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

<div><p>ArtinM, a D-mannose binding lectin from <i>Artocarpus heterophyllus</i>, 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 <i>Leishmania major</i> and <i>Leishmania amazonensis</i>. 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 <i>Leishmania</i>, without exacerbating inflammation. For this purpose, human neutrophils treated with ArtinM and infected with <i>Leishmania major</i> 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 <i>L</i>. <i>major</i> 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 <i>L</i>. <i>major</i>. 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.</p></div

ArtinM inhibits NET formation induced by <i>L</i>. <i>major</i>.

<p>Human neutrophils were incubated with ArtinM, PMA, ArtinM+PMA, or medium (untreated), and were either infected or not infected with <i>L</i>. <i>major</i> promastigotes (3:1). <b>A–DNA quantitation.</b> 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>B–NET immunofluorescence detection.</b> Neutrophils were plated onto poly-l-lysine coated slides, treated or untreated with PMA or ArtinM, and either infected or not infected with <i>L</i>. <i>major</i> 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.</p

ArtinM increases the leishmanicidal capacity of human neutrophils: a model.

<p>Once infected with <i>L</i>. <i>major</i>, 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, ArtinM-treated 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.</p

ArtinM stimulates the degranulation of <i>L</i>. <i>major</i>-infected or uninfected neutrophils.

<p><b>A–Intracellular levels of myeloperoxidase.</b> Human neutrophils were incubated for 20 h with ArtinM or medium only (untreated), and infected or not infected with <i>L</i>. <i>major</i> promastigotes (MOI 3:1); cells were permeabilized and reacted with anti-MPO^PE. Cells were analyzed by flow cytometry and data were expressed as normalized mean ± SD of the median fluorescence intensity. * p<0.02, **p<0,01. Student's <i>t</i> test. <b>B–Elastase releasing.</b> Human neutrophils were stimulated with ArtinM or medium only (untreated); Cell supernatants were monitored for 30 min for enzymatic activity, using the substrate N-succinyl-Ala-Ala-Val-p-nitroanilide. The NE activity was expressed as the area under curve (AUC) ± SD. ** p<0.05 in comparison to untreated cells. Student's <i>t</i> test. <b>C–Elastase leishmanicidal activity.</b> Supernatants from neutrophils treated for 1 h or not treated with ArtinM were incubated for 30 min (or not incubated) with anti-MPO or anti-NE antibodies (both 1:500). Next, supernatants were added to <i>L</i>. <i>major</i> culture (1x10⁵), in Schneider’s medium. After 24 h, live parasites were quantified by reaction with Alamar Blue. Data are expressed as the number of viable parasites ± SD. * p<0.05 in comparison to the supernatant from untreated cells. One way ANOVA, followed by Bonferroni's post-test. Each assay was carried out in duplicate. The shown data are representative from three different experiments.</p

ArtinM stimulates the production of inflammatory cytokines by <i>L</i>. <i>major</i>-infected or uninfected neutrophils.

<p>Human neutrophils were treated with or without ArtinM or PMA, and either infected or not infected with <i>L</i>. <i>major</i> promastigotes (MOI 3:1). At 20 h post-infection, the supernatant of these cells was collected for cytokine measurement by ELISA. <b>A–TNF, B–TGFβ-1 and C–IL-1β</b> concentrations are expressed as mean ± SD. * p<0.05; **** p<0.001 in comparison to untreated cells, or as indicated. One way ANOVA followed by Bonferroni's post-test. ND stands for “not detected”. ELISA assays were carried out in triplicate. The shown data are representative from three different experiments.</p

ArtinM enhances <i>L</i>. <i>major</i> uptake and killing by human neutrophils.

<p>Human neutrophils were treated or were not treated with ArtinM, and infected with <i>L</i>. <i>major</i> promastigotes (MOI 3:1). <b>A–<i>L</i>. <i>major</i> uptake.</b> 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>B–<i>L</i>. <i>major</i> killing.</b> 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 <i>t</i> test. Each assay was carried out in triplicate. The shown data are representative from three different experiments.</p

ArtinM treatment postpones apoptosis of uninfected neutrophils.

