This study was approved by the Institutional Review Board of Human Ethical Research Committee of Fundação Oswaldo Cruz-Bahia under the protocol number 1.213.510.

Leishmania amazonensis MHOM/Br/00/BA125 strain was used for DCs infection. L. amazonensis promastigotes were maintained in Schneider culture medium (sigma^®) supplemented with 10% fetal calf serum (sigma^®), 2 mm L-glutamine, 100 μg/ml penicillin and 100 μg/ml streptomycin (sigma^®) in an incubator at 24°c. During the culture period, promastigotes were counted in the Neubauer chamber with a 100x dilution in saline in order to measure parasite growth dynamics. In all experiments, stationary phase parasites were used which are enriched by infective stage of parasite, called metacyclic promastigotes.

Monocytes were obtained from the peripheral blood of healthy donors from the Bahia State Blood Center (HEMOBA). Blood was diluted in saline (1:1) and processed using the HISTOPAQUE^® 1077 separation gradient (Sigma Aldrich, St Louis, MO) for 30 minutes at 360xg at room temperature. After the centrifugation, a ring consisting of mononuclear cells was formed, collected and washed 3 times with saline at 200xg, 4°C. For differentiation into DCs, mononuclear cells were passed through magnetic separation columns for isolation of CD14⁺ cells (monocytes). These CD14⁺ cells were cultured for 7 days at a concentration of 1x10⁶/mL in complete RPMI medium (Gibco Invitrogen Corporation, Carlsbad, CA, USA) supplemented with 2mM L-glutamine, 100U/mL of penicillin and 100μX/mL streptomycin (complete medium) in the presence of IL-4 (100UI/mL) and GM-CSF (50ng/mL; both from PeproTech, Rocky Hill, NJ, USA) in 24- wells plates. Every 3 days of culture, 500 μL of medium containing growth factors were removed and replaced with the same amount of fresh medium.

As previously described, stationary phase L. amazonensis promastigotes were used for in vitro infection of human dendritic cells. Infection assays were performed at the rate of 10 parasites for each dendritic cell (10: 1) in complete culture medium at 37°C, 5% CO₂ for 24 hours. Subsequently, cells were centrifuged twice with saline solution to remove non-internalized parasites.

PMNs were obtained from the peripheral blood of healthy donors from the Bahia State Blood Center (HEMOBA). Blood was processed using the Polymorphprep separation gradient as per manufacturer’s instructions (Axis-Shield Poc AS, Oslo, Norway). Briefly, the blood and gradient were centrifuged for 45 minutes at 300xg at room temperature. After centrifugation, PMNs were collected and washed three times with saline (0.9% NaCl) at 4°C for 10 minutes at 200xg. After washing, cells were counted in a Neubauer chamber with a final dilution of 100x in Trypan Blue stain. Cells were resuspended at a concentration of 5x10⁵/mL and cultured at 37°C in 5% CO2 in RPMI-1640 medium (Gibco Invitrogen Corporation, Carlsbad, CA, USA) supplemented with 2mM L-glutamine, 100U/mL of penicillin and 100μL/mL streptomycin (complete medium) (Gibco Invitrogen Corporation) for 96-well plate culture (Corning Incorporation, Costar, NY, USA).

Neutrophil activation was induced by exposure of cells to culture plate surfaces coated with fibronectin extracellular matrix proteins. The activation protocol was adapted from Pinheiro et al. (17). Briefly, sterile 96-well culture plates (Corning Incorporation, Costar, NY, USA) were previously coated with 50μl of a 300μg/ml collagen solution (BD Pharmingem, San Diego, CA) and incubated for 12 hours at 4°C. To increase fibronectin binding to plate wells, 100μl of a 10μg/ml solution of human recombinant fibronectin (Sigma-Aldrich, St Louis, MO) was added to wells previously covered with collagen for 1 hour at 37°C. After sensitization with fibronectin, the wells were washed three times with saline and 2x10⁶ PMNs in RPMI medium were added to each well and incubated for 1 hour at 37°C and 5% CO₂. After incubation with the matrix proteins, PMNs were collected and centrifuged with saline for 100xg 10 minutes at 4°C. Next, cells were counted and used in co-cultures with infected dendritic cells. Neutrophil treatment with enzyme inhibitors of human PMNs were plated at 5x10⁵ per well, and appropriate experimental groups were treated with myeloperoxidase (MPO) inhibitor (benzoic acid hydrazide analog, 0.1 mg/ml), TIMP (matrix metalloproteinase 9 [MMP-9] inhibitor, 30 ng/ml)or neutrophil elastase inhibitor (r & d, systems, Mineapollis, USA).

