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Journal Pre-proof Toxoplasma gondii induces extracellular traps release in cat neutrophils Luciana Carvalho Lacerda, Jane Lima dos Santos, Amanda Brito Wardini, Aisla Nascimento da Silva, Andréa Gonçalves Santos, Herbert Pina Silva Freire, Danielle Oliveira dos Anjos, Carla Cristina Romano, Érica Araújo Mendes, Alexandre Dias Munhoz PII:
S0014-4894(19)30108-0
DOI:
https://doi.org/10.1016/j.exppara.2019.107770
Reference:
YEXPR 107770
To appear in:
Experimental Parasitology
Received Date: 8 March 2019 Revised Date:
3 September 2019
Accepted Date: 30 September 2019
Please cite this article as: Lacerda, L.C., dos Santos, J.L., Wardini, A.B., da Silva, A.N., Santos, André.Gonç., Silva Freire, H.P., dos Anjos, D.O., Romano, C.C., Mendes, É.Araú., Munhoz, A.D., Toxoplasma gondii induces extracellular traps release in cat neutrophils, Experimental Parasitology (2019), doi: https://doi.org/10.1016/j.exppara.2019.107770. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Inc.
Toxoplasma gondii induces extracellular traps release in cat neutrophils
1 2 3 4 5 6 7 8 9 10
Luciana Carvalho Lacerda1, Jane Lima dos Santos2, Amanda Brito Wardini3, Aisla Nascimento da
11
Silva1, Andréa Gonçalves Santos2, Herbert Pina Silva Freire4, Danielle Oliveira dos Anjos2, Carla
12
Cristina Romano4, Érica Araújo Mendes5, Alexandre Dias Munhoz1*
13 14 15 16
1
17
BA, Brasil,
18
2
19
Santa Cruz, Ilhéus, BA, Brasil,
20
3
Médica Veterinária da Clínica Veterinária Associação Bichos Gerais, Belo Horizonte, MG, Brasil
21
4
Laboratório de Imunologia, Departamento de Ciências Biológicas, Universidade Estadual de Santa
22
Cruz, Ilhéus, BA, Brasil,
23
5
24
São Paulo, SP, Brasil.
25
* Corresponding author: A.D. Munhoz. E-mail: [email protected]
26 27 28 29 30 31 32 33 34 35
Departamento de Ciências Agrárias e Ambientais, Universidade Estadual de Santa Cruz, Ilhéus,
Laboratório de Imunobiologia, Departamento de Ciências Biológicas, Universidade Estadual de
Lab. de Virologia Clínica e Molecular, Departamento de Virologia, Universidade de São Paulo,
36
Abstract
37
Neutrophils respond differently to violations of the body's physiological barriers during infections.
38
Extracellular traps comprise one of the mechanisms used by these cells to reduce the spread of
39
pathogens to neighboring tissues, as well as ensure a high concentration of antimicrobial agents at
40
the site of infection. To date, this innate defense mechanism has not been previously demonstrated
41
in neutrophils of cats exposed to Toxoplasma gondii. The aim of this study was to characterize the
42
in vitro release of neutrophil extracellular traps (NETs) when neutrophils isolated from cats were
43
exposed to T. gondii. First, cellular viability was tested at different time points after parasite
44
exposure. The production of reactive oxygen species (ROS) and lactate dehydrogenase and the
45
amount of extracellular DNA were quantified. In addition, the number of parasites associated with
46
neutrophils was determined, and the observed NETs formed were microscopically characterized.
47
Results showed that (i) in culture, neutrophils isolated from cats presented diminished cellular
48
viability after 4 hours of incubation, and when neutrophils were incubated with T. gondii, they
49
displayed cytotoxic effects after 3 hours of interaction; (ii) neutrophils were able to release
50
structures
51
immunofluorescence, and electron scanning microscopy, when stimulated with T. gondii; (iii) only
52
11.4% of neutrophils were able to discharge NETs during 3 hours of incubation; however, it was
53
observed through extracellular quantification of DNA that this small number of cells were able to
54
display different behavior compared to a negative control (no parasite) group; (iv) significant
55
differences in ROS production were observed in neutrophils exposed to T. gondii. In conclusion,
56
our results showed that neutrophils isolated from cats exposed to T. gondii release structures
57
composed of DNA and histones, similar to what has already been described in other neutrophil
58
species infected with the parasite.
