IL-17 Induction by ArtinM is Due to Stimulation of IL-23 and IL-1 Release and/or Interaction with CD3 in CD4+ T Cells.

ArtinM is a D-mannose-binding lectin extracted from the seeds of Artocarpus heterophyllus that interacts with TLR2 N-glycans and activates antigen-presenting cells (APCs), as manifested by IL-12 production. In vivo ArtinM administration induces Th1 immunity and confers protection against infection with several intracellular pathogens. In the murine model of Candida albicans infection, it was verified that, in addition to Th1, ArtinM induces Th17 immunity manifested by high IL-17 levels in the treated animals. Herein, we investigated the mechanisms accounting for the ArtinM-induced IL-17 production. We found that ArtinM stimulates the IL-17 production by spleen cells in BALB/c or C57BL/6 mice, a response that was significantly reduced in the absence of IL-23, MyD88, or IL-1R. Furthermore, we showed that ArtinM directly induced the IL-23 mRNA expression and the IL-1 production by macrophages. Consistently, in cell suspensions depleted of macrophages, the IL-17 production stimulated by ArtinM was reduced by 53% and the exogenous IL-23 acted synergistically with ArtinM in promoting IL-17 production by spleen cell suspensions. We verified that the absence of IL-23, IL-1R, or MyD88 inhibited, but did not block, the IL-17 production by ArtinM-stimulated spleen cells. Therefore, we investigated whether ArtinM exerts a direct effect on CD4+ T cells in promoting IL-17 production. Indeed, spleen cell suspensions depleted of CD4+ T cells responded to ArtinM with very low levels of IL-17 release. Likewise, isolated CD4+ T cells under ArtinM stimulus augmented the expression of TGF-β mRNA and released high levels of IL-17. Considering the observed synergism between IL-23 and ArtinM, we used cells from IL-23 KO mice to assess the direct effect of lectin on CD4+ T cells. We verified that ArtinM increased the IL-17 production significantly, a response that was inhibited when the CD4+ T cells were pre-incubated with anti-CD3 antibody. In conclusion, ArtinM stimulates the production of IL-17 by CD4+ T cells in two major ways: (I) through the induction of IL-23 and IL-1 by APCs and (II) through the direct interaction with CD3 on the CD4+ T cells. This study contributes to elucidation of mechanisms accounting for the property of ArtinM in inducing Th17 immunity and opens new perspectives in designing strategies for modulating immunity by using carbohydrate recognition agents

Recognition of TLR2 N-glycans: critical role in ArtinM immunomodulatory activity.

TLR2 plays a critical role in the protection against Paracoccidioides brasiliensis conferred by ArtinM administration. ArtinM, a D-mannose-binding lectin from Artocarpus heterophyllus, induces IL-12 production in macrophages and dendritic cells, which accounts for the T helper1 immunity that results from ArtinM administration. We examined the direct interaction of ArtinM with TLR2using HEK293A cells transfected with TLR2, alone or in combination with TLR1 or TLR6, together with accessory proteins. Stimulation with ArtinM induced NF-κB activation and interleukin (IL)-8 production in cells transfected with TLR2, TLR2/1, or TLR2/6. Murine macrophages that were stimulated with ArtinM had augmented TLR2 mRNA expression. Furthermore, pre-incubation of unstimulated macrophages with an anti-TLR2 antibody reduced the cell labeling with ArtinM. In addition, a microplate assay revealed that ArtinM bound to TLR2 molecules that had been captured by specific antibodies from a macrophages lysate. Notably,ArtinM binding to TLR2 was selectively inhibited when the lectin was pre-incubated with mannotriose. The biological relevance of the direct interaction of ArtinM with TLR2 glycans was assessed using macrophages from TLR2-KOmice, which produced significantly lower levels of IL-12 and IL-10 in response to ArtinM than macrophages from wild-type mice. Pre-treatment of murine macrophages with pharmacological inhibitors of signaling molecules demonstrated the involvement of p38 MAPK and JNK in the IL-12 production induced by ArtinM and the involvement ofPI3K in IL-10 production. Thus, ArtinM interacts directly with TLR2 or TLR2 heterodimers in a carbohydrate recognition-dependent manner and functions as a TLR2 agonist with immunomodulatory properties

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

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

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

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

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