Neutrophils Contribute to the Protection Conferred by ArtinM against Intracellular Pathogens: A Study on Leishmania major

ArtinM, a D-mannose binding lectin from Artocarpus heterophyllus, has immunomodulatory activities through its interaction with N-glycans of immune cells, culminating with the establishment of T helper type 1 (Th1) immunity. This interaction protects mice against intracellular pathogens, including Leishmania major and Leishmania amazonensis. ArtinM induces neutrophils activation, which is known to account for both resistance to pathogens and host tissue injury. Although exacerbated inflammation was not observed in ArtinM-treated animals, assessment of neutrophil responses to ArtinM is required to envisage its possible application to design a novel immunomodulatory agent based on carbohydrate recognition. Herein, we focus on the mechanisms through which neutrophils contribute to ArtinM-induced protection against Leishmania, without exacerbating inflammation. For this purpose, human neutrophils treated with ArtinM and infected with Leishmania major were analyzed together with untreated and uninfected controls, based on their ability to eliminate the parasite, release cytokines, degranulate, produce reactive oxygen species (ROS), form neutrophil extracellular traps (NETs) and change life span. We demonstrate that ArtinM-stimulated neutrophils enhanced L. major clearance and at least duplicated tumor necrosis factor (TNF) and interleukin-1beta (IL-1β) release; otherwise, transforming growth factor-beta (TGF-β) production was reduced by half. Furthermore, ROS production and cell degranulation were augmented. The life span of ArtinM-stimulated neutrophils decreased and they did not form NETs when infected with L. major. We postulate that the enhanced leishmanicidal ability of ArtinM-stimulated neutrophils is due to augmented release of inflammatory cytokines, ROS production, and cell degranulation, whereas host tissue integrity is favored by their shortened life span and the absence of NET formation. Our results reinforce the idea that ArtinM may be considered an appropriate molecular template for the construction of an efficient anti-infective agent.


Introduction
Global immunization regimes have eradicated smallpox and controlled a large number of other infections [1,2]. Indeed, vaccines have been successful against infections caused by extracellular pathogens or those whose pathogenesis is mediated by toxins. Under these circumstances, vaccines confer protection by inducing antibodies that neutralize the inoculum and prevent the establishment of infections [3]. This effective formula is not applied to prevent infections with intracellular pathogens [4] because they require T-cell mediated immunity for elimination. Therefore, a research field has emerged that concerns the development of alternative prophylactic or therapeutic agents to enhance host cellular response. In this context, agonists of innate immunity receptors, especially Toll-like receptors (TLRs), provide promising approaches [5]. The interaction of agonists with TLRs triggers cell signaling and production of inflammatory and anti-inflammatory mediators [6]. This process, beyond inducing early mechanisms of host defense, primes and orchestrates antigen-specific adaptive responses [7]. The ability of activated TLRs to modulate adaptive immunity motivates ongoing trials of new drugs based on natural or synthetic TLR ligands for infectious diseases in humans [8].
Concerning neutrophils, the cell type focused in this work, they are known to participate in the protection against intracellular pathogens, through mechanisms that involve phagocytosis, cell degranulation, ROS production, release of lipid mediators, and formation of neutrophil extracellular traps (NETs) [22]. Further mechanisms known to favor host defense are the release of cytokines combined with changes in cell life span [23]. Our previous work showed that ArtinM induces neutrophil migration by haptotaxis [24], due to the concomitant interactions of ArtinM CRDs with N-glycans on neutrophil surface receptors, such as those linked to C-X-C chemokine receptor 2 (CXCR2), and glycoproteins of the extracellular matrix, such as laminin [25,26]. Also, ArtinM activates neutrophils, causing tyrosine phosphorylation, L-selectin shedding, and interleukin-8 (IL-8) and leukotriene B4 (LTB4) secretion. These responses result in the enhancement of phagocytic and microbicidal abilities of neutrophils [19,20] and indicate that ArtinM activates neutrophils hugely.
Although effective against pathogens, neutrophils also account for exacerbated inflammation and tissue injury [27], a fact that caused concerns regarding the possibility of using ArtinM to design a novel class of immunomodulatory agents acting through carbohydrate recognition. Although exacerbated inflammation was never observed in the ArtinM-treated animals, we always had concerns regarding the occurrence of inflammatory tissue injury. Indeed, it is unclear how ArtinM may take advantage of neutrophil activation to eliminate pathogens, without promoting tissue damage. Therefore, in this study, we focused on understanding the mechanisms through which neutrophils contribute to the protective effect of ArtinM against intracellular pathogens and how this process occurs without exacerbating inflammation.