composed
of
DNA
and
histones,
characterized
as
NETs
under
optical,
59 60
Keywords: neutrophils; Toxoplasma gondii; cats; extracellular traps
61 62
1. Introduction
63 64
Toxoplasma gondii is an obligatory intracellular parasite which causes toxoplasmosis, a
65
zoonotic disease with high prevalence around the world and responsible for deaths of
66
immunocompromised patients as well as fetal losses when the parasite is acquired during pregnancy
67
(Montoya and Liesenfeld, 2004; Wang et al., 2017). In its life cycle, T. gondii presents an asexual
68
phase which occurs in intermediate hosts, homeothermic mammals, and a gut sexual phase in the
69
definitive host, felines, resulting in excretion of oocyst in feces (Dubey et al., 1998; Tenter et al.,
70
2000; Gazzinelli et al., 2014).
71
In the host body, in the innate immune response developed against T. gondii, neutrophils
72
play an important role in controlling parasite replication (Miller et al., 2009). After activation, these
73
cells acquire microbicide activity through phagocytosis and the release of granules containing lytic
74
enzymes (Bliss et al., 2000; Mantovani et al., 2011; Sheshachalam et al., 2014).
75
Additionally, a new mechanism discovered by Brinkmann et al. (2004), the release of
76
extracellular traps by neutrophils (NETs) which are filaments containing DNA, histone, and
77
antimicrobial peptides, has also been described (Borregaard, 2010; Miralda et al., 2017). NETosis is
78
associated with drastic changes in neutrophil morphology (Fuchs et al., 2007). The event depends
79
on calcium mobilization, generation of reactive oxygen species (ROS) by the NADPH complex,
80
and transference of proteases such as myeloperoxidase and elastase from neutrophils nuclei
81
(Sheshachalam et al., 2014; Sollberger et al., 2018). These processes result in an extended net that
82
inhibits pathogen dissemination and lead to high concentrations of antimicrobials agents at the
83
infection site (Brinkmann et al., 2004; Brinkmann and Zychlinsky, 2007; Brinkmann and
84
Zychlinsky, 2012).
85
NETs have already been described in humans and other animal species such as mice, dogs,
86
cats, sheep, cows, fishes, donkeys, and seals (Palic et al., 2007; Wardini et al., 2010; Abi Abdallah
87
et al., 2011; Munoz-Caro et al., 2015; Wei et al., 2016; Yildiz et al., 2017; Yildiz et al., 2019). The
88
involvement of NETs in host defense against different pathogens such as Staphylococcus aureus,
89
Shigella flexneri, Salmonella typhimurium, Escherichia coli, Aspergillus sp., Candida albicans,
90
Leishmania sp. and T. gondii is well stablished (Brinkmann et al. 2004; Urban et al., 2006; Grinberg
91
et al., 2008; Bianchi et al., 2009; Bruns et al., 2010; Gabriel et al., 2010; Wardini et al., 2010; Abi
92
Abdallah et al., 2011; Guimarães-Costa et al., 2014).
93
Several studies have demonstrated the discharge of NETs against parasites from the
94
Apicomplexa Phylum (Abi Abdallah et al., 2011, Hermosilla et al., 2014, Munoz-Caro et al., 2014,
95
Reichel et al., 2015, Villagra-Blanco et al., 2017, Yildiz et al., 2017; Yildiz et al., 2019) with a
96
directed effector mechanism (Hermosilla et al., 2014, Munoz-Caro et al., 2014), without evidence
97
of a species-specific immune response. However, as only a small proportion of the cells are able to
98
release NETs, it is possible the existence of different neutrophils subpopulations, both in vitro and
99
in vivo, that are programmed for this specific phenomenon (Fuchs et al., 2007, Yildiz et al., 2019).