Infected dendritic cells were co-incubated with fibronectin-pre-activated PMNs in RPMI medium supplemented with 2 mM L-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin (Gibco, Invitrogen Corporation, Carlsbad, CA) for 4, 12 or 18 hours. In the interaction experiments, L. amazonensis- infected DCs and PMNs, a ratio of 5: 1 (PMNs: DCs) was used. After these periods, the wells were washed with saline, cells were submitted to cytospin centrifugation, fixed with ice-cold methanol for 10 minutes, and subsequently stained with hematoxylin-eosin (H&E). To determine the infection rate (number of DCs harboring at least one amastigote) and Parasite load (the number of amastigotes found in 100 cells), a total of 200 cells were counted per condition and in duplicate coverslips. For Transwell assays, L. amazonensis infected dendritic cells were accommodated in the lower compartment of Transwell plates (Sigma Aldrich, St Louis, MO) containing RPMI medium supplemented with 2 mM L-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin (Gibco, Invitrogen Corporation, Carlsbad, CA). In parallel, activated human PMNs were accommodated at the top of the Transwell plate. Importantly, DCs and PMNs were cultivated in transwell plates at the same ratio of 5: 1 (PMNs: DCs). The coculture was incubated at 37°C for 18 hours. Subsequently, DCs present in the lower portion of the plate were collected, washed with saline and the coverslips fixed with methanol for 20 minutes and stained with hematoxylin-eosin (H&E). Determination of infection rates and parasite load were performed as described above.

After the determined interaction times between activated PMNs and L. amazonensis infected dendritic cells, the supernatants of such cocultures were collected to evaluate the production of cytokines and lipid mediators. Supernatants were tested for the presence of interleukins IL-6, IL-8, IL-12p70 and TNFα by ELISA (BD Biosciences,San Diego, CA) according to the manufacturer’s instructions. LTB₄ and PGE2 lipid mediators were quantified by competition ELISA according to manufacturer’s instructions (CaymanChemicalCompany, MI, USA. Additionally, For MPO measurement, 50 µl fresh supernatant was added to a 96-well plate with 50 µl the developer solution ([5 ml] H2O2 30% + [10 ml] 1 mM citrate + 5 mg OPD) After 10–20 min, the reaction was stopped with 50 µl H₂SO₄ (8 N). MPO activity was measured by reading absorbance at 492 nm. For NE activity, 50 µl fresh supernatant was added in triplicate to ELISA plates, followed by 25 µl elastase reaction buffer (0.1 M HEPES, 0.5 M NaCl, 10% DMSO; all from Sigma-Aldrich; pH 7.5). Then, 0.2 mM elastase substrate I (MeOSuc-AAPV-pna) was added, and samples were incubated at 37°C for 3 d. NE activity was measured by reading absorbance at 410 nm.

For DC-sign neutralizing assays, human DCs were initially infected with L. amazonensis for 24 hours in RPMI medium supplemented with 2 mM L-glutamine, 100 U/mL penicillin and 100 µg/mL streptomycin (Gibco, Invitrogen Corporation, Carlsbad, CA). Subsequently, DCs were centrifuged at 100xg for 10 minutes at 4°C to remove non-internalized promastigotes. Finally, activated human PMNs were added together with either 50ng/ml of the DC-SIGN neutralizing antibody (Abcam). For TNF-α neutralization, 10 µM of αTNF neutralizing antibody were used (Sigma Aldrich, St Louis, MO).

