and cattle [31] and from polymorphonuclear neutrophils (PMNs) and monocytes in harbor seals [26]. NETs also have negative effects on the viability of entrapped tachyzoites in addition to the physical entrapment, which prevents invasion of host cells by tachyzoites [17,31]. Infection rates reduced significantly when T. gondii tachyzoites were exposed to PMNs and monocytes from harbor seals [26]. The donkey has become more popular with the increasing importance of its milk for children with allergies to cow's milk [32]. Some researchers have focused on the potential risk of jenny milk for foodborne toxoplasmosis [33]. As a member of the Equidae family, donkeys are generally accepted to be biologically similar to horses [34]. Although clinical toxoplasmosis has not been confirmed in horses, specific antibodies develop between 6 and 12 days after experimental infection with T. gondii [35]. There are almost no data about T. gondii infection in donkeys, apart from a few seroprevalence reports [36e38]. Toxoplasma gondii has the ability to infect a broad range of animal species [2]. Some hosts, such as the donkey, can be infected with the parasite but do not play an important role in parasite epidemiology. One of the reasons for this may be innate immunity. Following infection with T. gondii, the parasites are first exposed to innate immune cells, especially neutrophils. The main aim of the study was to detect the formation of extracellular traps in donkey PMNs when exposed to T. gondii tachyzoites in vitro. Activities of MPO and NE were measured during the incubation period in the donkey PMN-tachyzoite coculture. The effects of different parasite concentrations and incubation periods on NET development in donkey PMNs and the effect of NET structures on host cell invasion by T. gondii were studied. 2. Materials and Methods 2.1. Toxoplasma gondii Tachyzoites Toxoplasma gondii RH strain tachyzoites were passaged intraperitoneally in Swiss albino mice. The tachyzoites were obtained from the peritoneal fluid 48 hours after the experimental infection of mice. The fluid was centrifuged at room temperature (200 g for 10 minutes). The supernatant was centrifuged (1,000 g for 10 minutes), and the pellet was washed with a sterile saline solution (pH 7.2). After staining with Diff-Quick stain (Bio Optica, Italy), murine inflammatory cells were evaluated by light microscopy (Leica DM750), the pellet did not include any murine inflammatory
cells. Tachyzoites were counted using a Neubauer chamber with a light microscope and then resuspended in RPMI 1640. 2.2. PMN Isolation From Donkey Blood All animal handling procedures were approved by the Kirikkale University Local Ethics Committee for Animal Experiments (Protocol no: 16/53). This study was carried out according to Munoz Caro et al. [18], with some modifications. The blood samples (20 mL) were collected from clinically healthy donkeys (n ¼ 3) into heparinized tubes. The samples were layered into sterile tubes with Biocoll Separating Solution (Biochrom) after mixing with equal volumes of sterile 0.02% PBS-EDTA. The plasma, lymphocytes, and monocytes were removed after centrifugation (800 g, with no brake, 45 minutes, 22 C; Thermo Scientific 16S). Erythrocytes were lysed by addition of ultrapure sterile water (25 mL); 30 seconds later, osmolarity was adjusted by addition of 3 mL sterile HBSS (10; Sigma). Polymorphonuclear neutrophils were centrifuged with RPMI 1640 (without phenol red; Sigma) (400 g, 10 minutes, 4 C). After staining with Diff-Quick stain (Bio Optica, Italy), neutrophil purity was evaluated by light microscopy (Leica DM750) and was higher than 90%. Polymorphonuclear neutrophil viability was determined using the trypan blue dye test and was higher than 95%. Polymorphonuclear neutrophils were counted using a Neubauer chamber and then reconstituted as 1 105/100 mL in RPMI 1640. 