100
To date, this innate defense mechanism has not been demonstrated in neutrophils of cats exposed to
101
T. gondii.
102
Knowing that neutrophils play an important role as a mechanism of primary defense against
103
parasitic infections, the importance of felines during the T. gondii life cycle, and due to the absence
104
of knowledge regarding the mechanisms engaged by neutrophils during feline infection with T.
105
gondii, we sought to clarify how neutrophils derived from cats behave when in contact with
106
tachyzoites from T. gondii. Here, we show that cat neutrophils, when in contact with T. gondii, are
107
able to induce NETs formation.
108 109
2. Materials and methods
110
2.1. Ethics statement
111
All experiments were performed according to the University Animal Ethic Guidelines -
112
Ethics Committee for the use of Animals (CEUA – UESC), being approved with the protocol
113
number 024/15.
114 115
2.2. Animals
116
Samples from 12 healthy cats which did not present any abnormalities suggestive of
117
systemic disease such as vomiting, diarrhea, weight loss, nasal secretion, or neoplasms after clinical
118
examination were used (Collado et al., 2012). All animals were FIV/FeLV negative by
119
immunochromatographic kit for FIVac/FeLVag (Alere™, Waltham, USA) and lacking anti-IgG
120
antibodies against T. gondii by indirect immunofluorescent antibody test, with a cut-off of 1:16,
121
according to Pinto and co-workers (2009).
122 123
2.3. Parasite culture
124
Toxoplasma gondii tachyzoites from the RH strain were cultivated in Vero cells. The
125
cultures were maintained in RPMI-1640 + L-glutamine (Gibco, Carlsbad, USA), supplemented with
126
10% inactivated bovine serum (Gibco) and 1% antibiotic plus antimycotic (Gibco), at 37ºC in a
127
humidified incubator containing 5% CO2. The tachyzoites were purified using a 5 µm syringe filter
128
(Sigma–Aldrich, St. Louis, USA) and re-suspended in saline (NaCl 0.9%). Their viability was
129
evaluated by Trypan blue exclusion assays with 0.01% dye (Gibco) and using a hemocytometer.
130 131
2.4. Isolation of neutrophils from cats
132
Peripheral blood was collected from each cat by venipuncture of the cephalic vein into tubes
133
containing heparin (Vacutainer®; BD, Franklin Lakes, USA). Feline blood was placed on a
134
discontinuous Ficoll-histopaque gradient (densities 1.077/1.119) (Sigma–Aldrich) and centrifuged
135
at 400 g for 30 min at 22°C. Neutrophils were recovered from the 1.119/1.007 interface, re-
136
suspended in saline and washed at 400 g for 10 min, followed by re-suspension in RPMI-1640
137
without serum, and their viability was evaluated by 0.01% Trypan blue exclusion assays using a
138
hemocytometer. In the case of red blood cells contamination, the cells were incubated with ACK
139
lysis buffer (8.29 g 0.15 M NH4Cl, 1 g KHCO3 1 mM, 0.074 g 0.1 mM Na2 EDTA, pH 7.2-7.4) for
140
2 min, and thereafter washed again with saline.
141 142
2.5. Viability of neutrophils
143
2.5.1. MTT assay
144
The MTT methodology is an indirect assay that verifies cell death using a reaction that
145
induces reduction of tetrazolium formazan salt by mitochondrial enzymes, and by enzymes present
146
in the endosomal/lysosomal compartments (Lü et al., 2012; Van den Berg, 2015). Neutrophils (2 ×
147
105/well) were incubated with T. gondii tachyzoites (1:5 cell/parasites) at 34°C with 5% CO2 for 0,
148
2, 3 and 4 h. Five milligrams per milliliter of MTT solution (3-[4,5-dimethylthiazol-2-yl]-2,5-
149
diphenyltetrazolium bromide) (Sigma-Aldrich) was added to each well, and plates were further
150
incubated for 4 h (Mosmann, 1983). Formazan crystals were solubilized with a solution of
151
dimethylformamide + dodecyl sodium sulfate (SDS) 10% (1:1 v/v). Cellular viability was
152
determined by reading the plates in a spectrophotometer at 570 nm. The optical density of formazan
153
in each well is directly proportional to cell viability, and each value was normalized to the negative
154
control. Cell survival was expressed as the percentage of formazan absorbance.