Ethics statement
The Ethics Committee of the Clinical Hospital of the Faculty of Medicine of Ribeirão Preto, University of São Paulo, approved this study (Doc. Number: 10012/2009) and all the adult volunteers signed an informed consent form prior to blood and/or urine donation.

Leishmania major uptake by neutrophils
Human neutrophils (2 × 10 6 cells/mL) were previously treated with ArtinM for 30 min, and infected with L. major. Infected/untreated neutrophils were used as controls. After 3 and 20 h post-infection, the cultures were cytocentrifuged (50 × g, 5 min) and Diff-Quik-stained (Laborclin, Brazil). The number of neutrophils with intracellular parasites was determined by count among 200 cells/condition/time, using an Olympus BX50 microscope coupled to a Nikon DXM-1200 photographic system.

Parasite viability
To analyze the intracellular leishmanicidal activity, we assessed the parasite viability, as described by Tavares et al. (2014) [28], with slight modifications. Briefly, neutrophils that were treated with ArtinM and untreated controls were infected and cultured for 3 h with the parasites. Uninternalized parasites were discarded from cultures by washing twice for 5 min at 100 × g, and the culture was immediately fed with supplemented Schneider's medium. The cells were then cultured at 26°C for an additional 48 h. The intracellular leishmanicidal activity was determined by assessing the number of extracellular motile promastigotes produced.
To analyze if some degranulated content had leishmanicidal activity, we performed a free cell assay based on methodology previously demonstrated by Mikus and Steverding (2000) [29]. Neutrophils treated with ArtinM for 1 h and untreated controls were pelleted, and the supernatants of the cells were collected. The supernatants were incubated with antibodies to myeloperoxidase (anti-MPO; ab62141; Abcam; 1/100) or neutrophil elastase (anti-NE; ab21595; Abcam; 1/100) for 30 min. Control supernatants were not incubated with the antibodies. After the incubation, 1 × 10 5 parasites were added to the supernatants for 1 h, and then assayed for viability in Schneider's medium containing Alamar Blue (Life Technologies, USA; 1/10 dilution) for an additional incubation of 24 h. Fluorescence measurement was performed on a FLx800 Fluorescence Microplate Reader (BioTek Instruments, USA; excitation, 590 nm; emission, 635 nm). Fluorescence count data from unchallenged, serially diluted parasites were used to obtain a standard curve of viable parasites.

ELISA
Freshly isolated human neutrophils (2 × 10 6 cells/mL) were previously treated with ArtinM for 30 min and infected with L. major. Neutrophils without ArtinM treatment and uninfected with L. major were used as negative controls. PMA stimulated (50 nM) neutrophils were used as the positive control. Cytokine levels were quantified in the neutrophil supernatants 20 h after infection and at the same time in the supernatants of controls. TNF, IL-1β (BD-Biosciences, USA), and TGFβ-1 (R&D Systems, USA) were measured by sandwich ELISA, according to the manufacturer's instructions.

Measurement of ROS production
The neutrophil chemiluminescence assay was performed in 96-well microplates using the procedure described by Lucisano-Valim et al. (2002) [30], with slight modifications. Freshly isolated human PMNs (2 × 10 5 cells/well in HBSS) were mixed with the chemiluminescent probes luminol (0.1 mM) or lucigenin (0.1 mM). The mixture was incubated at 37°C for 3 min, and the reaction was initiated after adding ArtinM, phorbol myristate acetate (PMA, 0.1 μM), formyl-Met-Leu-Phe (fMLP, 1 μM), or HBSS. Some assays were performed by first incubating the cells with ArtinM during 30 minutes, and then by adding fMLP, PMA or L. major (MOI 3:1). The luminol-and lucigenin-enhanced chemiluminescence was measured in a microplate luminometer (LB 960 Centro, Berthold Technologies, Germany), and light emission was recorded in photon counts per second (CPS) for 30-60 min, at 37°C. AUC represents the area under the time-course curve, which was used to determine the total amount of measured ROS.

Evaluation of Neutrophil Elastase (NE) activity
Neutrophil elastase activity was evaluated as previously described [31], with some modifications. Briefly, 1 × 10 5 neutrophils were treated for 30 min with different concentrations of ArtinM (2.5 μg/mL to 312 ng/mL), fMLP (1 μM), or medium only, and NE activity was detected by using the substrate N-succinyl-alanine-alanine-valine-p-nitroanilide (SAAVNA) (1 mM), which is cleaved by the enzyme released in the supernatant, forming p-nitroaniline as one of the products, which was spectrophotometrically quantified, using the microplate reader PowerWaveX (BioTek Instruments, USA; 405 nm).

