Primary human neutrophils were isolated from peripheral blood of healthy volunteers by Histopaque and Percoll gradient centrifugation as described elsewhere (21). The blood collection was conducted with the understanding and written consent of each participant and was approved by the ethical committee of the Medical Faculty of the University of Lübeck (18-187). Blood was layered on a two-layer density gradient consisting of lymphocyte separation medium 1077 (upper layer, PAA, Pasching, Austria) and Histopaque^® 1119 (bottom layer, Sigma-Aldrich, Deisenhofen, Germany) and centrifuged for 5 min at 300 × g followed by 20 min at 800 × g. Cells from the upper layer consisting mainly of lymphocytes and monocytes were discarded. The granulocyte-rich lower layer was collected leaving the erythrocyte pellet at the bottom of the tube. Granulocytes were washed once in PBS, resuspended in complete medium (RPMI 1640 medium, Sigma) supplemented with 50 µM 2-mercaptoethanol, 2 mM L-glutamine, 10 mM HEPES (all from Biochrom, Berlin, Germany) and 10% fetal calf serum (FCS, Gibco, Karlsruhe, Germany) and further fractionated on a discontinuous Percoll^® (Amersham Biosciences, Uppsala, Sweden) gradient consisting of layers with densities of 1.105 g/ml (85%), 1.100 g/ml (80%), 1.093 g/ml (75%), 1.087 g/ml (70%), and 1.081 g/ml (65%). After centrifugation for 20 min at 800 × g, the interface between the 80 and 70% Percoll^® layers was collected, washed once in PBS and resuspended in complete medium. All procedures were conducted at room temperature. The cell preparations contained > 99.9% granulocytes as determined by morphological examination of > 1,000 cells on Giemsa stained cytocentrifuge (Shandon, Pittsburgh, PA) slides. Cell viability was > 99%, as determined by trypan blue exclusion.

Neutrophils (500.000 cells/sample) were resuspended in FACS buffer [1x PBS (Thermo Fisher) supplemented with 1% bovine serum albumin and 1% human serum] in a V-bottom plate. After washing once with FACS buffer the cells were stained with FITC-conjugated mAb to human CD62L (clone DREG-56, IgG1, BD Biosciences) and PE-conjugated mAb to human CD11b (clone 2LPM19c, IgG1, Dako, Waldbronn, Germany) for 30 min at 4°C protected from light. After two wash steps with FACS buffer at 4°C the cells were resuspended in FACS buffer and measured with a BD FACS Canto II (BD) flow cytometer.

Apoptosis of neutrophils was determined by Annexin-V FLUOS binding as Annexin-V exhibits calcium-dependent binding to phosphatidylserine (PS) expressed in the outer leaflet of the cell membrane of apoptotic neutrophils. Assessment of membrane integrity and identification of necrotic cells was performed by counterstaining with propidium iodide (PI). For staining, 5×10⁵ cells/100 µl were transferred to a U-tube and 10 µl Annexin-V buffer (10 mM HEPES + 140 mM NaCl, pH 7.4), 1 µl 1 M CaCl₂, 1 µl PI (Sigma-Aldrich) and 1 µl Annexin-V FLUOS (Sigma-Aldrich) were added. Samples were incubated 10 min at 4°C protected from light. Analysis was carried out by flow cytometry.

Virulent L. donovani promastigotes (strain MHOM/IN/82/Patna 1) were cultivated in Schneider’s Drosophila Medium with L-glutamine (Genaxxon, Ulm, Germany) supplemented with 10% FCS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2% sterile filtered human urine at 27°C in a humidified air atmosphere containing 5% CO₂. For seeding of cultures, a parasite density of 1 × 10⁶ cells/ml was used. The parasite counting was conducted in a hemocytometer with a chamber depth of 0.02 mm (0.0025 mm², depth 0.02 mm, VWR). The culture was considered to be in the stationary phase 72 h after seeding. Serial passaging was conducted until passage 10.

Stationary phase L. donovani promastigotes were labeled with CFSE (Cell Trace CFSE, Invitrogen, Thermo Fischer Scientific) by incubating the parasites in a volume of 1 ml at a concentration of 1x10⁸/ml with 5 µM CFSE in PBS for 15 min. with shaking. 500 µl complete medium was then added and incubated for another 5 min with shaking. Cell were then centrifuged at 1300 x g and resuspended in complete medium.