155 156
2.5.2. Lactate dehydrogenase (LDH) assay
157
Neutrophils (1 × 106/well) were incubated with T. gondii tachyzoites (1:5 cell/parasites) at
158
34°C with 5% CO2 for 0, 2, 3 and 4 h. After incubation they were centrifuged at 400 g for 10 min to
159
remove the supernatant, followed by evaluation of LDH release in the supernatant. A positive
160
control was obtained by total lysis of neutrophils obtained with 0.2% Triton X-100. LDH activity
161
was determined according to the manufacturer’s instructions (LDH® Liquiform, Labtest
162
Diagnostica, Lagoa Santa, Brazil). To avoid any influence of T. gondii involving the LDH results,
163
cultures of T. gondii using the same conditions were used in parallel for comparison. The results
164
identified for interactions were deducted from the T. gondii culture results.
165 166
2.6. Superoxide detection
167
Neutrophils (2 × 105/well) were incubated with T. gondii tachyzoites (1:5 cell/parasites)
168
and/or phorbol myristate acetate (PMA) (600 µM), as a positive control, for 30 min at 34°C with
169
5% CO2. For superoxide detection, a dihydroethidium probe (DHE-Invitrogen, Carlsbad, USA) was
170
added (final concentration 3 µM). Cells were analyzed using a FC500 (Beckman Coulter™) flow
171
cytometer instrument in which 20,000 events were collected. Results were analyzed using the
172
software CXP 2.2. Data are represented as mean of fluorescence intensity.
173 174
2.7. Extracellular DNA detection
175
Neutrophils (2 × 105/well) were incubated with T. gondii tachyzoites (1:5 cell/parasites)
176
and/or PMA – 600 µM as positive control (Sigma-Aldrich) at 34°C with 5% CO2 for 0, 2 and 3 h.
177
Cells were centrifuged at 200 g for 8 min and the DNA present in the supernatant was quantified
178
using the kit dsDNA PicoGreen™ (Invitrogen) according to manufacturer’s instructions.
179
Extracellular DNA was measured using a spectrofluorometer at a wavelength of 480 nm, with
180
emission at 520 nm. Reference DNA from salmon sperm (Sigma-Aldrich) was used as a standard.
181 182
2.8. Neutrophil-parasite interactions
183
Neutrophils (1 × 105/well) were incubated with T. gondii tachyzoites (1:5 cell/parasites) under
184
glass cover slips (13 mm2) pre-treated with 0.001% of poly-l-lysine (Sigma-Aldrich). Infected cells
185
were incubated for 3 h at 34°C with 5% CO2. Cells were then stained with Diff-Quick™ (Sigma-
186
Aldrich) and observed under an optical microscope. One hundred neutrophils were counted to
187
determine the percentage of neutrophils with T. gondii tachyzoites trapped in networks, the
188
percentage of neutrophils with parasites in the cytoplasm (phagocytosis or active penetration), as
189
well as the relationship of T. gondii/neutrophils in both events.
190 191
2.9. NETs characterization
192
Neutrophils (1 × 105/well) were incubated with T. gondii tachyzoites, as previously
193
described (sub-section 2.8). Cells were stained with Diff-Quick™ solution and NETs were observed
194
under an optical microscope using bright-field illumination. For indirect immunofluorescence
195
microscopy, neutrophils (1 × 105/well) were incubated with T. gondii tachyzoites and fixed with 4%
196
paraformaldehyde. To characterize the components of NETs, the slides were incubated with an anti-
197
histone H2A antibody (diluted 1:800, Sigma-Aldrich), followed by a FITC anti-mouse antibody
198
diluted 1:50 (Sigma-Aldrich) and/or DAPI 5 µg/mL (Sigma-Aldrich). Slides were mounted with
199
SlowFade (Molecular Probes; Invitrogen) and observed under an immunofluorescence microscope.
200
For scanning electron microscopy, neutrophils (2 × 106/well) were incubated with T.
