Contents lists available at ScienceDirect Veterinary Parasitology journal homepage: www.elsevier.com/locate/vetpar Research paper Canine neutrophils activate effector mechanisms in response to Leishmania infantum Maria Pereiraa,b, Ana Valério-Bolasa,1, David Santos-Mateusa,1, Graça Alexandre-Piresc, Marcos Santosc, Armanda Rodriguesc, Hugo Rochad, Ana Santosd, Catarina Martinse, Ana Tomasf, Filipe Passerog, Isabel Pereira da Fonsecac, Gabriela Santos-Gomesa,⁎ a Global Health and Tropical Medicine, GHMT, Instituto de Higiene e Medicina Tropical, IHMT, Universidade Nova de Lisboa, UNL, Rua da Junqueira 100, 1349-008 Lisbon, Portugal b Polytechnic Institute of Portalegre, Higher Agrarian School of Elvas, Edifício do Trem Auto, Avenida 14 de Janeiro s/n Apartado 254, 7350-903 Elvas, Portugal c Interdisciplinary Animal Health Research Centre, Faculty of Veterinary Medicine, University of Lisbon, Av. Universidade Técnica, 1300-477 Lisbon, Portugal d Division of Veterinary Medicine, National Guard, Largo do Carmo 1200-92 Lisbon, Portugal e Chronic Diseases Research Center, Immunology, NOVA Medical School, New University of Lisbon, Rua Câmara Pestana n° 6, 6-A Edifício CEDOC II, 1150-082 Lisbon, Portugal f I3S, Instituto de Investigação e Inovação em Saúde. IBMC, Instituto de Biologia Molecular e Celular and ICBAS, Instituto de Ciências Biomédicas Abel Salazar, University do Porto, Rua Alfredo Allen, 208, 4200-135 Porto, Portugal g São Paulo State University (Unesp), Institute of Biosciences, São Vicente, Praça Infante Dom Henrique, S/N, 11330-900 São Vicente, SP, Brazil A R T I C L E I N F O Keywords: Canine leishmaniosis Neutrophils Superoxide Neutrophil elastase Neutrophil extracellular traps Electron microscopy A B S T R A C T Canine leishmaniosis caused by L. infantum is a severe zoonotic disease. Although macrophages are the definitive host cells, neutrophils are the first cells to encounter the parasite soon after its inoculation in the dermis by the phlebotomine vector. To study the interaction of dog neutrophils and L. infantum promastigotes, blood neu- trophils were isolated from healthy donors and the infection was established in vitro. In the majority of the dogs, L. infantum was efficiently phagocytized by neutrophils, and oxidative (superoxide production) and non-oxi- dative (neutrophil elastase exocytosis) intracellular effector mechanisms were activated, but the release of neutrophil extracellular traps was minimized. Furthermore, promastigotes and culture supernatants induced neutrophil migration, but the prior contact with Leishmania inhibits chemotaxis, which might contribute to neutrophil retention at the inoculation site. Neutrophil-parasite interaction resulted in a decrease in parasite viability, although some intracellular promastigotes survive and maintain their proliferative capacity. These findings indicate that dog neutrophils are competent effector cells able to control the initial L. infantum infection. However, some parasites evade intracellular effector mechanisms and can be transferred to the definitive host cell, the macrophage, contributing to the development of canine leishmaniosis. 1. Introduction Adaptive immune response is crucial for the resolution of Leishmania infection. However, innate immune mechanisms have been gained importance, in particular the activity of neutrophils (or poly- morphonuclear cells, PMN) as effector and immunomodulatory cells (Faria et al., 2012; Carlsen et al., 2015; Hurrell et al., 2016). PMN ra- pidly reach Leishmania injection site (Pompeu et al., 1991; Beil et al., 1992; Santos-Gomes et al., 2000; Matte and Olivier, 2002; Thalhofer et al., 2011; Ribeiro-Gomes and Sacks, 2012), recruited by parasite-, host- and sand fly-derived chemotactic factors (Hurrell et al., 2016), and seems to be the first cells to internalize the parasite (Ribeiro-Gomes et al., 2012; Hurrell et al., 2015). Despite the high microbicidal potential of PMN, represented by the presence of granules full of enzymes and antimicrobial molecules and by the ability to produce reactive oxygen species (ROS), some inter- nalized promastigotes survive (van Zandbergen et al., 2004). Indeed, promastigote uptake via lysosome-independent pathway leads to the formation of tight phagosome and promotes parasite survival, while internalization through lysosome-dependent pathway generates large phagosome that kills the parasite (Gueirard et al., 2008). Furthermore, some Leishmania species seem to be able to modulate PMN apoptosis, http://dx.doi.org/10.1016/j.vetpar.2017.10.008 Received 22 February 2017; Received in revised form 25 September 2017; Accepted 17 October 2017 ⁎ Corresponding author. 1 These authors contributed equally to this paper. E-mail address: santosgomes@ihmt.unl.pt (G. Santos-Gomes). Veterinary Parasitology 248 (2017) 10–20 0304-4017/ © 2017 Elsevier B.V. All rights reserved. T http://www.sciencedirect.com/science/journal/03044017 https://www.elsevier.com/locate/vetpar http://dx.doi.org/10.1016/j.vetpar.2017.10.008 http://dx.doi.org/10.1016/j.vetpar.2017.10.008 mailto:santosgomes@ihmt.unl.pt https://doi.org/10.1016/j.vetpar.2017.10.008 http://crossmark.crossref.org/dialog/?doi=10.1016/j.vetpar.2017.10.008&domain=pdf prolonging cell life span or accelerating cell death, which could pro- mote parasite transference to macrophages that reaches inoculation site later on (Aga et al., 2002; Gueirard et al., 2008; Carlsen et al., 2013; Falcão et al., 2015). Neutrophil release Neutrophil Extracellular Traps (NET) in response to L. amazonensis, L. donovani, L. infantum, L. major, L. donovani and L. mexicana (es-Costa et al., 2009, 2014; es-Costa et al., 2009, 2014; Gabriel et al., 2010; Hurrell et al., 2015). However, some parasite species are protected from NET-mediated killing by lipophosphoglycan (LPG) (Gabriel et al., 2010), enzyme 3′-nucleotidase/nuclease (Guimarães-Costa et al., 2014) or by other factors not yet identified (Hurrell et al., 2015). Beyond microbicide and limiting spread effects, NET may also contribute to modulate the host immune system towards a pro-inflammatory response (Luo et al., 2014). Thus, PMN seem to have a protective or a permissive impact at the early phase of Leishmania infection, depending on parasite species and host genetic background (Hurrell et al., 2016). Since innate immune mechanisms developed by the dog in the context of a L. infantum in- fection are largely unknown, this study aims to characterize the effector functions developed by peripheral blood PMN when in contact with L. infantum virulent promastigotes, investigating its contribution to the control of infection or, by the contrary, to disease progression. 