Intracellular Myeloperoxidase (MPO) detection
Myeloperoxidase, as well as elastase, are azurophilic granule contents of neutrophils. Their intracellular levels were measured in neutrophils after 20 h of incubation with or without ArtinM, and challenged or not with L. major. To detach ArtinM-treated neutrophils, it was necessary to use EDTA-glucose (10 μM/5 μM) containing PBS (pH 7.2). Then, the cells were washed twice with PBS, and immediately fixed and permeabilized using the Cytofix/Cytoperm kit (BD-Biosciences, USA), following the manufacturer's instructions. Next, the cells were incubated with anti-MPO PE or anti-Hamster IgG1 PE isotype control antibodies (BD-Biosciences, USA), for 20 min. Immunofluorescent staining was analyzed by flow cytometry using Guava EasyCyte Mini (Millipore, USA).

NET-DNA release measurement
Neutrophils (2 × 10 5 cells/well in HBSS) were incubated with or without ArtinM, PMA (50 nM), or both together (ArtinM+PMA) in a 96-well plate for 2 and 4 h at 37°C, in 5% CO 2 . NET-DNA was quantified using a modified version of a previously published method [32]. Bacterial endonuclease EcoRI (200 unit/mL) was added for 10 min to partially digest any released NETs. Next, the cells and debris were pelleted by centrifugation at 600 × g for 10 min. A sample of the supernatant was added to SYTOX green nucleic acid stain (Invitrogen; 1 μM) in a black 96-well plate and incubated at 37°C for 10 min. NET-DNA fragments were quantified using a FLx800 Fluorescence Microplate Reader (BioTek Instruments, USA; excitation 485 nm, emission 528 nm) and the results have been expressed as Δ fluorescence values.

NET immunofluorescence detection
Neutrophils (2 × 10 6 cells/mL in HBSS) were incubated on poly-L-lysine-treated glass coverslips in a 24-well plate and treated or not with ArtinM or PMA, followed by incubation at 37°C for 6 h. Samples were collected, by gently removing coverslips, fixed with 3% paraformaldehyde (20 min at room temperature), and blocked in PBS supplemented with 3% FCS for 1 h at room temperature. Coverslips incubated overnight at 4°C with anti-neutrophil elastase antibody (ab21595; Abcam; 1:200) were washed and incubated for 1 h at room temperature with Alexa Fluor488 goat anti-rabbit IgG secondary antibody (Molecular Probes; 1:1000), and then washed and mounted with ProLong Antifade containing DAPI (Molecular Probes). Images were obtained using a Leica CTR 6000 fluorescence microscope, with Leica Application Suite software (Wetzlar, Germany).

DNA fragmentation analysis
DNA electrophoresis was performed to determine the effects of ArtinM treatment on neutrophils DNA degradation. The analysis was performed using a kit, the Wizard SV genomic DNA purification system (Promega Corporation, USA). Briefly, after 24 and 48 h of incubation, 4 × 10 6 cells were washed twice with PBS and lysed, and genomic DNA was isolated. The extracted DNA was quantified using the NanoVue Plus spectrophotometer (GE Healthcare, USA). A sample of 1 μg of DNA was analyzed using 1.5% agarose gel electrophoresis and stained with ethidium bromide. The DNA was then visualized under UV light on ChemDoc MP Imaging System and photographed using ImageLab software v.4.0 (both from Bio-Rad Laboratories, USA). Regarding the analysis of DNA fragmentation, the band area was quantified using ImageJ Software, and represented graphically in pixels 2 .

Phosphatidylserine exposure assessment
Neutrophil apoptosis was assessed by Annexin V staining [33]. Human neutrophils (2 × 10 6 cells/mL in RPMI-1640), were treated with ArtinM, IL-8, Lysis Buffer, or non-treated, and coincubated or not with L. major during 3 and 20 h at 37°C in 5% CO 2 . After incubation, the cells were detached, as described above (MPO detection), washed once and suspended in 100 μL of annexin V binding buffer (140 mM NaCl, 2.5 mM CaCl 2 , 1.5 mM MgCl 2 , and 10 mM HEPES, pH 7.4), containing annexin V-FITC or PE (1 μg/mL) for 15 min. Some assays were performed on neutrophils infected with green-fluorescent L. major forms (mβT3-LV39 strain), in order to detect if the dying cells are the ones infected. Immunofluorescence staining was analyzed by flow cytometry, using a Guava EasyCyte Mini instrument (Millipore, USA).