201
gondii tachyzoites. After 3 h incubation, cells were washed with saline and fixed with 2.5%
202
glutaraldehyde in a 0.1 M sodium cacodylate buffer for 1 h, at 4°C, followed by 3 washes with the
203
same buffer, and then gradually dehydrated with ethanol (30, 50, 70, and 100%, followed by
204
acetone (70, 80 and 100%) for 10 min at each concentration. Lastly, the slides were metalized and
205
fixed in stubs and analyzed under a Quanta 250 microscope (FEI Company™; Hillsboro, USA).
206 207
2.10. Statistical analysis
208
Data are represented as mean ± standard deviation. Data were analyzed using Prism GraphPad
209
5.0 software. After normality tests, the parametric Tukey test and/or Student’s t-test were
210
performed, and for non-parametric analysis, the Kruskal Wallis test was used. The results were
211
considered significant with p < 0.05.
212 213 214 215 216
3. Results 3.1. Animal health All cat samples used in the present study came from clinically healthy cats which tested negative for FIV/FeLV and T. gondii.
217 218
3.2. Cytotoxicity of T. gondii for neutrophils
219
Purified neutrophils derived from cats displayed diminished viability over time in culture,
220
with a significant reduction observed after 4 h of incubation (Figure 1A). Because of this, we
221
proceeded using a maximum period of 3 h for immunological assays.
222
In viability assays, the cytotoxic effect caused by T. gondii involving neutrophils was
223
observed after 3 h of interaction (Figure 1A). However, there were no significant changes in the
224
release of the LDH enzyme when compared with cells exposed and not exposed to T. gondii. LDH
225
activity in the supernatant was markedly induced by the use of the lysis buffer only (Figure 1B).
226 227
3.3. Neutrophils induce ROS when exposed to T. gondii
228
Cat neutrophils, when in contact only with T. gondii, produced ROS after 30 min of
229
interaction, leading to a significant augmentation of the intracellular production of superoxide
230
anions, with a difference of approximately three-fold compared to pure neutrophils (Figure 2,
231
Supplementary data 1). This result was similar to that in the PMA positive control, and when PMA
232
and T. gondii were simultaneously incubated with neutrophils (Figure 2).
233 234
3.4. Cat neutrophils release NETs when exposed to T. gondii
235
It is interesting to highlight that the presence of T. gondii led to a significant liberation of
236
DNA from the nuclei of neutrophils to the extracellular milieu, which was quantitatively directly
237
proportional to the period of parasite exposure. Similar results were also observed when the positive
238
control PMA was used instead of tachyzoites (Figure 3).
239 240
3.5. Toxoplasma gondii tachyzoites are trapped by NETs
241
Knowing that tachyzoites of T. gondii are trapped by NETs, we sought to determine the
242
extent of parasite association with NETs, as well as the internalization of T. gondii by neutrophils. It
243
was observed that a small percentage of neutrophils released NETs after 3 h of interaction when T.
244
gondii was present (11.14%), and these were also able to internalize the parasite (7.14%). The ratio
245
of parasites (trapped in the networks)/neutrophils was 1.21, and the ratio of parasites/neutrophils
246
with internalized parasites was 1.52.
247 248
3.6. NETs characterization
249
After 3 h of interaction, net structures trapping T. gondii tachyzoites were observed (Figure
250
4A), as well as parasites in neutrophils’ cytoplasm (Figure 4B). Aiming to demonstrate that the
251
released NETs are composed of DNA and histone, cells were incubated with DAPI, and anti-histone
252
antibody results showed that the NETs observed in cats were composed of nucleus material, DNA,
253
and histone (Figure 4C and 4D). Ultra-structural observation of the NETs was possible using the
254
scanning electron microscopy (Figure 4E and 4F), in which was possible to identify filaments
255
discharged by neutrophils, with a diameter of 17 nm (Supplementary data 2), that join into larger
256
nets able to trap parasites.
257 258
4. Discussion
259
Neutrophils are known for their complex roles during T. gondii infection. These cells
260
harbor mechanisms to control the spread of parasites; however, they are also able to protect the
261
parasite against damage caused by the host immune system since the parasite finds a safer
262
environment to survive in their cellular cytoplasm (Wilhelm, Yarovinsky, 2014). To date, there are
263
no descriptions regarding how neutrophils isolated from cats, the parasite’s definitive host, are able
264
to promote NETs release when exposed to T. gondii.