<p>Human neutrophils were incubated (indicated period) with medium (untreated), lysis buffer, ArtinM or IL-8. <b>A–Neutrophil morphology.</b> Neutrophils were cytocentrifuged and stained for evaluation by light microscopy. Arrows indicate neutrophils with nuclear condensation. <b>B–Phosphatidylserine exposure.</b> Neutrophils were labeled with Annexin V^-FITC and analyzed by flow cytometry. 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”. <b>C</b>–<b>JC-1 monomer detection.</b> 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. <b>D and E—Electrophoretic detection of DNA fragmentation.</b> 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 <b>D.</b> Panel <b>E</b> represents the band area in pixels², 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.</p

Electrophoretic profile of rPCN_exon4 and rPCN_full.

<p><b>Panel A:</b> Induction time/response rPCN_exon4 detected in the lysates of <i>E. coli.</i> The time elapsed since IPTG induction is shown in hours. <b>Panel B:</b> The bound material to glutathione-sepharose was separated by 12% SDS-PAGE Coomassie blue staining revealed a single band (lane 1), which was recognized by specific antibodies against GST (lane 2) and PCNprep (lane 3). <b>Panel C:</b> rPCN_full was purified and evaluated for its ability to bind to <i>N</i>-acetylglucosamine. The bound material was separated by 10% SDS-PAGE under reducing conditions, and then stained with Coomassie blue. Lane 1, material before refolding; Lane 2, material after refolding (a single band was detected with an apparent molecular mass of 28 kDa). Molecular markers were a mixture of pre-stained proteins (Fermentas). <b>Panel D:</b> The rPCN_full band was recognized by an anti-paracoccin antibodies (lane 2) and not by antibodies from pre-immune sera.</p

Cloning strategies for cloning the paracoccin ORF for expression.

<p>The left panel shows the standard strategy, with PCR amplification of the largest exon, restriction endonuclease digestion, and cloning into the expression vector pGEX-4T-1. The positions of the exons are displayed on the map of the gene. Genomic DNA template was extracted from <i>P. brasiliensis</i> strain Pb18. Agarose gel electrophoresis (mid-left) shows the corresponding band amplified by PCR. The exon 4 amplicon was cloned by <i>Bam</i>HI and <i>Eco</i>RI digestion. The right panel shows the strategy for synthesis of the predicted paracoccin sequence fused with the 5′-UTR elements for transcription in the vector pUC57. Green arrow, T7 promoter; black box, lacO (lac operator); and red box, the phage T7 trailer sequence for ribosome binding.</p

Biological and enzymatic properties of rPCN_exon4 and rPCN_full.

<p><b>Panel A:</b> production of TNF-α, <b>Panel B:</b> production of NO by induced murine macrophages following <i>in vitro</i> stimulation with PCNprep and rPCN_exon4 or PCNprep and rPCN_full. Cells were harvested from the peritoneal cavity of C57BL/6 mice and induced with thioglycollate. Adherent cells were incubated for 48 h with different recombinant proteins (0.25 mg/mL), medium (negative control), or LPS+IFNγ (positive control). The standard deviation was calculated based on tests performed in triplicate. The activity of the samples was compared to that of the medium alone. <b>Panel C:</b> The PCNprep, rPCN_exon4 and rPCN_full were assayed for NAGase activity. A colorimetric assay was performed in spectrophotometer set at λ = 405 nm. The standard deviation was calculated by analysis of experiments performed in triplicate. <b>Panel D:</b> Binding of rPCN_full to laminin. Different amounts of biotinylated recombinant protein were incubated with laminin (250 ng) coated in the microplate wells. The binding of the biotinylated protein was detected with a neutravidin-peroxidase conjugate and a chemiluminescent substrate. Luminescence readings are reported as relative luminescence units (RLU). <b>Panel E:</b> Inhibition of rPCN_full binding to laminin by sugars. Different concentrations of GlcNAc, d-glucose, and d-galactose were pre-incubated with the recombinant protein (100 ng), and the mixture was then added to the laminin-coated wells. The margin of error was calculated by analysis of triplicate experiments. Each sample with sugar was compared to a sample without sugar.</p