Disruption of mitochondrial transmembrane potential
Human neutrophils (2 × 10 6 cells/mL in RPMI-1640) were treated with ArtinM, IL-8, Lysis Buffer, or non-treated, and incubated during 3 and 20 h at 37°C in 5% CO 2 . JC-1 staining solution (10 μM) was then added to the wells and incubated at 37°C for 15 min. The fluorescent intensity for monomeric forms of JC-1 was measured using the FLx800 Fluorescence Microplate Reader (BioTek Instruments, USA; excitation 485 nm, emission 528 nm).

Statistical analysis
Statistical analyses were performed by Student t test, one way ANOVA followed by Bonferroni's post-test, and two way ANOVA followed by Bonferroni's post-test (all using GraphPad Prism software version 6; GraphPad), as indicated at the graph. The p values <0.05 were deemed statistically significant.

Results
ArtinM enhances uptake and killing of L. major by neutrophils To evaluate whether neutrophil activation by ArtinM contributes to its protective effect against intracellular pathogens [21], we compared the internalization and elimination of L. major by neutrophils that were pre-treated or were not pre-treated with ArtinM.
We assessed the effect of ArtinM treatment on the frequency of neutrophils with internalized parasites. At 3 h after in vitro infection, the number (15±0%) of ArtinM-treated neutrophils with internalized parasites was 88% higher than the number (7.5±0.7%) verified in untreated neutrophils (Fig 1A). The difference increased 140% (14.5±0.7% vs 34±2.8%) when the same assay was performed 20 h after infection (Fig 1A).
We also evaluated the leishmanicidal activity of the ArtinM-treated neutrophils. Cells were washed at 3 h post-infection and cultured in Schneider`s medium for additional 48 h. At this point, the number of mobile parasites (2.9x10 5 ±0.7x10 5 ) recovered from the culture of ArtinM-treated neutrophils was, on average, 50% lower than the number of viable parasites (5.8x10 5 ±1.3x10 5 ) recovered from untreated neutrophils (Fig 1B).
In conclusion, our data show that ArtinM treatment increased the ability of neutrophils to eliminate L. major, a fact that favors the idea that neutrophils can contribute to the protective effect of ArtinM against infection.
ArtinM alters the pattern of cytokine production by L. major-infected neutrophils The prominent production of inflammatory or anti-inflammatory cytokines by neutrophils contributes to an appropriate microenvironment for L. major elimination or survival, respectively [34,35]. We quantified the TNF, TGF-β, and IL-1β cytokines in the supernatant of nontreated or ArtinM-treated neutrophils that were infected or not with L. major. Twenty h after ArtinM-treatment, the supernatant contained 7-fold higher concentration of TNF (241.7±5.59 ρg/mL) than the supernatant of untreated neutrophils (65.65±5.47 ρg/mL). A similar level was detected when the assay was performed with L. major infected neutrophils (245.77±3.93 ρg/mL and 93.77±0.68 ρg/mL- Fig 2A). On the other hand, TGF-β production was 3-fold augmented in L. major-infected neutrophils (169.6±32.01 ρg/mL), while ArtinM-treated cells, infected (56.99±16.73 ρg/mL) or not (27.51±3.14 ρg/mL), released levels that were similar to the ones verified for untreated cells (54.94±15.27 ρg/mL- Fig 2B). IL-1β was not detected in the supernatant of untreated or L. major infected neutrophils. In contrast, we detected IL-1β production by ArtinM-treated neutrophils (14.37±2.19 ρg/mL), which increased by 6-fold when these ArtinM-treated cells were infected with L. major (94.67±7.01 ρg/mL- Fig 2C). Taken together, our results show that ArtinM treatment enhances the production of TNF and IL-1β, but not of TGF-β by human neutrophils. Instead of the high levels of TGF-β produced by L. major-infected neutrophils, the ArtinM pre-treated neutrophils released higher levels of TNF and IL-1β.