265
In this work, we selected healthy cats which were also negative for FeLV because previous
266
studies have demonstrated that animals that are FeLV-positive display reduced production of ROS
267
and therefore diminished assembly of NETs. Moreover, FeLV-positive symptomatic animals induce
268
spontaneous stimulation of neutrophils (Wardini et al., 2010), which could have influenced our
269
results.
270
Due to the shortage of information regarding cat neutrophils, we first evaluated cell viability
271
over time using MTT. Our results showed diminished cell viability over time, with increased death
272
rates being observed after 4 h of cellular culture. According to Brinkmann and Zychlinsky (2012),
273
in culturing human neutrophils, an incubation time > 4 h can reflect other mechanisms of cell death
274
which are different from NETosis. Therefore, we decided to use up to 3 h of incubation time for all
275
experiments. Our results showed significant reduction in viability of neutrophils after 3 h of
276
interaction due to the presence of T. gondii.
277
In relation to LDH concentration, only a slight augmentation (~ 20%) was observed over
278
time, what was not significant (p > 0.05), corroborating previous studies in cattle and dogs (Aulik et
279
al., 2010; Wei et al., 2016). These results could be related to the fact that only 10-33% of
280
neutrophils release NETs and consequently die after stimulation (Brinkmann, Zychlinsky, 2007;
281
Fuchs et al., 2007), or to the possibility that activated neutrophils release nuclear fragments and
282
chromatin without damaging the plasma membrane (Pilsczek et al., 2010). It seems that the discrete
283
release of LDH may be directly connected only to the neutrophil population that dies due to
284
NETosis. More, these results allow us to conclude that the dosage of extracellular DNA was more
285
sensitive than LDH for NETosis characterization.
286
Reactive oxygen species are a defense mechanism used by neutrophils to kill invaders
287
(Brinkmann, Zychlinsk, 2007) trapped in phagolysosomes (Brinkmann, Zychlinsky, 2012). During
288
T. gondii infection it seems that ROS do not directly affect the parasite (Denkers et al., 2004),
289
although they act as second messengers, promoting signaling, which first induces the dissociation of
290
primary granules through hydrogen peroxide, allowing the release of myeloperoxidase and elastase
291
to the cytoplasm. These proteases migrate to the nucleus and cleave histones, allowing the
292
chromatin to relax, and the NETosis process commences (Brinkmann, Zychlinsky, 2007; Fuchs et
293
al., 2007; Wardini et al., 2010; Brinkmann and Zychlinsky, 2012; Sollberger et al., 2018).
294
Our results showed rapid production of ROS when neutrophils were incubated with T.
295
gondii. Based on the studies cited above, it is probable that neutrophils initiate signaling for NETs
296
release immediately after their exposure to the parasite. These findings corroborate with the study
297
by Reichel et al. (2015) that described NETs 10 min after interaction with T. gondii. Also,
298
Brinkmann and Zychlinsky (2007) observed neutrophil-induced respiratory explosion in less than
299
an hour under their experimental conditions.
300
Li and Tablin (2017) verified that 100 nM PMA is a potent inductor of canine neutrophil
301
histone hypercitrullination, with significant production of ROS and consequently NETs release. Our
302
positive control presented a mean of fluorescence intensity similar to that of neutrophils incubated
303
with T. gondii. An augmented NETs release was expected when associating PMA with the parasite;
304
however, this interaction between neutrophils, T. gondii, and PMA was significant only when
305
compared with purified neutrophils. In cellular culture of donkey neutrophils, the interaction of T.
306
gondii plus neutrophils presented high release of ROS compared to the control group of PMA plus
307
neutrophils(Yildiz et al., 2019), which was different than what we observed.
