Immunological intervention with MRPs controls cutaneous Leishmaniasis.

<p>(<b>A</b>, <b>line graph</b>) BALB/c mice were injected with 2 mg of α-MRPs 8/14 one day before infection with <i>L. major</i> in the footpad and subsequent inoculation of the antibodies three times/week during four weeks after infection (…). Footpad sizes were measured every week for 8 weeks. (*) indicates significant difference P<0.01 between groups (n = 6). (<b>A, bar graph</b>) Parasites from footpads were extracted, and the parasitic burden was measured by limiting dilution assay, (*) denotes: P<0.05 between groups (n = 6). (<b>B, line graph</b>) BALB/c mice were injected with 10 µg of rMRPs 8/14 during 4 weeks (weeks 8 to12) directly into the infected footpad three times per week. Footpad sizes were measured every week for 12 weeks. (*) indicates significant difference P<0.01 between groups (n = 6). (<b>B, bar graph</b>) Parasites from footpads were extracted, and the parasitic burden was measured by limiting dilution assay, (*) denotes P<0.05 between groups (n = 6).</p

<i>Leishmania</i> induces leukocytes recruitment and MRP secretion in the murine air-pouch model.

<p>Air-pouches were raised in BALB/c mice during 6 days with sterile air, at day 7 they were stimulated with LPS or infected with <i>L. major</i> for 6 hr. Pouches were washed and total cell recruitment (<b>A</b>) and MRP secretion by ELISA were measured (<b>B</b>). BALB/c mice were treated with α-MRP 16 hr before infection with <i>L. major</i> for 6 hr. Total cell recruitment (<b>A</b>) and MRP secretion by ELISA (<b>B</b>) were measured. (*) denotes P<0.05 between the <i>L. major</i> infected group (n = 6) and the <i>L. major</i> infected group treated with α-MRPs.</p

MRPs decrease the parasitic load in macrophages at 24<i>L. major</i>.

<p>B10R macrophages were primed with 5, 10 and 25 µg/ml of MRP8/14 for 1 hr, and then infected for 6 hr (<b>A</b>) and 24 hr (<b>B</b>) with <i>L. major</i> luciferase. Level of infection was measured by luciferase assay. The dashed line represents the infected cells at 6 hr (<b>A</b>) or 24 hr (<b>B</b>) without MRP treatment (RLU value = 100%). (*) denotes P<0.05 between the untreated 24 hr infected cells and cells primed with MRPs before 24 hr of infection. One representative experiment of three is shown.</p

Impact of <i>Leishmania</i> infection on MRPs–induced ERK and JNK signaling.

<p>(<b>A</b>, <b>C</b>) B10R macrophages were only treated or primed with MRPs 8/14 (5, 10, 25 µg/ml) for 30 min before infection with <i>L. major</i> (1 hr). (<b>B</b>, <b>D</b>) B10R macrophages were only treated or MRP-stimulated (30 min) after 1 hr <i>L. major</i> infection. Protein lysates were assessed for phosphorylation of ERK 1/2 kinase (<b>A</b>, <b>B</b>) and JNK (<b>C</b>, <b>D</b>) kinase by immunoblotting. One representative experiment of three is shown.</p

Nuclear translocation and binding of transcription factors (TFs).

<p>B10R macrophages were treated with MRPs 8/14 (5, 10, 25 µg/ml) for 1 hr or MRP-stimulated before infection with <i>L. major</i> (<b>A</b>, <b>C</b>) or treated 1 hr with MRPs after ON infection (<b>B</b>, <b>D</b>). Nuclear proteins were extracted and subjected to EMSA for NF-κB (<b>A</b>, <b>B</b>), and AP-1 (<b>C</b>, <b>D</b>). Consensus oligonucleotides non-specific competitors (Ins Co100×) and specific competitor (Spec Co100×) were used in and 100× molar excess. One representative experiment of three is shown.</p

Impact of <i>Leishmania</i> infection on the production of nitric oxide (NO) and iNOS expression by MRPs 8/14.

<p>B10R macrophages were stimulated with different doses of MPRs 8/14 (5, 10 and 25 µg/ml) before (<b>A</b>) or after (<b>B</b>) of infection (24 hr) with <i>L. major</i>. NO production (after 24 hr) was assessed by measuring nitrates in the supernatants. (<b>C</b>) B10R macrophages were treated as in (<b>A</b>) and (<b>B</b>) and protein lysates were subjected to immunoblotting analysis with anti-iNOS. Equal protein loading is shown by β-actin. (*) denotes P<0.05 between the MRP-stimulated macrophages and <i>L. major</i> infected-MRP-stimulated macrophages. Mean of three different experiments is shown.</p

S100A9 treatment decreases the secretion of pro-inflammatory cytokines (A) 5 days after LPS injection, sera were collected and tested for IL-6 by ELISA.

<p>Values are the mean ± SEM of 10 mice. *p<0.05, t test (n = 10 serum samples/group) (B) Thirty micoliters from a pool of 5 paw homogenates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and tested for the presence of IL-6 and TNF-α. F: forepaw, H: hind paw.</p

Neutrophil migration across HUVECs in response to S100A9.

<p>A) Increasing concentrations of S100A9 were added in the upper wells with neutrophils. IL-8 (5 ng/ml) or buffer was added to the lower wells and neutrophils were allowed to migrate for 2 h at 37°C. Data are the mean ± SEM of 3 experiments using neutrophils from different donors. *p<0.05, **p<0.01, one-way ANOVA, Dunnett’s multiple comparison test. B) S100A9 prolongs the time for neutrophil migration across endothelial cells. S100A9 (40 µg/ml) was added to the upper well with neutrophils. IL-8 (5 ng/ml) or buffer was added to the lower well and every 30 min for up to 2 h the upper wells were moved to new lower wells. The number of transmigrated cells was determined as described in Materials and Methods. Data shown represent the mean ± SEM of at least 3 experiments using neutrophils from different donors.</p

Effect of anti-S100A9, TNFα, or isotype control Abs on the LPS-CIA clinical score.

<p>(A) Anti-collagen II Abs were measured in mouse serum 4 h after LPS injection. The results are expressed as µg of IgG Ab/ml of serum. (B) S100A8 and S100A9 expression in arthritic paws. Paw tissue sections were stained with rabbit pre-immune serum, rabbit anti-S100A8, or rabbit anti-S100A9 polyclonal IgGs. B: bone, Js: joint space. (Magnification 1000X). (C) Four hours after LPS injection, sera were collected and ELISA S100A8/A9 were performed *p<0.05, one-way ANOVA test (n = 10 paws/group), Tukey’s Multiple Comparison Test (D) Clinical score as assessed by two blinded observers. Data are the mean scores calculated from at least 15 mice per group until day 19.</p

Decreased neutrophil and monocyte antigen expression in the paws of anti-S100A9-treated mice.

<p>(A) Western blot analysis of 7/4 antigen (Ag), a neutrophil marker (top). Quantification of 7/4 Ag on an immunoblot by densitometry analysis (bottom). (B) Western blot analysis of Gr-1 Ag, a marker of granulocytes and monocytes (top). Quantification of Gr-1 Ag on an immunoblot by densitometric analysis (bottom).</p