ArtinM induces neutrophil degranulation
Releasing of the contents of neutrophil granules contributes to elimination of pathogens [36]. In the case of L. major infection, elastase released by azurophilic granules of neutrophils plays an outstanding protective role in host responses [37]. Here, we examined the release of neutrophil elastase (NE) and myeloperoxidase (MPO) by cells that were treated or not with ArtinM. The occurrence of neutrophil degranulation was verified by detection of (1) decreased Fig 1. ArtinM enhances L. major uptake and killing by human neutrophils. Human neutrophils were treated or were not treated with ArtinM, and infected with L. major promastigotes (MOI 3:1). A-L. major uptake. After incubation for 3 and 20 h, the cells were centrifuged on slides and stained for evaluation by light microscopy. Arrows indicate internalized parasites. In the graphic, data are expressed as the mean of neutrophils with intracellular parasites ± SD. * p<0.05; ** p<0.01 in comparison to untreated cells at the same period. One way ANOVA followed by Bonferroni's post-test. B-L. major killing. After 3 h of infection the cultures were washed to discard uninternalized parasites, fed with Schneider`s medium and cultured for additional 48 h. Motile promastigotes were counted, and the data are expressed as mean of viable parasites ± SD. ** p<0.002: compared to untreated, Student t test. Each assay was carried out in triplicate. The shown data are representative from three different experiments. The intracellular MPO content in ArtinM-treated neutrophils was 4-fold lower than that verified in untreated cells (Fig 3A). Consistent with this, 2-fold higher levels of NE activity were detected in the supernatant of ArtinM-treated neutrophils in comparison to that observed in the supernatant of untreated cells (Fig 3B). This last assay was actually performed to verify the period necessary for occurrence of neutrophil degranulation in response to ArtinM, and the highest NE activity was detected shortly (5-60 min) following treatment, even when the concentrations used were as low as 312 ng/mL of lectin, as demonstrated by a time-course assay (S2 Fig). We also examined the neutrophil degranulation after L. major infection. Regarding MPO intracellular content, we verified that the infection per se was able to inhibit the degranulation of untreated cells (70%- Fig 3A). In contrast, the intracellular levels of MPO decreased drastically in the ArtinM-treated neutrophils (3-fold), showing that the lectin promotes degranulation even in L. major infected neutrophils (Fig 3A). Therefore, ArtinM induces degranulation of both uninfected and infected neutrophils.

Elastase contributes to the augmented leishmanicidal activity induced by ArtinM on neutrophils
To assess the leishmanicidal activity of the neutrophil secreted products, we incubated parasites with the supernatant of cells that were pre-stimulated with ArtinM. The number of viable L. major was 66% lower following incubation with the supernatant of ArtinM-treated neutrophils (2.0x10 4 ±0.6x10 4 ) than with the supernatant of untreated cells (6.0x10 4 ±6.4x10 4 ). In order to evaluate the specific contribution of MPO and NE, released by azurophilic granules, to the observed leishmanicidal activity, the supernatant of ArtinM-treated neutrophils was pre-incubated with antibodies specific to MPO or NE, and then added to the parasite suspensions. Our results show that anti-MPO had no effect on the number of viable L. major (1.5x10 4 ±0.07x10 4 ), whereas anti-NE antibodies inhibited the leishmanicidal activity provided by the supernatant of ArtinM-treated neutrophils once the number of parasites recover in this condition (5.5x10 4 ±0.2x10 4 ) was similar to that found on the supernatant coming from untreated neutrophils (Fig 3C). These data suggest that the leishmanicidal activity of ArtinMtreated neutrophils is due, at least partially, to an NE-mediated mechanism.

ArtinM inhibits the formation of Neutrophil Extracellular Traps induced by Leishmania major
Besides killing microbes through its direct enzymatic activity [38][39][40], NE also contributes to the formation of neutrophil extracellular traps (NET) [41], which constitutes a mechanism for capture and kill microorganisms [42] and for host tissue damage [43]. To investigate whether neutrophil treatment with ArtinM could result in NET formation, the DNA concentration was measured in the supernatant of neutrophils, 2 and 4 h after ArtinM-treatment. The DNA levels detected in the supernatant of ArtinM-treated cells were as low as those found in untreated neutrophils. In contrast, neutrophil stimulation with PMA (positive control) resulted in the detection of high DNA levels (> 5-fold, Fig 4A), at both periods analyzed. When we assayed L. major-infected neutrophils, we observed that untreated cells formed NET, in concordance with previous reports [44], but, in contrast, ArtinM-treated cells do not form NET (Fig 4A). The microscopic observation of the assayed cells was consistent with the DNA measurement in the neutrophil supernatant (Fig 4B). Therefore, we concluded that the ArtinM treatment does not induce NET formation and inhibits the formation of NET that follows L. major infection.

ArtinM induces ROS production only by infected neutrophils
Once we had verified that ArtinM triggers intense neutrophil activation not associated with NET formation, we evaluated whether lectin induces the production of ROS, which is directly implicated in NET formation [45]. We monitored the levels of ROS following exposure to the ArtinM stimulus, through reactions with luminol or lucigenin, which are oxygenated by H 2 O 2 (and its derived species), or by superoxide anion, respectively [46].
As shown in Fig 5, ArtinM-treated neutrophils, as well as untreated control cells, did not produce ROS since low and stable levels were demonstrated by both luminol-and lucigeninchemiluminescence detection. In contrast, neutrophils stimulated with PMA or fMLP (positive controls) triggered high ROS production (Fig 5A and 5B).
Although ArtinM did not induce ROS production, it did not inhibit the production induced by other agents, a fact that was demonstrated by the observation that following the incubation with ArtinM, neutrophils responded to PMA-or fMLP-stimulus, with ROS levels similar to the ones measured in cells that were not pre-incubated with ArtinM ( Fig 5C and S3 Fig).

