The IL-23 effect on the IL-17 production by spleen cells under ArtinM stimulus.

<p>(<b>A and B</b>) Murine spleen cells (2 × 10⁶/mL) from C57BL/6 (WT), IL-4 KO, IL-10 KO, IFN-γ KO, IL-6 KO, IL-23 KO, and IL-22 KO mice were incubated for 48 h at 37°C with ArtinM (1.25 μg/mL) or medium alone as an additional negative control. (<b>B</b>) Spleen cells from WT and IL-23 KO mice were stimulated for 48 h with ArtinM alone or in association with IL-6 or IL-23. As controls, IL-6 and IL-23 were used alone, and these cytokines were associated with IL-1β as positive control. In all cases, cytokine concentration was 10 ng/mL. (<b>C</b>) Peritoneal macrophages (2 × 10⁶/mL) from C57BL/6, TLR2 KO and CD14 KO mice were incubated with ArtinM (1.25 μg/mL) for 7 h and then the extracted RNA was used for real-time quantitative PCR of IL-23 mRNA, as described in Materials and Methods. Medium and LPS (1 μg/mL) were used as negative and positive controls, respectively. The results are expressed as the relative expression of IL-23 normalized to β-actin expression. (<b>A—C</b>) The results are expressed as mean ± SEM, and the levels of IL-17 and the expression of IL-23 were compared to that of the unstimulated cells (Medium); and additional comparison was established between the ArtinM stimulus in cells of WT and KO mice (<b>A</b>) and between the ArtinM stimulus and ArtinM plus IL-23 (<b>B</b>). Differences were considered significant when p < 0.05 (*).</p

The effect of ArtinM on IL-17 production by murine spleen cells.

<p>(<b>A</b>) Spleen cell suspensions (2 × 10⁶/mL) from C57BL/6 or BALB/c mice were incubated at 37°C for 12, 24 and 48 h with boiled (Boil) or native (not boiled) samples of ArtinM (1.25 μg/mL). (<b>B</b>) Spleen cell suspensions (2 × 10⁶/mL) from C57BL/6 or BALB/c mice were incubated at 37°C for 48 h with <i>Canavalia ensiformis</i> (ConA), <i>Phaseolus vulgaris</i> erythroagglutinin (E-PHA), <i>Phaseolus vulgaris</i> leukoagglutinin (L-PHA), <i>Sambucus nigra</i> agglutinin (SNA), <i>Maackia amurensis</i> leukoagglutinin (MAL), <i>Ulex europaeus</i> agglutinin (UEA), or Jacalin in concentrations of 0.625, 1.25 or 2.5 μg/mL. In both cases (<b>A</b> and <b>B</b>), positive and negative controls were incubated with PMA plus ionomycin (81 nM + 5 μM; Ctrl+) and medium, respectively. The IL-17A concentration in the culture supernatants was measured by ELISA and compared to the levels detected in the supernatant of medium. (<b>C</b>) Fixed spleen cells from C57BL/6 or BALB/c mice were incubated with 25 μg/mL biotinylated lectins (ArtinM, ConA, E-PHA, L-PHA, SNA, MAL, UEA, or Jacalin). Lectin binding was revealed by reaction with strp/FITC (5 μg/mL), and the fluorescence was measured by flow cytometry. The median fluorescence intensity (MFI; left Y axis) and the percentage of fluorescent cells (Positive cells—%; right Y axis) were determined for each lectin. (<b>A</b>, <b>B,</b> and <b>C</b>) The results are expressed as mean ± SEM. *Significant differences with p < 0.05 in relation to Ctrl-.</p

The effect of anti-CD3 antibody in the IL-17 production induced by ArtinM.

<p>Isolated CD4⁺ T cells (2 × 10⁶/mL) obtained from IL-23 KO mice were incubated at 37°C for 40 min with the anti-CD3 antibody (15 μg/mL; clone 17A2) or IgG Isotype control (15 μg/mL; A19-3 clone). Cell suspensions were stimulated for 48 h with ArtinM (1.25 μg/mL), with a mixture of IL-1β/IL-6/IL-23 (20 ng/mL; 20 ng/mL; 20 ng/mL), with IL-23 alone (20 ng/mL) or with medium alone. IL-17 level in the cell supernatants was measured by ELISA. The results are expressed as mean ± SEM. The values were compared to unstimulated cells (Medium), or between ArtinM-stimulated cells that were pre-incubated or not with anti-CD3 antibody. Differences were considered significant when p < 0.05 (*).</p

IL-1R signaling contributes to IL-17 production induced by ArtinM.