Neutrophils and L. donovani promastigotes were co-incubated in complete medium at a parasite-neutrophil ratio of 10:1 and a neutrophil density of 5 × 10⁶ cells/ml for 3 h at 37°C in a humidified air atmosphere containing 5% CO₂. Subsequently, the infection rate was assessed on Giemsa-stained cytocentrifuged samples. The cells were washed six times in 1 × PBS at 400 × g for 10 min to remove extracellular Leishmania. Infected cells were again adjusted to 5 × 10⁶ cells/ml.

The luminol-based chemiluminescence assay was used to detect the sum of intra- and extracellular ROS. 4×10⁵ neutrophils (2x10⁶ cells/ml) were seeded per well in a 96-well LUMITRAC™ 600 plates (Greiner Bio-One, Frickenhausen Germany) and mixed with a final concentration of 60 µM luminol (Sigma-Aldrich). Neutrophils were exposed to ATP (500 µM), UTP (500 µM), or LPS (100 ng/ml) or fMLP (1 µM). ROS-dependent luminol chemiluminescence was assessed using an infinite 200 reader and the Tecan i-control 1.7 Software (Tecan, Crailsheim, Germany). ROS release was monitored for 2 h every 2 min at 37°C. For statistical analysis, the area under the curve (AUC) value of each sample was calculated.

To assess the effect of ATP and UTP on the ROS production of Leishmania-exposed neutrophils, neutrophils were co-incubated with L. donovani promastigotes in the presence or absence of ATP or UTP and the ROS production was assessed for 60 min by using the luminol based chemiluminescence assay.

Intracellular Ca²⁺-concentration was determined by using a flow cytometry-based method with the fluorescent Ca²⁺-sensitive indicator Fluo-4 AM as described (22). Briefly, neutrophils, 1x10⁷/ml, were labeled with 10 µM Fluo-4 AM (AAT Bioquest, Sunnyvale, CA) for 30 min in the dark. After washing with PBS cells were resuspended in complete medium at a concentration of 5x10⁶/ml. Neutrophils were infected with Leishmania by co-incubating neutrophils with L. donovani promastigotes at a neutrophil to Leishmania ratio of 1:10 for 2h at 37°C. ATP (500 µM) or UTP (500 µM) was added to the infected neutrophils. Fluorescence intensity was assessed for 3 min in FITC channel by using a BD FACS Canto II flow cytometer (BD). The stimuli were added at the time point 30 sec of the flow cytometry analysis. Fluo-4-labeled non-infected cells were used as negative controls, Fluo-4-labeled non-infected cells exposed to ionomycin (10 µM) were used as positive control.

Neutrophils (5×10⁶ cells/ml) were exposed to ATP (500 µM), to Leishmania donovani promastigotes (Leishmania to neutrophil ratio 10:1) or a combination of ATP with Leishmania donovani promastigotes for 15 min at 37°C. In samples with the P2-antagonist PPADS (Tocris, Bio-Techne, Wiesbaden, Germany) the neutrophils were pretreated with the inhibitor (30 µM) for 15 min. Whole cell lysates were prepared as described (23). Western blot analysis was carried out by using antibodies against human phospho Akt (Thr308), phospho-p44/42 MAPK (ERK1/2, thr202/Tyr204), phospho p38 MAPK (Thr180/Tyr182) and beta-actin (all from Cell Signaling Technology) and probed with HRP-conjugated anti-rabbit IgG secondary antibody (New England Biolabs, USA). The signals were detected by using Immobilon Western Chemiluminescence HRP substrate (Millipore, USA) with a Fusion Fxt Chemiluminescence reader (Vilber Loumat, Germany). Signals of pAkt, pERK1/2, or pp38 were related to beta-actin signals on the same blots or of the same sample by using ImageJ software (NIH, USA). Data were then normalized by sum of all data point in a replicate as described (24).