ArtinM treatment prolongs neutrophil survival
The sustained neutrophil activation induced by ArtinM, which was detected even 20 h after treatment (S4 Fig), as well as the absence of NET formation and ROS production, motivated us to evaluate the survival rate of ArtinM-treated neutrophils. Several methods were used to assess the occurrence of cell death, such as the analysis of neutrophil morphological changes, fragmentation of genomic DNA, phosphatidylserine (PS) exposure, and disruption of the mitochondrial transmembrane potential.
The morphology of neutrophils was evaluated by optical microscopy. At 3h post-treatment, the microscopic features of ArtinM-or IL-8-treated cells, as well as of untreated cells, were typical of live neutrophils. At the same time point, cells treated with lysis buffer, as expected, showed remarkable nuclear condensation, as usually observed in apoptotic neutrophils [5,6]. After 20 h, nuclear condensation was verified in untreated cells, while the ArtinM-or IL-8-treated ones preserved their original morphology (Fig 6A).
ROS production, mostly mitochondrial, is implicated in the initiation of cell apoptosis [47]. Since disruption of the mitochondrial trans-membrane potential (ΔC) is one of the earliest intracellular events occurring in apoptotic cells, we examined mitochondrial instability through the detection of JC-1 monomer. Fig 6C shows that the treatment of neutrophils with ArtinM (5091±276.5 MFI) or IL-8 (4446±800.8 MFI) favored mitochondrial stability, since at 3 h posttreatment, the levels of JC1 monomer were at least half of those detected in untreated (10020 ±105.4 MFI) or lysis buffer-treated neutrophils (12186±1037 MFI). At 20 h, although JC1 monomer levels increased in ArtinM (9331±211.2 MFI) or IL-8 (8066±635 MFI) treated neutrophils, they remained significantly lower (-15%) than those detected in untreated neutrophils (11035±891 MFI). JC1 monomers were not detected in the rare neutrophils remaining 20 h after treatment with lysis buffer.
The electrophoresis analysis of DNA showed that ArtinM-treated neutrophils displayed at least two-fold less fragmented DNA than untreated cells at 24 h of incubation. At 48 h of incubation, treated and untreated neutrophils displayed similar DNA fragmentation. (Fig 6D and 6E).
To summarize, these data demonstrate that ArtinM treatment of human neutrophils postponed apoptosis, as shown by delayed disruption of the mitochondrial trans-membrane potential, DNA fragmentation, nuclear condensation and PS exposure. Altogether, these results revealed that ArtinM treatment prolongs neutrophil survival.

ArtinM triggers early apoptosis of L. major infected neutrophils
We found that ArtinM prolongs neutrophil survival, a fact that is considered to facilitate L. major infection [48]. Then, we analyzed the PS exposure on ArtinM-treated neutrophils that were infected with L. major. The ArtinM effect, exerted 3 hours after stimulation on the ArtinM inhibits NET formation induced by L. major. Human neutrophils were incubated with ArtinM, PMA, ArtinM+PMA, or medium (untreated), and were either infected or not infected with L. major promastigotes (3:1). A-DNA quantitation. DNA released at 2 and 4 h after treatment/infection was quantified in the cell supernatants by fluorescence detection (Ex./Em. 480/520 nm) after reaction with SYTOX green. Data are expressed as a mean of fluorescence intensity ± SD. **p<0.01; ***p<0.001; **** p<0.0001. Two way ANOVA followed by Bonferroni's post-test. DNA quantitation assays were performed in triplicate and, the data shown are representative of four independent experiments. B-NET immunofluorescence detection. Neutrophils were plated onto poly-l-lysine coated slides, treated or untreated with PMA or ArtinM, and either infected or not infected with L. major promastigotes (MOI 3:1). After 6 h, the cells were stained with DAPI (DNA blue stain) and with anti-NE antibody (green). Merged images confirmed NETs by colocalization of staining. NET immunofluorescence detection assays were performed in duplicate and the data shown are representative of three independent experiments. doi:10.1371/journal.pntd.0004609.g004  Data are expressed as mean of percentage of AnnexinV + neutrophils ± SD. *** p<0.001 in comparison with the untreated cells, two way ANOVA followed by Bonferroni's post-test. ND stands for "not detected". C-JC-1 monomer detection. Neutrophils were incubated with JC-1 probe and green fluorescence detection (Ex/Em = 485/528) was performed at 3 and 20 h after treatment. Data are expressed as mean of fluorescence intensity ± SD. *** p<0.01 comparing with untreated curve, two way ANOVA followed by Bonferroni's post-test. D and E-Electrophoretic detection of DNA fragmentation. Neutrophils were incubated for 24 and 48 h with ArtinM or medium (untreated). Their genomic DNA was analyzed by gel electrophoresis. Images are shown on panel D. Panel E represents the band area in pixels 2 , regarding the electrophoretic detection of DNA fragmentation (ImageJ Software). Each assay was carried out in triplicate. The shown data are representative from three different experiments.
Some assays performed by infecting neutrophils with fluorescent L. major forms allowed verifying that 91% of the AnnexinV labeled cells were infected. A close proportion (89%) was verified among ArtinM-treated cells as well (S5 Fig). We therefore concluded that apoptotic neutrophils were most frequently L. major infected cells, and that ArtinM did not change this distribution.