2.3. Fluorescence Microscope Examination of NET Structures Against T. gondii Just before starting the experiment, to facilitate their observation within NETs, tachyzoites were stained with CellTrace CFSE Cell Proliferation Kit (5 mM; Thermo Fisher Scientific) according to the manufacturer's instructions. Briefly, CFSE and tachyzoites were incubated (37 C, 10 minutes). Then, ice-cold PBS (containing 10% FCS) was added. CFSE-stained tachyzoites were protected from light until the start of the experiment after centrifugation for three times (10 minutes, 400 g). Donkey PMNs and CFSE-stained tachyzoites (1:3) were placed on poly-L-lysine-coated coverslips (Corning, BioCoat) and incubated for 1 hour (37 C, 5% CO2). After fixing with paraformaldehyde (4%) and treatment with Triton X-100 (0.1%), the cells were blocked with 1% BSA. To detect MPO, NE, and histone (H3) in NET structures, the samples were incubated with anti-MPO (1:1000, sc-52707, Santa Cruz), anti-NE (1:1000, sc-55548, Santa Cruz), and antihistone (H3) monoclonal antibodies (1:1000, sc-374669; Santa Cruz; 1 hour, 37 C, 5% CO2). After three washes, secondary antibodies (IgG1-FITC, 1:100, sc-358546; Santa Cruz for MPO and NE, and IgG2b-FITC, 1:100, ABIN 637988; antibodies.online for histone) were added for 1 hour (37 C, 5% CO2). Then, the cocultures were stained with SYTOX orange dye (Invitrogen) at room temperature (1:30,000, 5 minutes, in dark). The coverslips were placed gently onto microscope slides using a drop of anti-fading buffer (Mowiol). The slides were examined with the fluorescence microscope (Leica DM IL Led, 470 nm excitation/515 nm emission). Composite images were generated using Image J (version 2.0.0-rc-43/1.50 g). The isotype controls, mouse IgG1 (1:1000, sc-3877; Santa Cruz), were used to confirm the MPO and NE signals, and mouse IgG2b (1:1000, sc-3879; Santa Cruz) for histone (H3) signals. 2.4. Detection of NE, ROS, and MPO Activities in PMN-Tachyzoite Cocultures Tachyzoite and donkey PMN (1:1) cocultures were placed into the 96 wells of a flat bottom immunoplate (Nunc, Sigma-Aldrich). To detect NE and ROS activities, respectively, MeOSuc-Ala-Ala-
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Pro-Val-chloromethyl-ketone (the chromogenic substrate, 3 mg/ mL; Sigma-Aldrich) and 20 ,70 -dichlorofluorescin diacetate (DCFHDA, 10 mg/mL; Sigma Aldrich) were added into the wells. To detect MPO activity, Amplex red hydrogen peroxide/peroxidase assay kit (Thermo Fisher) was used according to manufacturer's protocol. The immunoplate was incubated in the fluorometer, and NE, MPO, and ROS activities were measured every 10 minutes during the incubation period (37 C, 150 minutes) (485 nm excitation/538 nm emission). In the experiments, phorbol 12-myristate 13-acetate (PMA)einduced PMNs and the samples containing untreated PMNs were used as the positive and negative controls, respectively.
2.5. Host Cell Invasion Assay During host-cell invasion assays, three groups were classified as follows: Donkey PMNs and tachyzoite cocultures (1:2) were incubated for 3 hours (37 C, 5% CO2). The cocultures were treated with deoxyribonuclease I (DNase I, 90 U; Sigma) added 15 minutes before the end of the incubation period. The cocultures were seeded onto a confluent
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monolayer of Vero cells in a 6-well plate (group 1). Donkey PMNs and tachyzoite cocultures (1:2) were incubated for 3 hours (37 C, 5% CO2). The cocultures were seeded onto a confluent monolayer of Vero cells in a 6-well plate (without treatment with DNase I) (group 2). As a control, equal numbers of tachyzoites were seeded onto a confluent monolayer of the Vero cells in a 6-well plate (group 3). After 2 hours of incubation, the plates were washed twice with prewarmed Dulbecco's Modified Eagle Medium to remove extracellular tachyzoites and PMNs. The plates were incubated for 22 hours under the same conditions (37 C, 5% CO2), the percentage of infected cells was determined quantitatively in 10 randomly selected microscopic areas (Olympus CKX41 inverted microscope). The percentage of the infected cells was accepted as 100% in the control group (group 3). The assay was performed at least three independent times. The arithmetic means of the results were calculated.