2. Material and methods 2.1. Experimental design To analyze neutrophil-L. infantum interactions during an early in- fection, blood PMN were isolated from 10 healthy dogs and cell purity and viability were determined. Then cells were in vitro exposed to virulent promastigotes and PMN effector functions were evaluated. The effect of parasite in PMN migration was assessed by a chemotaxis assay and the activation of intracellular (oxidative burst) and extracellular (granule exocytosis and NET emission) microbicide mechanisms were evaluated. Superoxide production was estimated by a colorimetric assay, exocytosis of neutrophil elastase (NE) was determined through a colorimetric enzyme/substrate reaction, NET structures were observed by electron microscopy and the exclusion of DNA and histone (NET constituents) from PMN nucleus were followed by fluorescent micro- scopy. The physical contact of PMN with promastigotes was char- acterized by flow cytometry, optical and electron microscopy and, the parasite fate tracked. Furthermore, the parasite effect on PMN natural viability was also evaluated by flow cytometry, investigating possible parasite modulation of PMN life span. 2.2. Selection of healthy dogs A group of apparently healthy dogs belonging to Grupo de Intervenção Cinotécnico da Guarda Nacional Republicana and to Faculdade de Medicina Veterinária, Universidade de Lisboa (FMV/ ULisboa) was selected. Dogs were subjected to a complete physical examination and blood collection for a complete blood count, bio- chemical profile, ionogram and proteinogram to confirm their health status and the absence of vector borne diseases. Detection of anti- Leishmania antibodies by indirect immunofluorescence assay (Kit Leishmania Spot IF®, BioMerieux, France) with a cut off of 1:80, and blood and lymph node evaluation by q-PCR (Helhazar et al., 2013) was used to exclude L. infantum infection. The absence of Dirofilaria immitis microfilariae was confirmed by Knott test. Other hemoparasites, such as Babesia spp., Ehrlichia spp., Anaplasma spp. and Ricketsia spp. were ruled out by microscopic observation of blood smears and q-PCR. In addition, the presence of Mycoplasma spp. was evaluated by micro- scopic observation of blood smear. Ten clinically and analytically healthy dogs were chosen. Sample size was calculated on the basis of decided sample size, taking into account statistical significance and the minimum use of animals (Charan and Biswas, 2013). These dogs aged between 2 and 4 years (2.5 ± 0.9), weigh between 24 and 40 kg (32.3 ± 5.2) and included pure breeds (1 Rafeiro do Alentejo Portu- guese breed, 3 German Shepherd, and 1 Belgian Shepherd) and mixed breeds (5) of which four were females and six were males (Supple- mentary data 1). Before inclusion in this study, dog’s tutors were informed about the objective and the nature of this work and official consents were ob- tained. The study is in conformity with the institutional guidelines and the EU requirements and was approved by the Ethics and Welfare Committee of FMV/ULisboa. 2.3. Parasites L. infantum zymodeme MON-1 (MHOM/PT/89/IMT151) virulent promastigotes collected from the stationary phase of growth of sub- cultures with less than five passages (Santos-Gomes and Abranches, 1996) and parasites expressing green fluorescent protein (GFP) (Marques et al., 2015) were used for PMN infection. Parasite con- centration was assessed in a Neubauer-counting chamber (Marienfeld, Germany). 2.4. PMN isolation and purification Each time, 20 mL of blood were collected into tubes containing ci- trate phosphate dextrose adenine (CPDA)-1 solution as anticoagulant (Kawasumi, Germany). PMN isolation was achieved using a Histopaque double-density gradient, according to the technique described by Strasser et al. (1998) and contaminant red blood cells were eliminated by osmotic lysis. Cells were resuspended in Hanks’ Balanced Salt So- lution (HBSS) (Sigma-Aldrich, USA) and its viability and concentration assessed by trypan blue exclusion in a Neubauer-counting chamber. Cell purity was determined by flow cytometry analysis and microscopic observation (Supplementary data 2). 2.5. In vitro infection Dog PMN (5 × 105 cells/well) were seeded in 96-well plates (Nunc, Denmark) with L. infantum or L. infantum-GFP promastigotes at a ratio parasite-PMN of 5:1 in 300 μL of HBSS supplemented with 5% heat- inactivated fetal bovine sera (FBS) (v/v). Plates were incubated at 37 °C in a humidified atmosphere containing 5% of CO2 for 1.5 h and 3 h. In parallel, PMN cultures were also established to be used as negative controls. Infected and uninfected cultures were used in the following experiments. 2.6. Interaction between L. infantum promastigotes and PMN The interaction between L. infantum-GFP parasites and PMN was evaluated by flow cytometry. After 1.5 h, 3 h and 4.5 h of incubation, resting-PMN (control) and PMN exposed to L. infantum-GFP were ac- quired by flow cytometry. L. infantum-GFP parasites were also acquired. The frequency of PMN-parasite association was assessed on one para- meter histogram, plotting as FL1-H/GFP vs the number of events. Additionally, the contact between parasites and PMN was observed under an optical microscope (OM). 