308
Results from extracellular DNA measurements showed improvement in fluorescence
309
intensity over time, with two-fold elevation in comparison to the initial values, corroborating with
310
results obtained from mice, sheep, donkeys, seals, and bovine neutrophils stimulated with T. gondii
311
(Abi Abdallah et al., 2011, Reichel et al., 2015; Yildiz et al., 2017, Yildiz et al., 2019). The same
312
was observed when Leishmania donovani, L. infantum, and Neospora caninum were used (Gabriel
313
et al., 2010; Guimarães-Costa et al., 2014; Wei et al., 2016), although it has not been observed in
314
the interactions with Besnoitia besnoiti (Munoz-Caro et al., 2014). However, an important
315
difference observed is the impact on the viability of T. gondii according to the host involved.
316
Studies with mice, seals, bovines and donkeys showed the lethality or diminished parasite ability to
317
invade cells from the host after NETs exposure (Abi Abdallah et al., 2011; Reichel et al., 2015;
318
Yildiz et al., 2017; Yildiz et al., 2019), opposed to what was observed in sheep neutrophils, in
319
which only parasite trapping was observed (Yildiz et al., 2017). These results reinforce the role of
320
the innate immune response in susceptibility or resistance to the parasite.
321
Using optical microscopy, we observed NETs trapping T. gondii, as well as parasites in the
322
neutrophils cytoplasm. Wardini et al. (2010) showed that cat neutrophils are able to release NETs
323
during Leishmania infection. In this study, the mean of phagocyted promastigotes, as well as the
324
number of parasites associated with neutrophils, were similar to our obtained data. In mice, NET
325
release was independent of T. gondii active invasion, which may be mediated by factors released by
326
the parasite and by contact between membranes of the host and parasite (Abi Abdallah et al., 2011).
327
NETs release by cat neutrophils presents DNA as the main structural component, which was
328
observed with DAPI staining and further confirmed by treatment with DNase (Supplementary data
329
2), which led to disintegration of the NETs - the same has been observed in other studies (Palic et
330
al., 2007; Gabriel et al., 2010). Histones were also identified in the extracellular environment,
331
similar to what Wardini et al. (2010) found. Although the microbicidal role of histones was
332
described a long time ago, their mechanism of action was unknown. However, with the NETs
333
description, their roles against bacteria and parasite were clarified (Wang et al., 2011).
334
Lastly, through scanning electron microscopy, it was possible to observe that the
335
neutrophils’ contours remained round, without projections on the cytoplasmic membrane in the
336
absence of stimulus. When cells where co-cultured with T. gondii tachyzoites, they presented
337
extended projections of cellular DNA towards the parasite and other neutrophils. The observed
338
NETs displayed approximately 17 nm, with the presence of globular domains which joined larger
339
segments, similar to what was found by Brinkmann et al. (2004), Pilsczek et al. (2010) and Wei et
340
al. (2016).
341
In conclusion, here we characterized NETs formation in neutrophils from the T. gondii
342
definitive host. We identified diminished neutrophils viability when they were in contact with the
343
parasite, with rapid and intense ROS production. Similar to previous studies, it was also observed
344
that NETs release were time dependent, leading to parasite trapping, signaling that the cat innate
345
immune response may contribute against T. gondii infection, it being important to determine how
346
this initial response influences the acquired immune response against the parasite.
347 348
Acknowledgements
349 350 351 352 353 354
To Fundação de Amparo à Pesquisa do Estado da Bahia – FAPESB (nº APP0028/2016) and Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq (nº 430375/2016-6) for funding the Project and to Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for the fellowship. The authors also thank the UESC’s Center for Electron Microscopy for the realization of the images and the UFMG’s Laboratory of Cell-cell Interactions for the use of the spectrofluorometer.
355 356
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Figure 1. Viability of cat PMNs exposed to Toxoplasma gondii through MTT and LDH. (A) PMN (2 × 105/well) were plated without and with T. gondii tachyzoites (1:5 cell/parasites) at 34°C/5% CO2. Absorbance was acquired at 570 nm. Viability was assessed 2, 3 and 4 h after incubation. Data are expressed as mean + standard deviation, and differences were considered significant when p < 0.05. (B) PMN (1 × 106/well) were stimulated or not with T. gondii tachyzoites (1:5 cell/parasites). After parasite addition and three hours of incubation, cell cultures were centrifuged and the supernatant collected for LDH assays. A positive control was prepared by total lysis with Triton X-100 at 0.2%. Results are expressed in U/L as mean + standard deviation. Significant differences were observed between the positive control and purified PMNs, and between PMNs plus T. gondii. * p < 0.05, ** p < 0.01, *** p < 0.001. PMN-polymorphonuclear. The assay was performed at least three independent times with the same results.