2.6. Quantitative Analysis of NETs Against T. gondii Donkey PMN-tachyzoite cultures (1:1 and 1:3) were incubated for four different incubation times (30, 60, 120, and 180 minutes;
Fig. 1. Fluorescence micrographs of extracellular trap (NET) structures triggered by Toxoplasma gondii tachyzoites. Cocultures of donkey polymorphonuclear neutrophils (PMNs) and tachyzoites stained with CellTrace CFSE Cell Proliferation Kit (5 mM; Thermo Fisher Scientific) (1:3) were stained with SYTOX orange dye for extracellular DNA backbone (A, D, G) and probed for histones (B), myeloperoxidase (MPO) (E), and neutrophil elastase (NE) (H) using anti-histone, anti-MPO, and anti-NE antibody, respectively. Composite images (C, F, I) were generated using Image J (version 2.0.0-rc-43/1.50 g). Tachyzoites to be entrapped in NET structures (arrows in the composite images) (Bar: 20 mm).
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37 C, 5% CO2). Micrococcal nuclease (5 U, NEB) was added, and then samples were incubated for 15 minutes under same conditions. The supernatants were transferred into 96-well flat bottom immunoplates after centrifugation (300 g, 5 minutes), each sample was tested in duplicate. SYTOX green dye (5 mM, Invitrogen) was added into the wells and incubated in the dark (15 minutes). Fluorescence levels were measured with a fluorometer (Fluoroskan Ascent; Thermo Scientific, 485 nm excitation/538 nm emission). Zymosan(1 mg/mL; Sigma Aldrich) and PMA- (50 nM; Sigma Aldrich) induced PMNs were used as positive controls and untreated PMNs as negative controls. Diphenyleneiodonium (DPI), a potent NADPH oxidase inhibitor, was used as an inhibitor in the experiment (10 mM; Sigma Aldrich). The assay was performed at least three independent times.
extracellular traps were observed simultaneously in some donkey neutrophils after 1-hour incubation. Some tachyzoites were also observed in PMNs that developed extracellular traps (Fig. 2). Light microscopic observation of donkey PMNs after being stained with Diff-Quick stain is submitted as Supplemental Fig. 1. Fluorescence micrographs of isotype controls for MPO, NE, and histone (H3) immunostainings are submitted as Supplemental File 2. 3.2. Detection of NE, ROS, and MPO Activities in PMN-Tachyzoite Cocultures
The data were analyzed using SPSS 14.01 (SPSS Inc, Chicago, IL, USA). Dose-dependent analyses were evaluated with one-way variance analysis and a Tukey post hoc test. Normality of data and homogeneity of variance were analyzed by the Shapiro Wilk test and Levene test, respectively. Time-dependent analyses of different parasite concentrations were evaluated with the General Linear Model for Repeated Measure. The terms of “dose (betweensubject factor),” “time (within-subject factor),” and “dose*time interaction” were used in the model. The Huynh-Feldt correction was used for sphericity. The Bonferroni test was used for simple effect analysis. Differences were considered significant at P < .05.
Neutrophil elastase activity was higher in the PMN-tachyzoite cocultures than in the positive controls in the experiments. This activity increased rapidly up to the 100th minute of incubation in all groups, after this time the activity slowed down and even began to decrease in the control groups (P < .001) (Fig. 3). Quantitative analysis revealed that ROS activity increased in a time-dependent fashion and was higher in tachyzoite-PMN cocultures than in control groups during the incubation periods (P < .001) (Fig. 4). Myeloperoxidase activity increased over the standard curve in PMN-tachyzoite cocultures during all incubation periods (Fig. 5). Surprisingly, MPO activity was higher in all groups in the experiments. In PMN-tachyzoite cocultures, MPO activity increases and peaked at 80 minutes, and then it decreased slowly until the end of the incubation. Similar results were recorded in the positive and negative controls (P < .04).
3. Results
3.3. Host Cell Invasion Assay
3.1. Fluorescence Microscopic Examination of Effect of NET Structures Against T. gondii
In this experiment, the invasion rate by tachyzoites in host cells not incubated with PMNs was set as 100% (the control group). The tachyzoite invasion rate decreased by 75% in the untreated groups when compared with the controls. Although the invasion rate increased in the DNase I treatment groups (53%), it was nearly half that of the control groups (Fig. 6).