2.7. Chemotaxis assay The migratory ability of resting-PMN and of PMN previously ex- posed to L. infantum was evaluated in a 96-well modified Boyden chamber (Neuroprobe Inc., USA) containing a polycarbonate 3 μm pore membrane. Wells of the lower compartment were filled with 29 μL of medium containing 5 × 105 L. infantum promastigotes, 29 μL of Leishmania chemotactic factor (LCF) (van Zandbergen et al., 2002) (positive control) or HBSS (negative control). In the upper compart- ment of the chamber, 25 μL of resting-PMN and PMN-L. infantum M. Pereira et al. Veterinary Parasitology 248 (2017) 10–20 11 cultures incubated for 1.5 h were added. The chamber was incubated for 60 min at 37 °C in a humidified atmosphere containing 5% CO2. PMN migration from the upper to the lower compartment was assessed in a Neubauer-counting chamber under OM. 2.8. Superoxide production O2 − production was measured using a colorimetric nitroblue tet- razolium (NBT) assay (Marques et al., 2015). Absorbance was measured at 570 nm using a microplate reader (Anthos 2010, Austria). Resting- PMN were used as negative control and, phorbol myristate acetate (PMA, 0.2 μg.mL−1) (VWR, International) stimulated PMN and PMA- stimulated PMN exposed to parasites were considered positive controls. Cell free-wells were used as a blank. 2.9. Neutrophilic elastase (NE) exocytosis NE exocytosis was measured in culture supernatants of resting-PMN (negative control), PMN stimulated with 1 μg.mL−1 of Escherichia coli lipopolysaccharide (LPS) (Sigma-Aldrich) (positive control) and of PMN exposed to L. infantum promastigotes, using a colorimetric reaction (Marques et al., 2015) that was quantified at 405 nm in a plate reader (TRIADTM 1065, DYNEX Technologies, EUA) at≈ 0 min (right away following the addition of substrate to the supernatants), 15 min and 30 min of incubation at 37 °C in a humidified atmosphere containing 5% of CO2. It was assumed that enzyme activity is directly proportional to color intensity. 2.10. Neutrophil extracellular traps (NET) release Resting-PMN (negative control), PMN incubated with L. infantum promastigotes, PMN stimulated with 1 μg.mL−1 of PMA (positive con- trol) and PMA-stimulated PMN exposed to L. infantum promastigotes (positive control) adhered to coverslips were incubated in HBSS 2% FBS and prepared for scanning electron microscopy (SEM) and immuno- fluorescence, according to Brinkmann et al. (2010). SEM samples were dried using the critical point drying method, coated with gold palla- dium and mounted on stubs. Cells were then observed under a scanning electronic microscope (JEOL 5200-LV) and images were acquired. The number of NET-generating cells was counted in 50 PMN of each con- dition. Furthermore, the orientation of promastigotes bounded to PMN (flagellum, aflagellar pole, or by any other place) was evaluated in 50 interactions. Immunofluorescence samples were stained with histone H1 (AE-4) FITC antibody (Santa Cruz Biotechnology, Germany) and DAPI, and observed under a fluorescence microscope (Nikon Eclipse 90i). 2.11. PMN cell death Cell viability, apoptosis and necrosis were assessed by flow cyto- metry analysis. Resting-PMN (negative control), PMN exposed to L. infantum promastigotes and PMN stimulated with 100 μg.mL−1 of (S)- (+)-camptothecin (Sigma-Aldrich) (Campto, apoptotic positive con- trol) were incubated in HBSS 10% FBS. Then, cultures were incubated with the commercial kit TACSTM Annexin V-FITC (R &D Systems, USA), according with the manufacturer’s instructions. Prior to the flow cytometry acquisition, cells were treated with 10 μL of propidium io- dine (PI, R & D Systems). Untreated and annexin V-FITC or PI treated cells were used to compensate for PMN autofluorescence and for the fluorochrome fluorescence overlapping emission spectra. Resting un- treated-PMN were used to delimit the quadrants for the evaluation of annexin V-FITC−/PI− (viable cells), annexin V-FITC+/PI− (apoptotic cells) and annexin V-FITC+/−/PI+ cells (total necrotic cells) popula- tions in a FL1-H (Annexin V-FITC) vs FL2-H (PI) dot-plot. Necrotic cells were also subdivided into annexin V-FITC−/PI+ (primary necrotic cells) and annexin V-FITC+/PI+ (secondary necrotic cells). 2.12. Viability of L. infantum promastigotes after PMN contact To study the impact of L. infantum exposition to dog PMN, parasite viability was assessed. L. infantum-PMN cultures were incubated for 2 h. Then, 300 μL of culture were seeded in a 24-wells plate containing 300 μL per well of Schneider medium (Sigma-Aldrich) supplemented with 10% FBS (complete SCHN) and two successive dilutions (1:4) were made. Cultures were incubated at 24 °C for 24 h. In parallel, cultures of L. infantum promastigotes were used as positive control. Viable pro- mastigotes (moving parasites) were counted in a Neubauer-counting chamber. 2.13. Ultrastructural PMN-parasite interrelation PMN-parasite ultrastructural interrelation was investigated by transmission electron microscopy (TEM). Cells exposed to promasti- gotes were centrifuged (300 × g for 10 min), fixed in PBS (Sigma- Aldrich) 2% glutaraldehyde (Sigma-Aldrich) and 0.1% tannic acid (Sigma-Aldrich) for 1 h at 4 °C and then, pellets were washed two times in PBS. Samples were then prepared according to Yamamoto et al. (2015) and images were acquired under a transmission electron mi- croscope (Jeol 1010). 2.14. Viability of PMN-phagocytized parasites After 3 h of incubation, extracellular promastigotes were removed from parasite-exposed PMN by positive selection, using MicroBeads conjugated with monoclonal rat anti-mouse/human CD11b (Mac-1α) antibodies (Miltenyl Biotec, Germany) according to manufacturer’s in- structions. A previous experiment showed that this antibody also binds dogs PMN, allowing the separation between PMN and extracellular parasites. Viability and concentration of PMN free of extracellular parasites were assessed by trypan blue staining using a Neubauer- counting chamber. Then, 5 × 105 PMN were resuspended in 300 μL of complete SCHN medium and incubated at 24 °C for 72 h to promote parasite release from the PMN. Viable parasites were estimated using a Neubauer-counting chamber after 24 h and 72 h of incubation. 