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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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ArtinM binding to TLR2 depends on sugar recognition.(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 materials and methods. 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 p-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.
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pone-0098512-g005: ArtinM binding to TLR2 depends on sugar recognition.(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 materials and methods. 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 p-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.

Mentions: To further characterize the interaction of ArtinM with TLR2, we evaluated the ability of an anti-TLR2 antibody to inhibit ArtinM binding to the surface of murine macrophages. Cells harvested from the peritoneal cavity of C57BL/6 mice were pre-incubated with anti-TLR2 antibody. Then, they were washed, incubated with biotinylated ArtinM, and reacted with a streptavidin-FITC conjugate. Flow cytometric analysis showed that pre-incubation of cells with anti-TLR2 antibody decreased the number of ArtinM-labeled cells by 30% (Figure 5A) and the median fluorescence intensity reduced 71.5% (Figure 5B), suggesting that TLR2 is a major ligand for ArtinM on the surface of murine macrophages.


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)

ArtinM binding to TLR2 depends on sugar recognition.(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 materials and methods. 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 p-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.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0098512-g005: ArtinM binding to TLR2 depends on sugar recognition.(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 materials and methods. 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 p-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.
Mentions: To further characterize the interaction of ArtinM with TLR2, we evaluated the ability of an anti-TLR2 antibody to inhibit ArtinM binding to the surface of murine macrophages. Cells harvested from the peritoneal cavity of C57BL/6 mice were pre-incubated with anti-TLR2 antibody. Then, they were washed, incubated with biotinylated ArtinM, and reacted with a streptavidin-FITC conjugate. Flow cytometric analysis showed that pre-incubation of cells with anti-TLR2 antibody decreased the number of ArtinM-labeled cells by 30% (Figure 5A) and the median fluorescence intensity reduced 71.5% (Figure 5B), suggesting that TLR2 is a major ligand for ArtinM on the surface of murine macrophages.

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
Related in: MedlinePlus