2.7. Statistical Analysis
Extracellular trap structures were visualized in tachyzoitesPMN cocultures (1:3) after incubation for 1 hour. Toxoplasma gondii tachyzoites stained with CFSE were seen to be entrapped within cloud-like extracellular trap structures (Fig. 1). The nuclei of some PMNs had almost disappeared. Extracellular DNA, the backbone of the NET structures, was observed after staining with SYTOX orange dye (Figs. 1A, 1D, and 1G). Histone (H3), MPO, and NE were observed in NETs after staining with primary and secondary antibodies (Figs. 1B, 1E, and 1H). NETs developed in donkey PMNs after 1-hour incubation with tachyzoites. Some tachyzoites were seen to be located near the nucleus of neutrophils. Degranulation and
3.4. Quantitative Analysis of NETs Efficacy Against T. gondii According to dose-dependent studies, the fluorescence intensities of extracellular DNA contents were increased in parallel with tachyzoite concentrations (P < .001) (Fig. 7). Quantities of T. gondiieinduced extracellular DNA were significantly increased in donkey PMNs compared with the negative controls (P < .001). The amount of
Fig. 2. Various functions of donkey neutrophils against Toxoplasma gondii tachyzoites. Cocultures of donkey polymorphonuclear neutrophils (PMNs) and tachyzoites stained with CellTrace CFSE Cell Proliferation Kit (5 mM; Thermo Fisher Scientific) (1:3) were probed for neutrophil elastase (NE) (A), myeloperoxidase (MPO) (B), and histones (C) using antihistone, anti-MPO, and anti-NE antibody, respectively. CFSE-stained tachyzoites were in crescent shape seen to be located near the nucleus of neutrophils (stars) (A). Some tachyzoites were seen to be located close to the nucleus of neutrophils that developed extracellular traps (arrow) (B). Degranulation and extracellular traps were observed simultaneously in some donkey neutrophils after 1-hour incubation (arrowhead) (C). The structure of the nucleus disappeared in these neutrophils during degranulation and NETosis. Granules were in spherical forms around neutrophils (Bar: 15 mm).
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Fig. 3. Neutrophil elastase (NE) activity in polymorphonuclear neutrophils (PMNs)-tachyzoite cocultures. Tachyzoite and donkey PMN (1:1) cocultures were placed into the wells. MeOSuc-Ala-Ala-Pro-Val-chloromethyl-ketone (3 mg/mL; Sigma-Aldrich) was added into the wells for detection of NE activity. Neutrophil elastase activities were measured every 10 minutes during the incubation period (37 C, 150 minutes) (485 nm excitation/538 nm emission). Phorbol 12-myristate 13-acetate (PMA)einduced PMNs and PMNs alone were used as the positive and negative controls, respectively. The assay was performed at least three independent times with the same results (P < .001) (arbitrary unit [AU]).
extracellular DNA in PMN-tachyzoite cocultures significantly increased with incubation time (P < .001). Phorbol 12-myristate 13acetate (50 nM) and Zymosan (1 mg/mL) were good inducers of extracellular traps for donkey PMNs. NET reactions were inhibited in the groups where DPI (10 mM; Sigma Aldrich) was added.
4. Discussion Toxoplasma gondii has a wide intermediate host range, and it can infect nearly all cell types of its hosts [2]. Following oral infection, infectious forms of T. gondii disseminate through the bloodstream
Fig. 4. Reactive oxygen species (ROS) activity in polymorphonuclear neutrophil (PMN)-tachyzoite cocultures. Tachyzoite and donkey PMN (1:1) cocultures were placed into the wells. 20 ,70 -Dichlorofluorescin diacetate (DCFH-DA) (10 mg/mL; Sigma Aldrich) was added into the wells for detection of ROS activity. ROS activities were measured every 10 minutes during the incubation period (37 C, 150 minutes) (485 nm excitation/538 nm emission). Phorbol 12-myristate 13-acetate (PMA)einduced PMNs and PMNs alone were used as the positive and negative controls, respectively. The assay was performed at least three independent times with the same results (P < .001) (arbitrary unit [AU]).