Neutrophils (1x10⁶ cells/100 µl) were exposed to ATP (500 µM), UTP (500 µM), L. donovani promastigotes or a combination of ATP and UTP with L. donovani promastigotes or medium alone for 18 h at 37°C, 5% CO₂. Cultures were centrifuged at 300 x g for 10 minutes to separate cytokine-containing supernatants from cells. Detection of IL-8 and IL-1β in the supernatants was conducted by using Duo-Set ELISAs (R&D Systems) according to the manufacturer’s instructions.

Leishmania-infected neutrophils were exposed to ATP (500 µM), UTP (500 µM) or LPS (100 ng/ml) as well as to the combination of LPS with ATP and UTP for 18 h at 37°C in a humidified air atmosphere containing 5% CO₂. A limiting dilution culture assay was then used to detect viable L. donovani parasites in neutrophils and survival of L. donovani parasites as described previously (25). Briefly, serial 1.5-fold dilutions of L. donovani-infected neutrophil suspensions (5 × 10⁶ cells/ml) were plated in three replicates in 96-well flat-bottom microtiter plates containing Schneider’s Drosophila Medium. The plates were incubated at 27°C in humidified air atmosphere containing 5% CO₂ for 7-10 days. The growth of L. donovani promastigotes was detected microscopically. The last dilution resulting in a growth of parasites in > 50% of the wells is given as a quantitative measure of the parasite load in the neutrophil cell suspension.

If not stated differently, the presented data were collected/generated from minimum of three independent experiments with neutrophils isolated from different blood donors. Statistical analysis was performed with the GraphPad Prism software 6 using the ordinary one-way ANOVA followed by Turkey’s multiple comparison. A p-value ≤0.05 was considered statistically significant.

Since activation of PMN leads to shedding of L-selectin (CD62L) from the cell surface (26), analysis of CD62L expression is a widely used method to distinguish CD62L^neg/low activated cells from CD62L^high non-activated neutrophils (27). To obtain information regarding the activation status of neutrophils upon exposure to ATP and UTP the expression of L-selectin (CD62L) was assessed via flow cytometry. As compared to untreated cells, a marked shedding of CD62L was observed in neutrophils upon a 30 min exposure to ATP and UTP ( Figure 1A ).

Granule exocytosis is an important function of activated neutrophils. During this process, the granule membrane incorporates into the cell membrane. Consequently, increasing expression of granule-membrane markers such as CD11b (28) can be observed on the cell surface of activated PMN. To investigate whether exposure to ATP and UTP affects granule exocytosis the cell surface expression of the degranulation marker integrin alpha M (CD11b) was assessed by using flow cytometry. The expression of CD11b was significantly enhanced on ATP- and UTP-treated neutrophils. ( Figure 1B ). These data indicate an activated state and granule exocytosis of neutrophils upon exposure to extracellular ATP and UTP.

The production of reactive oxygen species (ROS), the oxidative burst, is one of the most important mechanisms of neutrophil granulocytes to kill phagocytosed microbes. The ability to induce the production of ROS is characteristic for many neutrophil-activating agents. Therefore, we investigated the effect of ATP and UTP on the ROS production by human neutrophils. The production of MPO-derived ROS and extracellular superoxide was assessed by the luminol-based chemiluminescence assay. PMN were incubated in medium alone as negative control or in the presence of ATP (500 µM) or UTP (500 µM). Exposure to ATP and UTP did not induce a significant level of ROS production ( Figures 2A–F ).

A characteristic feature of low-level PMN activation is the priming, i.e., the amplification of the neutrophil’s functional response to other stimuli. We have investigated whether ATP and UTP can amplify LPS- or fMLP-induced ROS production of neutrophils. Treatment with LPS ( Figures 2A, B ) and fMLP ( Figures 2C, D ) resulted in ROS production. Importantly, exposure of LPS- and fMLP-preincubated cells to ATP and UTP markedly and significantly enhanced the production of ROS ( Figures 2A–D ). These data show that ATP and UTP alone do not induce significant level of ROS production but enhance LPS- and fMLP-induced ROS production in a synergistic manner.