Immunostaining of DCs for maturation molecules was performed by incubating cells with antiCD11c – PE-cy7, antiCD80- FITC, antiCD209- PE, antiHLA-DR – BV711, antiCD86 – PE-CY5, antiCD1a-AP antibodies (Bioscience), on ice, protected from light, for 30 minutes before to FACS analyses. Labelled DCs were acquired on BD LSR II flow cyometer (BD Biosciences). Supplementary Table 1 summarizes all the details of flow cytometric antibodies used in this study. We used single color controls to further performed fluorochrome spillover compensation. Supplementary Figure 3A depicts gating strategy applied to analyze DCs. Briefly, DCs were initially gated based on FSC (forward scatter) and SSC (side scatter) channels. Next, we applied two sequential doublet exclusion gates to ensure the selection of single events (FSC-a vs. FSC-H and SSC-W vs. SSC-H). Then, Cells were gated based on CD11C expression. Median fluorescence intensity was calculated for each marker. For high dimensional analysis, 30000 DCs were downsampled to obtain subgroups with equal number of events. FlowSOM (Flow Self-organizing maps), an unsupervised clusters technique was employed to identify subpopulations of DCs with varied expression of maturation markers. Then, a t-Distributed Stochastic Neighbor Embedding (t-SNE) analysis were performed on gated DCs. ROS production was determined by flow cytometry (Dihydroethidium ROS Assay Kit, abcam).

Statistical analysis of the data was performed using the Prism software version 5.0 (GraphPad Software^®). The data represent the medians obtained in each condition. Comparisons between groups were performed using Dunn’s post-test ANOVA test for analyzes with more than 3 groups and the Mann-Witney test (t-test) for the other comparisons. All experiments were performed with at least three repetitions and p-value <0.05 was considered statistically significant. The heatmaps shown in this study were generated with Pheatmap (pheatmap, RRID : SCR_016418) R package, version 1.0.12) in R language. Principal component analysis and lollipop plots presented in this study were constructed with factoextra and ggplot2 packages, respectively (18).

Initially, we assessed whether human PMNs were able to influence the outcome of L. amazonensis infection in DCs, and if such infection constrain was dependent on the state of neutrophil activation. Briefly, DCs were infected with L. amazonensis for 24 hours and subsequently cultivated with either fibronectin-activated or resting PMNs for 12 hours as depicted in our experimental design scheme ( Figure 1A ). We verified that DCs co-cultivated with resting or activated PMNs exhibited diminished rates of infection as well as decreased numbers of Leishmania amastigotes found per 100 DCs ( Figures 1B, C ). We did not find statistically significant differences between the groups of resting or activated PMNs co-cultivated with DCs, suggesting that induction of parasite elimination is independent of the neutrophil activation state. Therefore, to mimic the biological context in which PMNs and DCs interact during Leishmania infection, we decided to only use activated PMNs for the subsequent experiments.

Next, we sought to explore the mechanisms through which PMNs promote parasite elimination in infected DCs. Initially, we measured the levels of neutrophil enzymes produced in the co-culture of Leishmania-infected DCs and either resting or activated PMNs. We detected higher levels of both MPO and neutrophil elastase (NE) in the co-culture of DCs and activated PMNs ( Supplementary Figures 1A, B ). Surprisingly, the pharmacological inhibition of individual neutrophil enzymes did not alter the infection rate ( Figure 1D ), suggesting that contact-independent mechanisms are not responsible for the parasite elimination we observed in neutrophil-DC interaction. Thus, we aimed to investigate to what extent the constrain of Leishmania infection in DCs was mediated by contact-dependent mechanisms. Accordingly, to avoid physical contact between the cells, we co-cultured DCs and PMNs in different compartments of transwell plates. We observed that DCs co-cultured in such plates exhibited higher rates of infection and parasite load when contrasted to the groups in which PMNs and DCs were free to establish direct contact ( Figures 1E, F ). Thus, our findings suggest that parasite elimination is dependent on the physical contact mechanism.

Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN) is a major orchestrator in DCs-Neutrophil crosstalk (19). A wealth of evidence points that DC-SIGN, a C-type lectin present on DCs surface, recognizes Lewis X glycan moieties on PMN receptors, thus mediating the establishment of immunological synapses between these two types of cells. It is well established that the DC-PMN cross-talk culminates in reciprocal modulation of their immune functions (by influencing processes such as Cell activation, cytokine production, and resistance to apoptosis) (19). Therefore, we sought to elucidate whether DC-SIGN-mediated contact plays a role in Leishmania elimination. Initially, we measured the surface expression of DC-SIGN in infected DCs. Of note, we observed a significant reduction of DC-SIGN levels in L. amazonensis-infected DCs, and that co-culture with PMNs was able to rescue this receptor expression ( Figure 2A ). Additionally, we found elevated rates of infectivity and parasite burden upon in vitro treatment with DC-SIGN neutralizing antibody, further suggesting that such receptor is a paramount mediator of the physical contact-dependent parasite elimination ( Figures 2B, C ).

Next, we wished to evaluate the contribution of DC-SIGN-mediated crosstalk between DCs and PMNs in the production of soluble mediators (namely, chemokines, lipid mediators, and cytokines). Concerning eicosanoid production, we observed increased secretion of both LTB₄ and PGE₂ when DCs were cultured with PMNs, but we could only detect statistically significant enhanced amounts of the latter lipid mediator ( Figures 3A, B ). Additionally, upon DC-SIGN neutralization, PGE₂ levels were only marginally increased when contrasted to monocultures of infected DCs ( Figure 3B ). We also detected enhanced levels of TNF-α in DC-Neutrophil cultures, with decreased production upon DC-SIGN inhibition ( Figure 3C ). Interestingly, the amounts of CCL3 were substantially reduced in the presence of human PMNs, while no significant difference in CCL20 production was observerd among the experimental groups ( Figures 3D, E ). Notably, we observed that DC-SIGN neutralization was associated with augmented production of IL-6 when compared to other experimental conditions ( Figure 3F ). Additionally, the addition of PMNs to infected-DCs culture did not result in elevation of IL-12 levels ( Figure 3G ). Collectively, our observations suggest that the establishment of direct contact between DCs and PMNs orchestrates the production of several soluble mediators in the settings of L. amazonensis infection. Subsequently, we investigated whether such cell-cell engagement via DC-SIGN is necessary for the fine-tuning induction of reactive oxygen species (ROS). Of note, we observed that the addition of activated PMNs to infected DCs cultures promoted enhanced levels of ROS production and DC-SIGN neutralization had a reverse effect ( Figure 3H ). Additionally, we applied a heatmap with the median values of the soluble mediators as an approach to identify patterns among the experimental conditions ( Figure 3I ). To better understand to what magnitude the contact via DC-SIGN regulates soluble mediator production, we devised an approach comparing the fold changes in cultures where DC-SIGN was inhibited versus not inhibited. Interestingly, we found a 1.5-fold higher increase of both IL-6 levels and the number of amastigotes in experimental conditions where DC-SIGN was neutralized. Conversely, we detected a 1.5-fold decrease in PGE₂ and TNFα production in the aforementioned conditions ( Figure 3J ). Additionally, our principal component analysis showed little overlap between these groups. Importantly, we found that TNFα levels positively correlate with ROS production, while we could detect negative correlations between ROS levels and the amounts of amastigotes in infected DCs ( Supplementary Figure 2 ). Therefore, these observations suggest that the constrain of L. amazonensis infection promoted by the interaction with PMNs is dependent on the TNFα and may be associated with ROS activity. Collectively, our data indicate that DC-SIGN-mediated contact can substantially influence both production of soluble molecules and ROS induction with eventual impact in L. amazonensis elimination in DCs ( Figure 3K ).