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Fig. 5. Detection of myeloperoxidase (MPO) activity in polymorphonuclear neutrophils (PMNs)-tachyzoite cocultures. Tachyzoite and donkey PMN (1:1) cocultures were placed into the wells. Amplex red hydrogen peroxide/peroxidase assay kit (Thermo Fisher) was used according to manufacturer's instructions for detection of MPO activity. A standard curve was prepared with horseradish peroxidase (HRP) and 1 reaction buffer (1xRB) supplied by the kit. MPO activities were measured every 10 minutes during the incubation period (37 C, 150 minutes) (485 nm excitation/538 nm emission). Phorbol 12-myristate 13-acetate (PMA)einduced PMNs and PMNs alone were used as the positive and negative controls, respectively. The assay was performed at least three independent times with the same results (P < .04) (arbitrary unit [AU]).
and cause inflammation [39]. The inflammatory reaction is important for innate immunity. As one of the most important components of innate immunity, neutrophils are the first to reach affected areas [40] and fight pathogens by a variety of mechanisms including NETosis [41], where DNA augmented with granular
Fig. 6. Host cell invasion assay. Donkey polymorphonuclear neutrophils (PMNs) and tachyzoite cocultures (1:2) were incubated 3 hours. The cocultures were seeded in Vero cells after treated with deoxyribonuclease I (DNase I) (90 U; Sigma) 15 minutes before the end of the incubation period (group 1). Donkey PMN and tachyzoite cocultures (1:2) were incubated for 3 hours (37 C, 5% CO2). The cocultures were seeded onto a confluent monolayer of Vero cells in a 6-well plate (without treatment with DNase I) (group 2). As a control, equal numbers of tachyzoites were seeded onto a confluent monolayer of the Vero cells in a 6-well plate (group 3). After 2 hours of incubation, the plates were washed twice to remove extracellular tachyzoites and PMNs. The plates were incubated for 22 hours under the same conditions, and the percentage of infected cells was determined quantitatively in 10 randomly selected microscopic areas. The percentage of the infected cells was accepted as 100% in the control group. The assay was performed at least three independent times. The graphic shows the arithmetic means of three experiments.
contents is released extracellularly from neutrophils after exposure to a pathogen [7]. Studies [14e22] suggested that extracellular trap structures are from PMNs of many different animal species. There is only one report on NETosis from donkey neutrophils, in which extracellular trap structures developed against donkey spermatozoa in vitro [23]. Neutrophils possess three defense strategies: phagocytosis, degranulation, and NETosis [6]. There is no clear information on which defense strategy is used when they are exposed to pathogens [11]. Some authors suggested that neutrophils used defense strategies in a consecutive order after they are exposed to a pathogen [11]. Neutrophils first try to internalize pathogens by phagocytosis for a few minutes. After about 10 minutes, secretory vesicles secrete their substances into the extracellular space, followed by secretions from specific and azurophilic granules. Eventually, extracellular traps develop against pathogens [11]. This hypothesis has not been widely accepted because pathogens internalized in the phagosome will be released again into the extracellular space if the same neutrophil develops extracellular trap structures after phagocytosis of pathogens. Others suggest that microbial size is a critical factor in regulation of NETosis and NETs are released in response to larger pathogens [42]. Others have claimed that there are subsets of neutrophils, which possess different fighting capabilities, some of them being responsible for phagocytosis and others for NETosis [41]. This view is supported by some observations that suggest that extracellular traps develop from only some neutrophils in vitro [41]. In the present study, the extracellular traps developed in donkey PMN-tachyzoite cocultures at the end of the 1-hour incubation period. Moreover, some tachyzoites were observed on close localization to the nucleus of neutrophils. It was thought that these tachyzoites had entered donkey neutrophils during the incubation period. However, this observation has not previously been reported in sheep and cattle PMNeT. gondii tachyzoite cocultures [31].
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Fig. 7. Quantitative dose and time studies. Donkey polymorphonuclear neutrophils (PMNs)-tachyzoite cultures (1:1 and 1:3) were incubated for 30, 60, 120, and 180 minutes (37 C, 5% CO2). Then, SYTOX green dye (5 mM, Invitrogen) was added, and fluorescence levels were measured with a fluorometer (485 nm excitation/538 nm emission). Phorbol 12myristate 13-acetate (PMA)einduced and Zymosan-induced PMNs were used as positive controls, whereas PMNs alone were used as negative controls. Diphenyleneiodonium (DPI) was used as an inhibitor in the experiments. The assay was performed at least three independent times with the same results (dose P < .001, time P < .001, dose*time P < .001) (arbitrary unit [AU]).
