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

Mariano VS, Zorzetto-Fernandes AL, da Silva TA, Ruas LP, Nohara LL, Almeida IC, Roque-Barreira MC - PLoS ONE (2014)

Bottom Line: Stimulation with ArtinM induced NF-κB activation and interleukin (IL)-8 production in cells transfected with TLR2, TLR2/1, or TLR2/6.Notably,ArtinM binding to TLR2 was selectively inhibited when the lectin was pre-incubated with mannotriose.Thus, ArtinM interacts directly with TLR2 or TLR2 heterodimers in a carbohydrate recognition-dependent manner and functions as a TLR2 agonist with immunomodulatory properties.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos, Faculdade de Medicina de Ribeirão Preto, USP, São Paulo, Brasil.

ABSTRACT
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.

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TLR2 mediates the cytokine production induced by ArtinM.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.
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pone-0098512-g006: TLR2 mediates the cytokine production induced by ArtinM.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.

Mentions: To investigate the biological relevance of the interaction of ArtinM with TLR2, we compared cytokine production by macrophages fromTLR2-KO and WT mice after stimulation with ArtinM for 24 h. Cell suspensions of peritoneal macrophages or adherent spleen cells derived from TLR2-KO mice produced significantly lower levels of IL-12p40 (41%) and IL-10 (42%) than those obtained from WT mice, in response to ArtinM stimulation (Figure 6). These results indicate that the ArtinM-induced production of cytokines requires the TLR2signaling pathway. Although production of IL-12 and IL-10 was still detected in cells from TLR2KO mice after stimulation with ArtinM, cytokine production was not detected after stimulation with the control TLR2 agonist P3C4. This result suggests that, in addition to TLR2, whose biological relevance is indubitable, additional(s) receptor(s) may contribute to the cell signaling and cytokine production triggered by ArtinM.


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

Mariano VS, Zorzetto-Fernandes AL, da Silva TA, Ruas LP, Nohara LL, Almeida IC, Roque-Barreira MC - PLoS ONE (2014)

TLR2 mediates the cytokine production induced by ArtinM.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.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4043963&req=5

pone-0098512-g006: TLR2 mediates the cytokine production induced by ArtinM.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.
Mentions: To investigate the biological relevance of the interaction of ArtinM with TLR2, we compared cytokine production by macrophages fromTLR2-KO and WT mice after stimulation with ArtinM for 24 h. Cell suspensions of peritoneal macrophages or adherent spleen cells derived from TLR2-KO mice produced significantly lower levels of IL-12p40 (41%) and IL-10 (42%) than those obtained from WT mice, in response to ArtinM stimulation (Figure 6). These results indicate that the ArtinM-induced production of cytokines requires the TLR2signaling pathway. Although production of IL-12 and IL-10 was still detected in cells from TLR2KO mice after stimulation with ArtinM, cytokine production was not detected after stimulation with the control TLR2 agonist P3C4. This result suggests that, in addition to TLR2, whose biological relevance is indubitable, additional(s) receptor(s) may contribute to the cell signaling and cytokine production triggered by ArtinM.

Bottom Line: Stimulation with ArtinM induced NF-κB activation and interleukin (IL)-8 production in cells transfected with TLR2, TLR2/1, or TLR2/6.Notably,ArtinM binding to TLR2 was selectively inhibited when the lectin was pre-incubated with mannotriose.Thus, ArtinM interacts directly with TLR2 or TLR2 heterodimers in a carbohydrate recognition-dependent manner and functions as a TLR2 agonist with immunomodulatory properties.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Biologia Celular e Molecular e Bioagentes Patogênicos, Faculdade de Medicina de Ribeirão Preto, USP, São Paulo, Brasil.

ABSTRACT
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.

Show MeSH