2.15. Statistical analysis The non-parametric Wilcoxon Signed Ranks Test was used to com- pare variables of two dependent samples from resting-PMN and L. in- fantum-exposed PMN and, viability and replication levels of parasites that have contacted PMN and cultured parasites (that have not con- tacted PMN) in relation to all parameters studied and time points. Results are represented as median, percentiles, maximum and minimum values or by mean values ± standard error, according to the experi- mental procedures. Differences were considered significant with a 5% significance level (p < 0.05). Statistical analysis was performed with the SPSS 2.0 for Windows software (SPSS Inc., USA) using samples of 10 dogs evaluated in triplicate or values of three independent experiments. 3. Results 3.1. Dog PMN efficiently bind L. infantum promastigotes Resting-PMN cultures were used to identify the GFP− cells (Fig. 1A). After 1.5 h of incubation, the percentage of PMN associated with the parasite (GFP+ cells, Fig. 1B) was 33.7% (19.43%, 44.53%) [median (interquartile range)]. After 3 h (GFP+ cells, Fig. 1C), the amount of PMN bound to L. infantum-GFP slightly decreased to 28.75% (20.83%, 37.85%) and after 4.5 h (GFP+ cells, Fig. 1D) about 29.0% (21.10%, 36.90%) of PMN still were associated with parasites (Fig. 1E). However, the amount of PMN associated parasites did not present sig- nificant oscilations during the incubation period. Furthermore, the level of GFP fluorescence displayed by the subpopulation GFP+ PMN, M. Pereira et al. Veterinary Parasitology 248 (2017) 10–20 12 expressed as the GFP median, remained unchanged throughout the incubation period (Fig. 1F). Thus, the last time point (4.5 h) was abandoned, because it did not contribute to increase the percentage of infected PMN and was associated with a boost of cell death. 3.2. L. infantum promastigotes bind to dog PMN in an orientated manner and are engulfed via funnel-like pseudopods Parasites adhesion to dog PMN through the aflagellar pole (Fig. 2A and B) was uncommon, reaching only 16.7% of the interactions. The binding through the flagellum tip (Fig. 2C–F) was observed in 63.9% of the interactions and by another parasite body site in 19.4% (Fig. 2G and H) of the cases. The engulfment of promastigotes takes place via funnel- like extensions of the phagocyte surface over the parasite (Fig. 2A–C). 3.3. Dog neutrophils efficiently internalize L. infantum promastigotes The promastigote was the most frequent form observed inside PMN (Fig. 3A and B), but in some cells the parasite was found in the amas- tigote-like form (Fig. 3C–E). A high proportion of PMN internalized two (Fig. 3B and C), three (Fig. 3D), four (Fig. 3E) or more parasites. In a few dogs’s samples, the parasite was detected almost exclusively out- side the cell, closely associated with PMN membrane, forming con- centric layers (Fig. 3F and G). After 1.5 h of incubation only 6% (5%, Fig. 1. L. infantum interacts with dog PMN. Resting-PMN and PMN-L. infantum-GFP were incubated for 1.5 h, 3 h and 4.5 h and analyzed by flow cytometry. The histogram FL1-H/GFP vs number of events was applied to determine the frequency of GFP+ PMN in cultures incubated for 1.5 h (B), 3 h (C) and 4.5 h (D), using resting-PMN (A) as a GFP− control. Levels of parasite-PMN interaction (E) and the intensity of GFP fluorescence in the subpopulation of GFP+ PMN (F) at different incubation time points are expressed by medians, 75th percentile and 25th percentile, and whiskers representing the highest and lowest values. Fig. 2. Dog PMN attaches to and phagocyte L. infantum promastigotes. Coverslip adhered-PMN exposed to promastigotes were incubated for 1.5 h (A–D and G) and 3 h (E, F and H) and evaluated by scanning electron microscopy. Attachment and engulfment of L. infantum promastigote via their posterior pole can be seen in A and in more detail in B. Orientated attachment via the tip of the flagellum, showing a well-defined elongated tubular pseudopod advancing along the flagellum can be observed in C and also in more detail in D. Promastigote phagocytosis, revealing parasite internalization via the tip of the flagellum are shown at different angles (E and F). Attachment through flagellum (G) and/or the base of the flagellum (H) can be also seen. White arrow − promastigote. M. Pereira et al. Veterinary Parasitology 248 (2017) 10–20 13 8%) of PMN were parasitized, but later on 23.5% (16.25%, 26.75%) of cells internalized the parasite, showing a significant increase (p < 0.001) (Fig. 3H). 3.4. Previous contact with L. infantum decreases PMN migration Resting-PMN migration was significantly higher towards viable Leishmania promastigotes (p = 0.001) and LCF (p = 0.003) when compared stimuli absence (medium). Interestingly, the migration of PMN previously exposed to L. infantum promastigotes decreased sig- nificantly toward medium (p = 0.008), Leishmania promastigotes (p = 0.003) and LCF (p= 0.002) compared with resting-PMN in equivalent conditions (Fig. 4). 3.5. L. infantum stimulates dog PMN oxidative burst The intracellular production of O2 − by PMA-stimulated PMN (p < 0.001), L. infantum-exposed PMN (p < 0.001) and PMA-stimu- lated PMN exposed to L. infantum (p < 0.001) was significantly higher compared with resting-PMN at both 1.5 h (Fig. 5A) and 3 h (Fig. 5B) of incubation. As expected, double-stimulated PMN (by PMA and L. in- fantum) for 1.5 h produced significantly higher amounts of O2 − com- pared with PMA-stimulated PMN (p < 0.001). However, double- stimulated PMN exhibited significantly lower production of O2 − at 3 h compared with 1.5 h (p = 0.013). 