Discussion
The immunomodulatory lectin ArtinM induces Th1 immune response and confers resistance to intracellular pathogens [21], without causing apparent tissue damage. The response to ArtinM is induced by its interaction with glycoconjugates on several immune cells, namely, macrophages, dendritic cells, neutrophils, mast cells and lymphocytes [10,11,13,16,17,[24][25][26]49]. Considering that neutrophils constitute a two-edged sword, accounting for resistance to pathogens and also for tissue injury, a full exploration of the neutrophil responses is required to envisage a possible application of ArtinM, or its analogues, as immunomodulatory agent. In this study, we verified that ArtinM enhances the neutrophils ability of eliminating the intracellular pathogen L. major. The parasite killing was associated with strong neutrophil activation, not accompanied by NET formation, which is known to cause tissue damage.
Although macrophages are the definitive refuge for Leishmania species in the host, neutrophils are considered by many as transitional shelters for the few invader parasites that survive the toxic extracellular milieu [50]. This idea is based on the observation that in the early stages of L. major infection, caused by the bite of an infected sandfly, large amounts of neutrophils are attracted to the site. Since a low number of macrophages reside in the invaded tissue and the recruited neutrophils failed to kill L. major, the authors concluded that the invading parasites depended on the rapidly recruited neutrophils to survive [51]. Once inside the neutrophils, L. major promastigotes postpone neutrophil apoptosis until 2 days [48] and are silently transferred to macrophages, without activating the immune response [51]. In opposition to this "Trojan horse" mechanism of parasite evasion, ArtinM accelerates the death of L. majorinfected neutrophils, favoring the process of parasite elimination.
The production of the anti-inflammatory cytokine TGF-β favors the silent uptake of apoptotic cells by macrophages, whereas the production of pro-inflammatory mediators like TNF and IL-1β decreases after the uptake of apoptotic cells [52]. Consequently, the prominence of TGF-β correlates with permissibility to L. major infection, whereas high production of TNF is associated with resistance to infection [35]. Our observation that L. major-infected neutrophils largely augmented TGF-β production and diminished the secretion of pro-inflammatory mediators is consistent with previous demonstrations that neutrophils are important contributors toward providing the required microenvironment for parasite survival [34,53,54]. Notably, the treatment of neutrophils with ArtinM inverted this pattern by significantly diminishing TGF-β production and augmenting the secretion of pro-inflammatory cytokines. We found that ArtinM promoted IL-1β secretion by human neutrophils, which was maximum when the lectin was added to L. major infected neutrophils. This is a relevant finding because IL-1β maturation, as recently demonstrated, results from activation of the NLRP3 inflammasome, which is an innate platform that crucially restricts parasite replication. The NLRP3 inflammasome triggers inducible nitric oxide synthase (NSO2)-mediated production of NO, a potent leishmanicidal factor [55]. In addition, NLRP3 inflammasome activation is associated with ROS production [56], which is a major factor for Leishmania killing by human cells [57]. Indeed, macrophages derived from human monocytes do not produce NO after classical activation [58] or upon infection with Leishmania [59]. Thus, neutrophils, monocytes, and macrophages can control the parasites via ROS that are produced during the respiratory burst process [60]. By promoting ROS production and activating the NLRP3 inflammasome, ArtinM facilitates Leishmania killing, as shown in this work. Considering that the importance of IL-1β, derived from inflammasome, for conferring resistance to Leishmania infection was demonstrated for other species than L. major [55], we plan performing further experiments with L. braziliensis-infected neutrophils, to better assess the IL-1β relevance for the ArtinM-induced protection against the parasite.