Equine neutrophils possess different chemotactic and secretory stimuli responses from other animal species [43]. Donkey PMNs can possess varied subtypes, which have different fighting strategies against pathogens. It is well known that neutrophils are shortlived immune system cells with a life span of only 6e8 hours if there is no inflammation [44]. When neutrophils are exposed to pathogens, there is insufficient time for subtypes to develop; for this reason, they may be released into the blood stream as separate subtypes in the donkey. However, degranulation and extracellular traps were observed simultaneously in some donkey neutrophils after incubation for 1 hour in the present study. Toxoplasma gondii can invade and replicate in all nucleated cells, including immune system cells [3]. Neutrophils may contain T. gondii as a result of phagocytosis in addition to active invasion by the parasites [45]. Active invasion develops rapidly, and T. gondii penetrates into the host cell by the formation of a parasitophorous vacuole (PV) [46]. The identification of PVs is important in the discrimination of the route of entrance of tachyzoites into host cells, especially in phagocytic cells to discriminate active entry from phagocytosis [45]. The parasites survive in the PV, which avoids lysosome fusion and acidification in the host cell after invasion [46]. Neutrophils may therefore play a role in parasite dissemination within the host and being involved in fighting infection [45]. Neutrophils are actively invaded and harbor live tachyzoites in the small intestine of mice after oral infection [45]. In the present study, tachyzoites were located near the nucleus of some donkey neutrophils. The entry route for tachyzoites into neutrophils could not be established. Tachyzoites used in our experiment were part of the T. gondii RH strain known as a virulent strain. Hypervirulent T. gondii strains are known to enter cells using active penetration [47]. Neutrophils possess many enzymes and proteins in their granules [10,44]. These granular substances have antimicrobial effects on pathogens [10]. Neutrophil elastase is abundant in the azurophilic granules and is involved in oxygen-independent
microbicidal mechanism [48]. Neutrophil elastase may have a crucial role in the destruction of microorganisms via different routes such as microbicidal peptide activation and outer wall protein proteolysis [49,50]. In this study, NE concentrations increased rapidly up to the 100th minute of incubation, and then the activity slowed in all groups. Neutrophil elastase concentrations are higher in PMN-tachyzoite cocultures than those of positive controls. Neutrophil elastase can have some lethal effects on T. gondii tachyzoites in vitro experiments after 3 hours incubation in the present study. Myeloperoxidase originating from azurophilic granules is one of the most potent in vivo oxidant agents [51]. Myeloperoxidase catalyzes halide oxidation by hydrogen peroxide [52]. Myeloperoxidase participates actively in the destruction of microorganism within the phagocytic vacuole of neutrophils. Also, during NETosis, MPO is released from neutrophils to fight against microorganisms [51]. Neutrophil activation is generally associated with an inflammatory response in equine medicine [53]. In the horse, increased MPO concentrations are considered a marker of inflammatory pathologies such as laminitis and recurrent airway obstruction [54], which are common problems in horses [55]. Myeloperoxidase from stimulated neutrophils can lead to tissue damage and plays an important role in inflammation in the horses [53,54]. Increased MPO concentrations due to neutrophil degranulation have also been shown in plasma of horses, even following exercise [54,56]. Unlike horses, no data were found on MPO activity in donkeys. In our study, MPO activity increased over the standard curve during all incubation periods in PMN-tachyzoite cocultures. Surprisingly, MPO activity was lower in the PMN-tachyzoite cocultures in all experiments. In the host cell invasion assay, the tachyzoite invasion rate decreased by 75% in the untreated groups in comparison to the control. Tachyzoites entrapped by extracellular traps can be one of the factors contributing to this low invasion rate. NETs can be