3.6. L. infantum regulates neutrophil elastase (NE) exocytosis NE activity was significantly higher in supernatants of PMN-L. in- fantum cultures incubated for 1.5 h (p < 0.001) (Fig. 6A) and 3 h (p0 min = 0.001, p15 min, 30 min < 0.001) (Fig. 6B), and in supernatants of LPS-stimulated PMN for 1.5 h (p0 min, 15 min = 0.012, p30 min = 0.025) compared with resting-PMN. Furthermore, at 1.5 h the release of NE by L. infantum-exposed PMN was higher than the positive control (p = 0.012). However, NE exocytosis by PMN exposed to L. infantum for 3 h was lower compared with 1.5 h cultures (p0 min = 0.015). 3.7. L. infantum precludes NET formation NET were observed as thin filamentous structures or as large cloud- like formations. As expected resting-PMN did not release NET (Fig. 7A and G). L. infantum induced a lower NET emission at both time points (1.5 h and 3 h) (Fig. 7B and G). However, NET emission by PMA-sti- mulated PMN was intense (Fig. 7C and G) and PMA-stimulated cells exposed to promastigotes also showed an exuberant NET release (Fig. 7D–G). A close contact between NET and the parasite was ob- served in PMA-stimulated PMN exposed to L. infantum promastigotes (Fig. 8). Immunolabeling assays showed extracellular structures stained with DAPI and histone H1-FITC, indicating the emission of nuclear DNA and of nuclear proteins. The parasite induced NET release, but to a lesser extent than PMA-stimulated and PMA-stimulated PMN exposed to L. infantum. As expected, resting-PMN did not show histone or DNA re- lease (Fig. 9). 3.8. L. infantum impacts PMN cell death Campto stimulation (p1.5 h = 0.011, p3 h = 0.008) and L. infantum exposure (p1.5 h and 3 h < 0.001) had a negative impact on PMN via- bility at both time points (1.5 h and 3 h) compared with resting-PMN. The proportion of viable cells was significantly higher in Leishmania- exposed PMN compared with Campto-stimulated PMN at 1.5 h (p = 0.008), but later on (3 h) the situation was reversed (p = 0.038) (Fig. 10A). Campto (apoptosis inducer) caused a significant increase in PMN apoptosis compared with resting-PMN and parasite-exposed PMN (p = 0.008), but only in late cultures (3 h) (Fig. 10B). The proportion of Fig. 3. Promastigotes are uptake by PMN. Cultured PMN-L. infantum promastigotes were incubated for 1.5 h and 3 h, stained and observed under optical microscope. Intracellular promastigote (A and B) and amastigote-like (C, D and E) forms can be observed in PMN. Extracellular promastigote circumventing PMN membrane in concentric incomplete layers can also be observed (F and G) (×1000 magnification). The amount of parasitized PMN is expressed by medians, 75th percentile and 25th percentile, and whiskers representing the highest and lowest values. Statistical analysis of 10 dogs and triplicate samples was performed using the non-parametric Wilcoxon test (p < 0.05). Significant differences are indicated by * when comparing 1.5 h vs 3 h (H). Fig. 4. L. infantum induce chemotaxis of dog PMN. The amount of resting-PMN (white bars) and PMN previously exposed to L. infantum promastigotes (dot bars) that migrate from the upper to the lower compartment of a modified Boyden chamber filled with culture medium (M), alive Leishmania promastigotes (L), and Leishmania chemotactic factor (LCF) are indicated. Results of triplicate samples of 10 dogs are presented as mean ± standard error. Statistical analysis was performed using the Wilcoxon test (p < 0.05). Significant differences are represented by * when comparing M (negative control) vs the other conditions, and by “ when comparing resting-PMN vs PMN+L in equivalent conditions. M. Pereira et al. Veterinary Parasitology 248 (2017) 10–20 14 total necrotic cells presented important increases in parasite-exposed PMN (p1.5 h and 3 h < 0.001) and Campto-stimulated PMN (p1.5 h = 0.005, p3 h = 0.008) compared with resting-PMN at both time points. Furthermore, a higher time of contact (3 h) with the parasite increased the proportion of total necrotic cells compared with Campto- stimulation (p= 0.021) (Fig. 10C). Interestingly, secondary necrotic cells prevailed at all time points, regarding the variable conditions [resting-PMN (p1.5 h < 0.001), parasite-exposed PMN (p1.5 h and 3 h < 0.001) and Campto-stimulated PMN (p1.5 h and 3 h = 0.008)], ex- cept in resting-PMN incubated for 3 h. The proportion of primary and also secondary necrotic cells in parasite-exposed PMN (p1.5 h < 0.001, p3h = 0.005 and p1.5hand3h< 0.001, respectively) and Campto-stimulated PMN (p 1.5h = 0.001, p 3h = 0.028 and p 1.5hand3h = 0.008, respectively) was significantly higher in comparison with resting-PMN at both time points. While the proportion of primary necrotic cells was higher in Campto-stimulated PMN compared to parasite-exposed cultures (p < 0.001) at 1.5 h, the amount of secondary necrotic cells was su- perior in parasite-exposed cultures compared with Campto-stimulated PMN at both time points (p 1.5hand3h = 0.038) (Fig. 10D). 3.9. L. infantum delays apoptosis of parasitized neutrophils Microscopic observation of L. infantum-exposed PMN and of resting- PMN revealed the presence of apoptotic cells exhibiting the classical features, such as cell rounding and shrinking, and chromatin con- densation (pycnotic nuclei), which tends to marginate, acquiring a crescent form (Fig. 11A and B). Late stages of PMN apoptosis were also observed as nuclear fragmentation (karyorrhexis, apoptotic bodies) (Fig. 11B and C). Although some infected PMN exhibited apoptotic nuclear morphology, after 3 h of parasite exposure most of the apop- totic PMN were not parasitized. 