TNFα is an important pro-inflammatory cytokine involved in cell activation and elimination of pathogens (20). We next ascertained whether TNFα contributes to L. amazonensis elimination in the studied co-culture model. Thus, we treated cell cultures with TNFα neutralizing antibody and measured the percentages of infected DCs as well as the number of amastigotes. Of note, we observed that the treatment with TNFα neutralizing antibody resulted in enhanced infection rates and parasite load when contrasted to the PMN- infected DC cultures not treated with such antibody ( Figures 4A, B ). These findings underscore the importance of TNFα in the control of L. amazonensis infection in the setting of DCs-neutrophil interaction.

Presenting antigens to adaptive immune cells is a critical immunobiological function of DCs (21). Accordingly, several species of Leishmania developed mechanisms to mitigate expression of molecules involved in this process, thus enhancing the chances of parasite survival and proliferation (22). Along these lines, we employed a flow cytometry approach to investigate to what degree the interaction between DCs and PMNs via DC-SIGN impacts the process of DC maturation. To analyze the maturation profiles, we took into consideration the expression of HLA-DR and costimulatory molecules (CD1a, CD80, and CD86), as well as CD209 (DC-SIGN) ( Figure 5A ). Of note, we found that L. amazonensis infection is associated with increased numbers of CD1a⁺ DCs and that the presence of PMNs reduced the size of these cell population in a DC-SIGN independent manner ( Figure 5B and Supplementary Figure 3B ). For the other markers, we could not observe significant variation in percentage of cells expressing such molecules ( Supplementary Figure 3B ).

Nevertheless, by analyzing the bulky population of DCs, we found that L. amazonensis infection decreased CD80 surface density (measured as the median value of fluorescence intensity), and that PMNs could partially upregulate this co-stimulatory molecule depending on DC-SIGN -promoted contact ( Figure 5C ). Strikingly, we noticed a CD86 upregulation in infected DCs ( Figure 5D ). Most interestingly, we observed that PMNs are able to upregulate the expression of HLA-DR in a DC-SIGN dependent fashion ( Figure 5E ). Collectively, our data suggest that the engagement of DCs and neutrophil via DC-SIGN is associated with partial reconstitution of maturation-associated molecules in settings of L. amazonensis infection.

As environmental cues largely influence the ability of DCs to present antigens, we hypothesized that co-culture with PMNs would give rise to DC populations with varying levels of such molecules expression in the context of L. amazonensis infection. Thus, we applied a t-distribute stochastic neighbor embedding (tSNE) analysis to depict co-stimulatory molecule expression profiles found in the overall experimental groups ( Figure 5F ) and among the different conditions ( Supplementary Figure 4 ). Notably, our FlowSom clustering was able to detect 6 populations with distinct maturation profiles ( Figure 5F ). We then conduct a heatmap with hierarchical cluster analysis to better dissect these population in regard to marker expression and distribution across experimental conditions ( Figures 5G ). Interestingly, we observed significant enrichment of two population clusters (populations 1 and 5) in the co-culture of DCs and PMNs ( Figures 5H–M ). The frequency of DCs referred as population 1 (CD16^high, CD1a ^dim, CD80 ^dim, CD209 ^high, CD86 ^int, CD11c ^int, and HLA-DR ^high) was increased in L. amazonensis-infected DCs and PMNs were able to promote its frequency reduction in a DC-SIGN dependent manner( Figure 5H ). Conversely, we noticed that population 5 (CD16 ^int, CD1a ^high, CD80 ^dim, CD209 ^high, CD86 ^dim, CD11c ^int, and HLA-DR ^high) was increased in the condition co-cultured with PMNs ( Figure 5M ).

Additionally, our spearman correlation analysis revealed a positive correlation between the frequencies of FlowSom population 1 and parasite load, while population 5 was positively correlated with ROS production in infected DC cultured with PMNs ( Figures 6A, B ). Of note, we this was not observed in aDC-SIGN treated cell cultures. We were not able to find significant correlation between the frequency these populations and the level of soluble molecules produced during neutrophil and DC co-culture. Collectively, our data suggest that DC-SIGN inhibition in co-cultures of PMNS and infected DCs can influence the DC maturation landscape, giving rise to populations associated either with ROS production or parasite survival.