Since ROS production is regarded as an essential mechanism of killing of Leishmania by neutrophils (29) next we investigated the effect of ATP and UTP on the ROS production of neutrophils in response to L. donovani. Coincubation of neutrophils with L. donovani promastigotes induced a low and statistically not significant level of ROS production ( Figures 2E, F ). Simultaneous exposure of neutrophils to L. donovani and to ATP or UTP, however, led to strong ROS production ( Figures 2E, F ). These data show that ATP and UTP enhance Leishmania-induced ROS production in a synergistic manner.

Next, we investigated whether ATP and UTP, in addition to their effects on ROS production, also activate other antimicrobial effector functions of neutrophils. Since purinergic signaling was recently shown to enhance phagocytic capacity of macrophages (30) we hypothesized that ATP and UTP can exert an activating effect on phagocytosis by neutrophils. Neutrophils were exposed to ATP and UTP and subsequently phagocytosis of fluorescence-labeled latex beads was analyzed by flow cytometry. Exposure of neutrophils to ATP and UTP only slightly enhanced the phagocytosis of latex beads by neutrophils ( Figure 3A ). Exposure to LPS enhanced both the ratio of phagocytosing cells ( Figure 3A ) and the number of phagocytosed particles per neutrophil, shown as mean fluorescence intensity (MFI) ( Figure 3B ). Simultaneous exposure of LPS-stimulated neutrophils to ATP and UTP strongly and synergistically enhanced the LPS-induced phagocytic capacity of neutrophils ( Figures 3A, B ).

Prerequisite of intracellular killing of Leishmania by neutrophils is the ingestion of the parasites. The effect of ATP and UTP on the ingestion of Leishmania parasites was assessed by using CFSE-labeled L. donovani promastigotes. Exposure to both ATP and UTP enhanced the phagocytosis of Leishmania by neutrophils ( Figure 3C ).

Neutrophils can play both protective as well as disease promoting roles in leishmaniasis. On the one hand neutrophils can kill intracellular Leishmania. On the other hand, if intracellular Leishmania are not killed, neutrophils can provide an intracellular niche for the survival of these intracellular pathogens. Our finding, that exposure to extracellular ATP and UTP induces ROS production ( Figure 2 ) suggests that neutrophils can kill Leishmania at an enhanced level. The enhanced ingestion of the parasites ( Figure 3C ), on the one hand may facilitate the killing of more parasites. However, on the other hand, if the parasites are not killed, enhanced ingestion of Leishmania may also lead to the survival of more parasites.

To test whether exposure to ATP and/or UTP can enhance antileishmanial activity of neutrophils Leishmania donovani-infected neutrophils were exposed to ATP and UTP and the intracellular killing of the pathogens was assessed. Exposure of neutrophils to ATP and UTP significantly and markedly increased the killing of L. donovani ( Figure 4 ). Exposure of L. donovani promastigotes to ATP and UTP did not affect the viability of the parasites indicating that ATP and UTP do not directly kill L. donovani promastigotes (data not shown). Therefore, the increased killing of L. donovani is due to activation of neutrophil anti-leishmanial activity by ATP and UTP.

As shown in Figure 2 , ATP and UTP synergize with other activating stimuli such as LPS and fMLP. Since in preliminary studies we observed that exposure to LPS leads to enhanced anti-laishmanial activity of neutrophils (data not shown) we investigated whether the ATP and UTP exert a synergistic effect with LPS regarding the capacity of neutrophils to kill intracellular Leishmania. Simultaneous exposure of Leishmania-infected neutrophils to LPS and ATP or UTP enhanced significantly the killing of the parasites in a synergistic manner ( Figure 4 ).

In addition to their antimicrobial effector functions neutrophils can regulate inflammatory and immune responses by releasing cytokines (31). After having seen that extracellular ATP and UTP can activate effector functions of neutrophils we investigated whether exposure to ATP and UTP can also influence the proinflammatory regulatory functions of neutrophils. Such a function can contribute to the antimicrobial effects of ATP and UTP in vivo.