3.10. PMN reduce L. infantum promastigote viability by extracellular and intracellular parasite killing L. infantum parasites previously exposed to PMN intracellular and extracellular killing mechanisms showed reduced viability when Fig. 5. PMN exposed to L. infantum promastigotes produce superoxide (O2 −). Resting-PMN (PMN), PMA-stimulated PMN (PMN-PMA), L. infantum-exposed PMN (PMN+L) and PMA-stimulated PMN exposed to L. infantum (PMN + L-PMA) incubated for 1.5 h (A) and 3 h (B) were used to measure O2 −. Results of triplicate samples of 10 dogs are presented by medians, 75th percentile and 25th percentile and, whiskers representing the highest and lowest values. Statistical ana- lysis was performed using the non-parametric Wilcoxon test (p < 0.05). Significant differences are indicate by * when comparing resting-PMN (negative control) vs the different conditions, ^ when comparing PMN + PMA vs PMN+L-PMA and by ̎ when comparing PMN+L-PMA at 1.5 h vs 3 h of incubation. Fig. 6. Enzymatic activity of PMN primary granules. Neutrophilic elastase (NE) exocytosis was quantified in supernatants of PMN, L. infantum-exposed PMN (PMN + L) and PMN stimulated by LPS (PMN- LPS) for 1.5 h (A) and 3 h (B). Supernatants were incubated for 0 min, 15 min and 30 min with the specific colorimetric substrate and read at 405 nm. Results of triplicate samples of 10 dogs are presented as mean ± standard error. Statistical analysis was performed using the Wilcoxon test (p < 0.05). Significant differences are indicated by * when comparing PMN (negative control) vs the different conditions, ^ when comparing PMN-LPS vs PMN + L and by " when comparing PMN+ L at 1.5 h vs 3 h. Fig. 7. Extracellular interaction between dog PMN and L. infantum promastigotes. Coverslips with adhered-PMN exposed to promastigotes (PMN+L) for 1.5 h and 3 h were observed by scanning electron microscopy (SEM). Resting-PMN were used as negative control. PMA-stimulated PMN (PMN-PMA) and PMA-stimulated PMN exposed to L. infantum (PMN + L-PMA) were used as positive controls. Representative SEM images of PMN (A), PMN+L (B), PMN-PMA (C) and PMN-PMA+L (D, E and F) incubated for 1.5 h (A, B, C and F) and 3 h (D and E) are shown at different magnification. White arrow – filamentous NET; Black arrow – cloud-like structure NET. The number of NET-generating cells was counted in 50 cells of each condition and the results are represented by the mean ± standard deviation of triplicates of three independent experiments (G). M. Pereira et al. Veterinary Parasitology 248 (2017) 10–20 15 compared with L. infantum promastigote cultures (p < 0.001) (Fig. 12). Optical microscopy revealed that some intracellular parasites preserved the structural integrity. However, images of PMN containing large vacuoles surrounding cellular debris that distort cellular mor- phology suggest parasite death (Fig. 13). Evidences of degraded para- sites were more frequent in some particular dogs. Some TEM images showed PMN interacting with extracellular parasites that have lost their normal ultrastructure: nucleus appeared more electro-dense and the presence of cytoplasmic blebs denotes loss of membrane integrity (Fig. 14A–D). Intracellular degraded parasites were observed within spacious phagosomes (Fig. 14E and F). Occa- sionally, the advanced intracellular degradation process prevented the identification of any parasitic organelle. 3.11. L. infantum maintains the viability and the capacity of multiplication after neutrophil phagocytosis In order to assess the viability of internalized parasites, PMN in- cubated with L. infantum were subjected to external parasite removal. After incubation, 5 × 105 (3.3 × 105, 8.3 × 105) moving parasites were counted, suggesting that the internalized parasites actively escape from PMN or were released after PMN death, maintaining the viability. Furthermore, after 72 h of incubation a significantly higher (p < 0.001) number of viable parasites were found) (Fig. 15). 4. Discussion The interaction between canine PMN and L. infantum is largely unknown. In a previous in vivo study our group demonstrated that the Fig. 8. Neutrophil extracellular traps limit parasite spread. Coverslips with adhered- PMN stimulated with PMA and exposed to L. infantum promastigotes for 3 h were ob- served by scanning electron microscopy. A close interaction between NET and the parasite (white arrows) evidence an en- trapping effect. Fig. 9. NET release evaluated by histone immunolabeling. Coverslips with adhered-PMN exposed to promastigotes (PMN+L) for 3 h were prepared for immunolabeling. Additional resting-PMN (PMN, nega- tive control), PMN stimulated with PMA (PMN-PMA) (positive con- trol) and PMN stimulated with PMA and exposed to L. infantum (PMN +L-PMA) were stained with anti-histone H1 FITC and DAPI. Representative images of cells stained with DAPI and anti-histone H1 and the respective merge images (×600 or ×1000 magnification) show the extracellular release of histone. M. Pereira et al. Veterinary Parasitology 248 (2017) 10–20 16 experimental injection of dogs with L. infantum promastigotes promotes a rapid dermal infiltration of PMN (Santos-Gomes et al., 2000). In the present study it becomes clear that live L. infantum promastigotes and culture supernatants induce a strong PMN migration, suggesting that some excretory/secretory substances released by the parasite are chemoattractant for PMN. Thus, Leishmania modulates leukocyte re- cruitment at the early phase of infection, favoring its internalization and consequently protection against soluble innate immune compo- nents, such as complement components that can destroy extracellular parasites. However, the previous contact with L. infantum inhibited PMN migration, which can promote the retention of infected cells at the inoculation site, ensuring parasite protection until macrophage arrival and its subsequent transference to the definitive host cell. Although promastigotes rapidly adhere to PMN surface, this is not a random interaction. Promastigotes preferentially adhere to PMN by the flagellum tip, possibly reflecting the main concentration of adhesion molecules (adhesiotopes) (Rittig and Bogdan, 2000). The attachment of L. infantum via the flagellum tip seems to promote the extend of sym- metrical pseudopods, that maintain the directional entry of the parasite into PMN (symmetrical phagocytosis). The type of phagocytosis seems to influence parasite fate with symmetrical phagocytosis favoring parasite