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destroyed by DNase activity [7]. In the present study, the cell invasion rate increased after destroying extracellular traps with DNase I, but it was still almost half that of the invasion rate in the control group. Granular enzymes and proteins released from donkey neutrophils may have a lethal effect on tachyzoites in vitro. Notably, NE concentration was higher in PMN-tachyzoite cocultures in this study (P < .001). Besides granular enzymes, extracellular histones released from neutrophils also play an important role in destruction of pathogens [57]. Histones, basic proteins, located in the neutrophil nucleus, are toxic to pathogens in the extracellular spaces [57]. In the present study, it is possible that histones had detrimental effects on tachyzoites. Histones are known to have harmful effects on promastigotes of Leishmania spp. [58]. Also, some tachyzoites were observed inside donkey neutrophils in addition to those entrapped within extracellular traps. These tachyzoites might have been removed from the cell culture together with neutrophils during washing procedures in the invasion assay. This may help explain the low invasion rate of both the untreated and the DNase I treatment groups. Extracellular trap structures are triggered by some parasitic protozoa [18,27e30]. Toxoplasma gondii tachyzoites are extracellular trap inducers for mouse and human PMNs [17], harbor seal PMNs and monocytes [26], and sheep and cattle PMNs [31]. Extracellular traps are formed a time-dependent manner in bovine and caprine PMNs against parasites such as Eimeria bovis, Cryptosporidium parvum, and Eimeria arloingi sporozoites ([19,28,59], respectively). However, no such time-dependent development was seen in cattle PMNeBesnoitia besnoiti tachyzoite cocultures [18]. Formation of extracellular traps increases in a time-dependent manner in relation to incubation time in mouse [17], sheep, and cattle PMNs [31] against T. gondii tachyzoites. In the present study, NET production from donkey PMNs was seen after exposure to T. gondii tachyzoites in vitro. The amount of extracellular DNA increased significantly in a time-dependent fashion in the donkey PMNs (P < .001). Parasite concentrations are an important factor for the induction of extracellular traps in vitro [18,28,30,60]. NETs are induced by Leishmania promastigotes and Entamoeba histolytica in dosedependent manner [30,60]. NETs released from bovine PMNs are dependent on the number of B. besnoiti tachyzoites and E. bovis sporozoites [18,28]. Toxoplasma gondiieinduced NETs are also dose dependent in harbor seal PMNs and monocytes [26] and sheep and cattle PMNs [31]. In the present study, a parasite concentrationdependent relationship was detected in donkey PMNs after encounters with T. gondii tachyzoite in vitro (P < .001). Phorbol 12-myristate 13-acetate is used for in vitro studies as a potential inducer of neutrophils from mice and humans, sheep, and cattle [17,31]. Zymosan are used as a potential inducer of neutrophils from different animals and human in vitro experiments [18,19,61]. In the present study, PMA and Zymosan were used as positive controls, and they were better extracellular trap inducers for donkey PMNs than T. gondii tachyzoites. In previous studies, PMA has been shown to be a worse NETosis inducer in human, sheep, and cattle PMNs than T. gondii tachyzoites [17,31]. 5. Conclusion To the authors' knowledge, this is the first report regarding the formation of extracellular traps in donkey PMNs when exposed to T. gondii tachyzoites in vitro. Some tachyzoites were seen to be located close to the nucleus of neutrophils. Degranulation and extracellular traps were observed simultaneously in some donkey neutrophils after incubation for 1 hour. Toxoplasma gondiieinduced extracellular DNA contents increased with increasing tachyzoite concentrations and incubation time. The host cell invasion rate