<p>(<b>A</b>) Spleen cells (2 × 10⁶/mL) from C57BL/6, IL-17R KO, MyD88 KO, TLR2 KO, IL-1R KO, TLR4 KO, CD14 KO, and IL-33R KO mice were stimulated with ArtinM (1.25 μg/mL) for 48 h at 37°C. PMA plus ionomycin (81 nM + 5 μM) and medium were used as positive and negative controls, respectively. The IL-17 levels in the cell supernatants were determined and compared to that of the unstimulated cells (Medium); the IL-17 levels induced by ArtinM in WT and KO mice were also compared. PMA plus Ionomycin (positive control) induced cells of all mice strains to produce IL-17, a response that was significantly reduced in cells from IL-1R KO mice (not shown). <b>(B)</b> Murine spleen cells (2 × 10⁶/mL) from WT mice were stimulated for 48 h with ArtinM (1.25 μg/mL) alone or in association with IL-1β (10 ng/mL). As a control, IL-1β (10 ng/mL) was used alone, and this cytokine was associated with IL-6 (10 ng/mL) and IL-23 (10 ng/mL) as positive control. Medium alone was an additional negative control. (<b>C and D</b>) Peritoneal macrophages (2 × 10⁶/mL) obtained from C57BL/6 (white bars), TLR2 KO (gray bars) and CD14 KO (black bars) mice were incubated for 48 h with ArtinM (1.25 μg/mL). LPS (1 μg/mL) and medium alone were used as positive and negative controls, respectively. IL-1α (<b>C</b>) and IL-1β (<b>D</b>) were determined by ELISA. (<b>A-D</b>) Statistical comparisons were established between unstimulated and stimulated cells, and (<b>A</b>, <b>C</b> and <b>D</b>) additional between the cytokines levels induced by ArtinM in WT and KO mice. The results are expressed as mean ± SEM; differences were significant when p < 0.05 (*).</p

Dectin-1 role in the peritoneal macrophages activation induced by ArtinM.

<p>(<b>A</b>) Total RNA of splenic adherent cells (2 × 10⁶/mL), BMDMs (1 × 10⁶/mL), and peritoneal macrophages (1 × 10⁶/mL), from C57BL/6 mice that were incubated with ArtinM (1.25 μg/mL; for 7 h at 37°C), was used for real-time quantitative PCR of Dectin-1 mRNA as described in Materials and Methods. Medium and LPS (1 μg/mL) were used as negative and positive controls, respectively. The results are expressed as the relative expression of Dectin-1 normalized to β-actin expression, and compared to medium values. (<b>B</b> and <b>C</b>) Peritoneal macrophages (1 × 10⁶/mL) obtained from C57BL/6 (white bars) and Dectin-1 KO (black bars) mice were incubated for 48 h with ArtinM (1.25 μg/mL). LPS plus IFN-γ (1 μg/mL + 2 ng/mL) and medium alone were used as positive and negative controls, respectively. The cell supernatants levels of IL-6 (<b>B</b>) and IL-12p40 (<b>C</b>) were determined by ELISA. (<b>A-C</b>) Statistical comparisons were established between unstimulated and stimulated cells and between the IL-17 levels induced by ArtinM in WT and KO mice. The results are expressed as mean ± SEM; differences were significant when p < 0.05 (*) and in other cases were nonsignificant (ns).</p

ArtinM triggers TLR2-mediated cell activation.