killing (Hsiao et al., 2011). Although a relative high proportion of PMN rapidly interacts with the parasite, promastigote internalization requires a longer contact time. Moreover, in some dogs the parasite was detected almost ex- clusively outside the cell. Taking into account that parasite undergoes phagocytosis as a passive partner, these findings point towards different PMN responses to L. infantum in a host dependent manner. Dog genetic background, breed, age, sex and lifestyle may account for the different Fig. 10. Levels of viable, apoptotic and ne- crotic dog PMN after exposure to L. infantum promastigotes. PMN exposed to L. infantum (PMN+L) were treated with annexin V FITC and propidium iodine (PI) and analyzed by flow cytometry. Resting-PMN and (S)- (+)-camptothecin-stimulated PMN (PMN- C) were used as negative and apoptotic po- sitive controls, respectively. Frequency of viable (A), apoptotic (B), total necrotic (C) and of primary and secondary necrotic cells (D) of triplicate samples of 10 dogs is pre- sented by medians, 75th percentile and 25th percentile and, whiskers representing the highest and lowest values. Statistical ana- lysis was performed using the Wilcoxon test (p < 0.05). Significant differences are re- presented by * when comparing PMN (ne- gative control) vs the other conditions, ̎ when comparing PMN+L vs PMN+C (A–C) and ^ when comparing primary vs secondary necrotic cells in the same condition. Black and gray connecting lines indicate sig- nificant differences in primary and sec- ondary necrotic cells, respectively (D). Fig. 11. L. infantum delays apoptosis of parasitized PMN. PMN-L. infantum promastigotes incubated for 3 h were citocentrifuged, stained and observed under optical microscope. Non- parasitized early (black arrow) (A, B, C) and late (arrow head) apoptotic cells (C, D) can be observed (× 1000 magnification). Fig. 12. Exposure to dog PMN has a negative impact in L. infantum survival. After in- cubation with PMN for 24 h (PMN+L) active L. infantum promastigotes were quantified in a Neubauer-counting chamber. Promastigotes not exposed to PMN (L) were used as positive control. The amount of viable parasites is express by medians, 75th percentile and 25th percentile and, whiskers representing the highest and lowest values. Statistical analysis of triplicate samples of 10 dogs was performed using the Wilcoxon test (p < 0.05). Significant differences are represented by * when comparing L vs PMN+L. M. Pereira et al. Veterinary Parasitology 248 (2017) 10–20 17 phagocytic capacity of PMN. The majority of parasites internalized by dog PMN keeps an elon- gated appearance, suggesting that promastigotes did not differentiate into the amastigote form as observed by other authors (van Zandbergen et al., 2004). Probably, intracellular environment of PMN does not provide the required conditions for the differentiation process and/or the time that the parasite remains inside PMN is not sufficient to complete this process. This observation reinforces the role of PMN as ordinary transitional host cells. Parasite phagocytosis induced a sudden and strong oxidative burst that with time turns out to be suppressed, probably due to PMN death or parasite derivative ROS-deactivating molecules (Longoni et al., 2013; Kima, 2014). O2 − production may have a negative impact on L. in- fantum survival, as already described in L. donovani and L. major pro- mastigotes (Pearson and Steigbigel, 1981; Laufs et al., 2002). Indeed, internalized parasites showing no structural integrity were found inside large vacuoles, pointing towards phagocytosis dependent killing. However, this kind of images were more frequent in some dogs, in- dicating different responses depending on the cell donor. Similar to previously described by Gueirard et al. (2008), two types of phago- somes were found in the same PMN: large spacious phagosomes con- taining degraded parasites and tight phagosomes with intact parasites, suggesting that different parasites induce different PMN responses. Promastigotes used in the present study were in the stationary growth phase, which comprise a heterogeneous population of parasites with different abundance of virulent factors, namely gp63 and LPG (Ueno and Wilson, 2012). Thus, it can be hypothesized that non-virulent or less virulent promastigotes can be killed by dog PMN while virulent parasites may subvert the effector machinery of PMN, surviving and giving origin to a Leishmania infection in the dog. Extracellular effector mechanisms, such as NET release and enzyme exocytosis also can contribute to parasite killing. Although it is already Fig. 13. Large phagolysosomes ensure parasite killing. PMN exposed to L. infantum promastigotes for 3 h were citocentrifuged, stained and observed under optical microscope. Parasites that lost the structural integrity can be found inside large phagosomes (arrow) (×1000 magnification). Fig. 14. Parasite killing by dog PMN. PMN incubated with L. infantum promastigotes for 3 h were observed by transmission electron microscopy. Extracellular (A–D) and intracellular (E–H) parasite killing were observed. A degraded parasite can be seen next to an activated (A) and a necrotic (B) PMN. Different amplifications of the contact between PMN and promastigotes reveal extracellular parasite killing (C and D). A PMN containing a large phagolysosome with a degraded parasite and a tight parasitophorus vacuole containing an intact parasite (E and F) can be seen. Amplification of the parasitophorous vacuole showing subpellicular microtubules and the flagellar cytoskeleton constituted by a canonical 9 + 2 microtubular axoneme can be observed in G and H. NPMN – necrotic PMN; IP – intact parasite; DP – degraded parasite; white arrow – subpellicular microtubules; white star – flagellum. M. Pereira et al. Veterinary Parasitology 248 (2017) 10–20 18 described that several Leishmania species induce human PMN to emit NET (es-Costa et al., 2009, 