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Figure 2. Mean fluorescence intensity after PMN exposure to Toxoplasma gondii. PMNs (2 × 105/well) were incubated with T. gondii tachyzoites (1:5 cell/parasites) and/or PMA at 600 µM for 30 min and maintained at 34°C/5% CO2. After addition of the DHE probe (3 µM) the mean of fluorescence intensity was measured. Data are representative of mean + standard deviation. Significant differences (p < 0.05) occurred between the stimulated cells compared to purified PMNs. * p < 0.05, ** p < 0.01, *** p < 0.001. PMN-polymorphonuclear. The assay was performed at least three independent times with the same results.
552 553 554 555 556 557 558 559 560
Figure 3. Quantification of the release of extracellular DNA in PMNs exposed to Toxoplasma gondii. PMN (2 × 105/well) were plated with T. gondii tachyzoites (1:5 cell/parasites) or PMA at 600 µM at 34°C/5% CO2. After 2-3 h of incubation, cells were centrifuged, and cell supernatant was collected for evaluation of extracellular DNA with the Picogreen dsDNA kit. Data are representative of mean + standard deviation. Significant differences (p < 0.05) occurred between time zero of parasite exposure and 2 and 3 h, as well between 2 and 3 h of incubation. * p < 0.05, ** p < 0.01. PMN-polymorphonuclear. The assay was performed at least three independent times with the same results.
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Figure 4. Characterization of NETs from cat neutrophils after incubation with Toxoplasma gondii. Optical (A-B) and immunofluorescence microscopy (C-E). Neutrophils (1 × 105/well) were incubated with T. gondii tachyzoites (1:5 cell/parasites) for 3 h at 34°C/5% CO2. Slides (A and B) were stained using Diff-Quick methodology. (A) Tachyzoites associated with NETs – arrows,
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(B) Tachyzoites present inside neutrophils – arrows. (C) NETs ( ) and neutrophils nuclei ( ) stained with a monoclonal antibody against anti-H2, (D) NETs ( ) and neutrophils nuclei ( ) stained with DAPI, (E) expanded area within the red box stained with DAPI. Electron scanning microscopy (F-G). Neutrophils (2 × 106/well) were incubated with T. gondii tachyzoites (1:5 cell/parasites) for 3 h at 34°C/5% CO2. After fixation with 2.5% glutaraldehyde in 0.1 M of Nacacodylate buffer, cells were further processed for image acquisition (F-G) NETs formation – arrows, with trapping of T. gondii. N – neutrophils, T – Toxoplasma gondii tachyzoites.
A
PMN viability (%)
120
PMN PMN + T. gondii
*
100
** 80
**
60 40 20 0 0
2
4
Time (h)
B 450
3
*** ***
***
***
LDH (U/L)
400 350 100 50 0 0
3
Time (h)
Figure 1.
Lysis PMN PMN + T. gondii
Mean Fluorescence
20
*** 15
***
***
PMN + T. gondii PMN + PMA + T. gondii
10 5 0
Time (3h)
Figure 2.
PMN PMN + PMA
DNA released (ng ml-1)
200 180 160 140 120 100 80 60 40 20 0
**
*
**
0
*
2
3
0
Time (h)
Figure 3.
PMN + PMA PMN + T. gondii
**
2
3
Figure 4
Highlights • • • • •
Here, we demonstrated that cat neutrophils exposed to T. gondii can release NETs. Tachyzoites from T. gondii are trapped by extracellular NETs released by cat neutrophils. Toxoplasma gondii diminishes neutrophils viability after 3 hours of interaction. Cat neutrophils, when in contact with T. gondii, produce ROS after 30 min of interaction. The amount of extracellular DNA released by neutrophils after parasite interaction was time-dependent, with high concentrations being observed after 3 hours.