decreased in tachyzoites after exposure to donkey PMNs. NETs can negatively affect the viability of entrapped tachyzoites in addition to their role in physical entrapment. Our results suggest that NE may play a role in the viability of T. gondii in donkeys. Further studies focusing on signaling pathways may shed light on what determines the behavior of donkey neutrophils after exposure to T. gondii. Acknowledgments Portion of data was presented at the fifth European Immunology & Innate Immunity, Berlin, Germany, July 21e23, 2016. Supplementary Data Supplementary data related to this article can be found at https://doi.org/10.1016/j.jevs.2018.11.002. References [1] Tenter AM, Heckeroth AR, Weiss LM. Toxoplasma gondii: from animals to humans. Int J Parasitol 2000;30:1217e58. [2] Dubey JP. Toxoplasmosis of animals and humans. Second Edition. Boca Raton: CRC Press; 2010. [3] Weiss LM, Kim K. Toxoplasma gondii. The model apicomplexan: Perspective and methods. San Diego: Academic Press; 2007. [4] Hill DE, Chirukandoth S, Dubey JP. Biology and epidemiology of Toxoplasma gondii in man and animals. Anim Health Res Rev 2005;6:41e61. [5] Mennechet FJ, Kasper LH, Rachinel N, Li W, Vandewalle A, Buzoni-Gatel D. Lamina propria CD4þ T lymphocytes synergize with murine intestinal epithelial cells to enhance proinflammatory response against an intracellular pathogen. J Immunol 2002;168:2988e96. [6] Kumar V, Sharma A. Neutrophils: cindirella of innate immune system. Int Immunopharmacol 2010;10:1325e34. [7] Papayannopoulos V, Zychlinsky A. NETs: a new strategy for using old weapons. Trends Immunol 2009;30:513e21. [8] Brinkmann V, Reichard U, Goosmann C, Fauler B, Uhlemann Y, Weiss DS, et al. Neutrophil extracellular traps kill bacteria. Science 2004;303:1532e5. [9] Steinberg BE, Grinstein S. Unconventional roles of the NADPH oxidase: signaling, ion homeostasis, and cell death. Sci STKE 2007;379:pe11. [10] Guimaraes-Costa AB, Nascimento MT, Wardini AB, Pinto-da-Silva LH, Saraiva EM. ETosis: A microbicidal mechanism beyond cell death. J Parasitol Res 2012;2012:929743. [11] Kaplan MJ, Radic M. Neutrophil extracellular traps: double-edged swords of innate immunity. J Immunol 2012;189:2689e95. [12] Fuchs TA, Abed U, Goosmann C, Hurwitz R, Schulze I, Wahn V, et al. Novel cell death program leads to neutrophil extracellular traps. J Cell Biol 2007;176: 231e41. [13] Papayannopoulos V, Metzler KD, Hakkim A, Zychlinsky A. Neutrophil elastase and myeloperoxidase regulate the formation of neutrophil extracellular traps. J Cell Biol 2010;191:677e91. [14] Palic D, Ostojic J, Andreasen CB, Roth JA. Fish cast NETs: neutrophil extracellular traps are released from fish neutrophils. Dev Comp Immunol 2007;31: 805e16. [15] Chuammitri P, Ostojic J, Andreasen CB, Redmond SB, Lamont SJ, Palic D. Chicken heterophil extracellular traps (HETs): novel defense mechanism of chicken heterophils. Vet Immunol Immunopathol 2009;129:126e31. [16] Wardini AB, Guimaraes-Costa AB, Nascimento MT, Nadaes NR, Danelli MG, Mazur C, et al. Characterization of neutrophil extracellular traps in cats naturally infected with feline leukemia virus. J Gen Virol 2010;91:259e64. [17] Abi Abdallah DS, Lin C, Ball CJ, King MR, Duhamel GE, Denkers EY. Toxoplasma gondii triggers release of human and mouse neutrophil extracellular traps. Infect Immun 2011;80:768e77. [18] Munoz Caro T, Hermosilla C, Silva LMR, Cortes H, Taubert A. Neutrophil extracellular traps as innate immune reaction against the emerging apicomplexan parasite Besnoitia besnoiti. PLoS One 2014;9:e91415. [19] Silva LMR, Munoz Caro T, Gerstberger R, Vila-Vicosa MJM, Cortes HCE, Hermosilla C, et al. The apicomplexan parasite Eimeria arloingi induces caprine neutrophil extracellular traps. Parasitol Res 2014;113:2797e807. [20] Jeffery U, Kimura K, Gray R, Lueth P, Bellaire B, LeVine D. Dogs cast NETs too: Canine neutrophil extracellular traps in health and immune-mediated hemolytic anemia. Vet Immunol Immunopathol 2015;168:262e8. [21] Wei Z, Hermosilla C, Taubert A, He X, Wang X, Gong P, et al. Canine neutrophil extracellular traps release induced by the apicomplexan parasite Neospora caninum in vitro. Front Immunol 2016;7:436. [22] Koiwai K, Alenton RR, Kondo H, Hirono I. Extracellular trap formation in kuruma shrimp (Marsupenaeus japonicus) hemocytes is coupled with c-type lysozyme. Fish Shellfish Immunol 2016;52:206e9.
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