2014; es-Costa et al., 2009, 2014; Gabriel et al., 2010), the present study demonstrates that L. infantum almost abolishes NET formation by dog PMN, indicating that the parasite ne- gatively modulate this effector mechanism, favoring parasite spreading and survival, and preventing the detrimental NET-induced pro-in- flammatory response. Furthermore, it is reasonable to assume that NE exocytosis at the early phase of infection contribute to parasite extra- cellular killing. However, enzyme decreased with infection time, pointing toward a late modulation parasite-induced. Indeed, inhibitors of serine peptidases that were identified in L. major and L. donovani (Eschenlauer et al., 2009; Alam et al., 2015) can play a role in L. in- fantum survival by negatively regulate the extracellular enzymatic ac- tivity. Although dog PMN reduced significantly parasite burden, a con- siderable proportion of intracellular parasites are viable and can re- plicate, probably using some of the evasion strategies already described (Laufs et al., 2002; Gueirard et al., 2008). These surviving parasites can be transferred to the definitive host cell and can perpetuate canine in- fection. In addition to the deleterious effects caused by PMN on the parasite, PMN viability is also negatively affected by the parasite, promoting secondary necrosis. This kind of death occurs when cells that undergo apoptosis experience subsequent necrosis and can occur after exposition to a strong stimulus (Iba et al., 2013). Secondary necrosis is char- acterized by the uncontrolled release of toxic components that promote pro-inflammatory responses, leading to tissue damage (Scaffidi et al., 2002) and generating an unfavorable environment for parasite survival. However, the absence of apoptotic cell removal by scavenger’s macro- phages can lead to cell elimination by necrosis (Silva, 2010) and justify the high levels of secondary necrosis found. Although it has been shown that some Leishmania were able to modulate PMN apoptosis, prolonging its life span or accelerating its death (Aga et al., 2002; Gueirard et al., 2008; Carlsen et al., 2013; Falcão et al., 2015), in the present study L. infantum infection did not increase apoptosis of dog PMN. In fact, the majority of apoptotic cells was not parasitized, suggesting that at early stages of infection in- tracellular parasites or even their products protect dog PMN, avoinding cell death. The mechanism implicated in this interaction is unknown, but LPG might be involved (Lüder et al., 2001). However, it is important to take into account that blood PMN may not act in the same way as PMN that have been recruited to the Leishmania inoculation site (Abebe et al., 2012). Furthermore, in vitro studies may not entirely reflect the in vivo conditions, since additional factors are involved as is the case of sand fly saliva and others com- ponents of the host immune system. The present study highlights the effector functions of dog PMN when exposed to L. infantum promastigotes, indicating that at least initially PMN promote the reduction of parasite load through intra and extracellular killing mechanisms. However, the inability of PMN to clear the parasite can favors dog infection. Additional studies are re- quired to clarify the diversity of effector functions found between PMN of different donors, correlating PMN phenotype with dog’s suscept- ibility to L. infantum infection. Funding This work was supported by the Portuguese Foundation for Science and Technology (FCT) through research grants (PTDC/CVT/113121/ 2009, PTDC/CVT/118566/2010, UID/Multi/04413/2013 and UID/ CVT/00276/2013) and PhD scholarships (SFRH/BD/73386/2010, SFRH/BD/77055/2011 and SFRH/BD/101467/2014).UID/Multi/ 04413/2013. Conflict of interest The authors declare no competing personal or financial interests. Acknowledgments A special acknowledge to Doutor MaríaVictoria Alarcón Sánchez from Centro de Investigaciones Científicas y Tecnológicas de Extremadura (CICYTEX) for her aid with fluorescence image acquisi- tion, to Telmo Nunes from Centro de Microscopia Eletrónica, Faculdade de Ciências, Universidade de Lisboa for his aid with SEM image ac- quisition, and to Amélia Fernandes for her help in image treatment. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.vetpar.2017.10.008. 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http://refhub.elsevier.com/S0304-4017(17)30445-4/sbref0210 http://refhub.elsevier.com/S0304-4017(17)30445-4/sbref0210 http://refhub.elsevier.com/S0304-4017(17)30445-4/sbref0210 http://refhub.elsevier.com/S0304-4017(17)30445-4/sbref0210 Canine neutrophils activate effector mechanisms in response to Leishmania infantum Introduction Material and methods Experimental design Selection of healthy dogs Parasites PMN isolation and purification In vitro infection Interaction between L. infantum promastigotes and PMN Chemotaxis assay Superoxide production Neutrophilic elastase (NE) exocytosis Neutrophil extracellular traps (NET) release PMN cell death Viability of L. infantum promastigotes after PMN contact Ultrastructural PMN-parasite interrelation Viability of PMN-phagocytized parasites Statistical analysis Results Dog PMN efficiently bind L. infantum promastigotes L. infantum promastigotes bind to dog PMN in an orientated manner and are engulfed via funnel-like pseudopods Dog neutrophils efficiently internalize L. infantum promastigotes Previous contact with L. infantum decreases PMN migration L. infantum stimulates dog PMN oxidative burst L. infantum regulates neutrophil elastase (NE) exocytosis L. infantum precludes NET formation L. infantum impacts PMN cell death L. infantum delays apoptosis of parasitized neutrophils PMN reduce L. infantum promastigote viability by extracellular and intracellular parasite killing L. infantum maintains the viability and the capacity of multiplication after neutrophil phagocytosis Discussion Funding Conflict of interest Acknowledgments Supplementary data References