Another trump against Leishmania infection provided by ArtinM treatment was the increased degranulation of human neutrophils. The intracellular levels of MPO were diminished in ArtinM-treated neutrophils, whereas the NE activity was augmented extracellularly. Both MPO and NE are stored in azurophilic granules, and their enzymatic activities are known to be implicated in the degradation of microorganisms in phagolysosomes. MPO is a key component of the oxidative burst, producing hypochlorous acid and other reactive oxidants [61], whereas NE is a serine protease that degrades the outer membrane of microorganisms, inducing their elimination [39]. NE and MPO have low capacity to be exocytosed from the neutrophils azurophilic granules during infectious and inflammatory processes [36,[62][63][64]. We found that treatment with ArtinM increased the exocytosis of these granules, even when the cells were infected with L. major. Many authors attribute an important role to neutrophil degranulation in mounting a response that culminates in pathogen elimination or control [36,65,66]. Regarding NE, recent data revealed that its release into the extracellular environment induces macrophage activation via TLR4, which culminates in augmented internalization and elimination of L. major [67]. In the current work, we did not investigate the signaling through TLR4, nor the relationship with macrophages, but we demonstrated that the ArtinM-induced NE release was followed by elimination of L. major by human neutrophils (Figs 1 and 2). The released NE accounts for the ArtinM leishmanicidal effect, once the NE presence in the extracellular space correlates with L. major elimination, as indicated by experiments using anti-NE neutralizing antibodies. Although NE is a serine protease that promotes microbe killing, our study apparently constitutes the first demonstration of its leishmanicidal activity, a finding that surely deserves further investigation.
ArtinM-treated neutrophils, in sterile conditions, did not produce ROS, while other neutrophil activators such as fMLP and PMA induced rapid and intense responses. The L. major infection of neutrophils increased ROS production, which was even higher in ArtinM-treated neutrophils. It is well established that ROS provides an important mechanism to combat L. major [68], but is also able to cause tissue injury. The ROS production induced by ArtinM was restricted to infected cells whose life span was shortened by ArtinM treatment, allowing us to Fig 8. ArtinM increases the leishmanicidal capacity of human neutrophils: a model. Once infected with L. major, non-stimulated neutrophils produce high levels of TGF-β, and low levels of TNF, and IL-1β, which are associated with decreased cell degranulation and postponed cell death. In contrast, ArtinMtreated neutrophils become activated, produce high levels of TNF, and IL-1β, while TGF-b is reduced, and enhance degranulation. Collectively, these ArtinM induced responses augments the leishmanicidal capacity of neutrophils.
doi:10.1371/journal.pntd.0004609.g008 postulate that the ability of ROS to cause tissue damage could be reduced. The effect of ROS on parasite elimination could be preserved, since it is effective in the early stages of infection [69].
ROS production accounts for NET formation, as demonstrated by the observation that neutrophils treated with NADPH oxidase, a pharmacological inhibitor of ROS production, do not form NET in response to conventional stimuli [70]. We showed that ArtinM-stimulated neutrophils, either uninfected or infected with L. major, did not form NET. This means that ArtinM inhibits the NET formation induced by the infection itself [44] and additionaly by PMA The inhibition occurred in spite of NE release and ROS production, which favor NET formation and were verified to occur in ArtinM-treated neutrophils. NET formation constitutes a mechanism whereby neutrophils eliminate pathogens, including L. amazonensis promastigotes [71]. Other Leishmania spp, namely, L. donovani, L. major, L. infantum, and L. mexicana, escape from their toxicity despite NET formation and trapping by these web-like structures [44,72,73]. The fact that ArtinM, although inhibiting NET formation, facilitates L. major clearance by human neutrophils reinforces the idea that NET formation is not required for L. major elimination. On the other hand, the absence of NETs can favor host tissue integrity, since NET-associated proteases and granular proteins have been shown to damage host tissue [74].
We conclude that ArtinM treatment of human neutrophils enhances the clearance of the intracellular pathogen L. major, through mechanisms that include: (1) production of inflammatory cytokines, i.e., high TNF and IL-1β, and virtual absence of TGF-β; (2) increased neutrophil degranulation; the released elastase promotes L. major elimination; (3) increased ROS production by infected neutrophils (4) shortened neutrophil survival. On the other hand, the host tissue integrity is favored by (1) the short period of ROS production and (2) the absence of NET formation.