IL-8 (CXCL8) is a major chemotactic factor for neutrophils. However, neutrophils not only respond to but also secrete IL-8 (32). This autocrine mechanism is thought to serve as positive feedback for neutrophil recruitment. Because neutrophil recruitment is generally considered to be proinflammatory, we tested the effect of extracellular ATP and UTP on the IL-8 release by human neutrophils. Neutrophils were exposed to L. donovani, ATP or UTP or to the combination of L. donovani with ATP or UTP for 18 h and the IL-8 content of the supernatants was analyzed by ELISA. Exposure to ATP or UTP alone did not induce the release of IL-8 ( Figure 5A ). Co-incubation with L. donovani promastigotes resulted in the release of high IL-8 levels ( Figure 5A ). Importantly, extracellular ATP strongly enhanced the L. donovani-induced IL-8 release ( Figure 5A ). Exposure to UTP enhanced slightly the Leishmania-induced IL-8 release. However, this effect was not statistically significant ( Figure 5A ).

Activation of purinergic receptors plays also a major role in the immune responses through the secretion of pro-inflammatory cytokines. Extracellular ATP was reported to activate the NALP3 inflammasome which initiates a cascade of proteolytic events commencing with the conversion of procaspase-1 to caspase-1 resulting in the processing and secretion of IL-1β (33). In our present study co-incubation of neutrophils with L. donovani did not induce the release of IL-1β ( Figure 5B ). Exposure to extracellular ATP or UTP also did not induce IL-1β release ( Figure 5B ). Simultaneous exposure to L. donovani and ATP, however, resulted in the release of IL-1β from primary human neutrophils ( Figure 5B ).

Purinergic signaling has been described to lead to Gα_q-dependent activation of phospholipase C-β (PLCβ) resulting in the hydrolysis of PtdIns (4,5)P₂ producing the second messengers Ins (1,4,5)P₃ which then in turn mobilized intracellular Ca²⁺ (30). In order to investigate whether this pathway can be involved in the enhanced killing of Leishmania in neutrophils upon exposure to ATP and UTP we next assessed the effect of extracellular ATP and UTP on Ca²⁺ mobilization in Leishmania-infected neutrophils. A strong intracellular Ca²⁺ mobilization was observed in infected neutrophils upon treatment with ATP and UTP ( Figure 6 ). Treatment with ionomycin was used in these experiments as positive control ( Figure 6 ).

Purinergic signaling has been described to lead to Gα_q-dependent activation of phospholipase C-β (PLCβ). Activation of PLCβ results in the hydrolysis of diacylglycerol (DAG) which activates protein kinase C (PKC) which then in turn activates the serine/threonine kinase ERK1/2 (34). Signaling events involving Akt, Erk1/2 and p38 MAPK are also involved in inflammasome activation and the subsequent processing of IL-1β. Having observed a synergistic effect of ATP and L. donovani on the IL-1β-release of human primary neutrophils ( Figure 5 ) we next investigated whether phosphorylation of Akt, Erk1/2 and p38 MAPK is affected upon simultaneous contact of neutrophils to L. donovani and ATP. Western blot analysis reveled an upregulation of the phosphorylation of Akt, Erk1/2 and p38 MAPK upon exposure to extracellular ATP in L. donovani-infected neutrophils ( Figure 7 ).

In order to demonstrate that the observed phosphorylation events are induced by signaling via purinergic receptors the effect of the non-selective P2 purinergic receptor antagonist PPADS was investigated. Treatment with PPADS strongly and significantly reduced the phosphorylation of ERK1/2 and Akt in neutrophils exposed to L. donovani and ATP ( Figures 7A, C ). The inhibitory effect of PPADS on the phosphorylation of p38 MAPK was apparent, however, statistically not significant ( Figure 7B ).

Since in our experiments a relatively high concentration (500 µM) of ATP and UTP was applied to neutrophils for a time period varying between 15 min and over night exposure, we tested the effect of ATP and UTP treatment on the apoptosis and viability of neutrophils. Neutrophils are short lived cells, they undergo spontaneous apoptosis within hours both in vivo and in vitro (35, 36). Apoptosis and viability of neutrophils were assessed after 1h, 4h and 20h exposure to 500 µM of ATP and UTP. Extracellular ATP and UTP neither enhanced apoptosis nor exerted any toxic effects ( Supplemental Figure 1 ). In contrary, exposure to ATP and UTP showed a tendency to reduce apoptosis and enhance viability. This effect was statistically significant after 20h exposure of neutrophils to ATP ( Supplemental Figure 1 ).