<p>HEK293A cells were transfected with the ectodomain of human TLR2 (A) or human TLR4 (B), and the necessary co-receptors (CD14, CD36, and MD-2), as well as an NF-κB reporter construct and a <i>Renilla</i> luciferase control reporter plasmid. The cells were then stimulated with different concentrations of ArtinM (15.6, 156, and 780 nM) at 37°C for 18 h. In (A), MALP-2 (50 ng/mL) was used as the positive control. In (B), LPS (1 µg/mL) was used as the positive control; the addition of polymyxin B (100 µg/mL) to LPS served as another control. In both A and B, medium and an empty vector were used as the negative controls. The luciferase activity was measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098512#s2" target="_blank">materials and methods</a>. Statistical comparisons between unstimulated and stimulated cells were performed using a one-way analysis of variance followed by Bonferroni's multiple comparison test. * p<0.05.</p

TLR2 mediates the cytokine production induced by ArtinM.

<p>IL-12p40 and IL-10 levels in the cell culture supernatants of adherent spleen cells (A and B) or peritoneal macrophages (C and D) from WT (white bars) and TLR2-KO (black bars) mice were measured by ELISA. Adherent spleen cells were stimulated with ArtinM (156 nM) or P3C4 (1 µg/mL) for 24 h, while the peritoneal macrophages were incubated with ArtinM (39 nM) or P3C4 (1 µg/mL) for 48 h. Statistical comparisons between unstimulated and stimulated cells were performed using a one-way analysis of variance followed by Bonferroni's multiple comparison test. *p<0.05.</p

ArtinM induces the activation of TLR2/1- and TLR2/6-expressing cells.

<p>HEK293A cells were transfected with TLR2/1 (A and C) or TLR2/6 (B and D), co-receptors, an NF-κB reporter construct, and a <i>Renilla</i> luciferase reporter plasmid as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098512#pone-0098512-g002" target="_blank">Figure 2</a>. The transfected cells were stimulated with ArtinM (15.6, 156, and 780 nM) at 37°C for 4 h. Medium and cells transfected with an empty vector were used as the negative controls. The positive controls were P3C4 (1 nM) for TLR2/1 activation (A and C) and FSL1 (0.1 nM) for TLR2/6 activation (B and D). The luciferase activity (A and B) was measured as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098512#s2" target="_blank">materials and methods</a>. IL-8 levels in the culture supernatants (C and D) were measured by ELISA. Statistical comparisons between the cells incubated with medium and the cells stimulated with ArtinM were performed with a one-way analysis of variance followed by Bonferroni's multiple comparison test. * p<0.05.</p

Enhanced TLR2 relative expression by ArtinM-stimulated macrophages.

<p>Peritoneal macrophages from C57BL/6 mice were incubated with ArtinM (39 nM) for 5 h. Medium was used as a negative control and P3C4 (1 µg/mL) was used as a positive control. RNA from macrophages were isolated and used for qRT-PCR as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098512#s2" target="_blank">Materials and Methods</a>. The results are expressed as the relative expression of TLR2 after quantification using the ΔΔCt method and normalized to β-actin expression. Statistical comparisons between stimulated cells and unstimulated were performed with one-way analysis of variance followed by Bonferroni's multiple comparison test. ** p<0.01.</p

ArtinM binding to TLR2 depends on sugar recognition.

<p>(A and B) Peritoneal macrophages from C57BL/6 mice were incubated with biotinylated ArtinM after pre-incubation with anti-TLR2 antibody or non-specific IgG. ArtinM binding was detected with streptavidin-FITC and analyzed by flow cytometry, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098512#s2" target="_blank">materials and methods</a>. Results are expressed as the percentage of cells positive for ArtinM binding (A) and MFI (median fluorescence intensity) (B). (C) The dependence of ArtinM-TLR2 binding on carbohydrate recognition used anti-TLR2 antibody coated onto 96-well microplates (5 µg/mL) to capture TLR2 from a cellular extract of peritoneal macrophages. Biotinylated ArtinM (40 µg/mL), previously incubated with the indicated concentrations of Manα1-3 [Manα1-6] Man or Galα(1,6)Galα(1,6)Gluα(1,2)Fru, was added to the wells. After washing, ArtinM binding was detected by neutravidin-AP, and signal was developed with <i>p</i>-nitrophenyl phosphate. Results are expressed in O.D as the mean ± SEM. Statistical comparisons between cells incubated or not with carbohydrates were performed with one-way analysis of variance followed by Bonferroni's multiple comparison test. *p<0.05.</p