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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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Cell signaling molecules that possibly mediate the macrophage activation induced by ArtinM.Peritoneal macrophages were pretreated with the inhibitors PD98059 (p42/44 MAPK or ERK 1/2), SB202190 (p38 MAPK), SP600125 (c-Jun N-terminal kinase), and LY-294002 (PI3K), or with medium alone (absence of inhibitor) and then stimulated with ArtinM (39 nM). After 48 h, IL-12p40 and IL-10 levels in the culture supernatants were assessed by ELISA. (A) Cytokine production reported as pg/mL (mean ± SEM) and statistical comparison were performed using a one-way analysis of variance followed by Bonferroni's multiple comparison test. *p<0.05. (B) Inhibition of ArtinM induced cytokine production after pre-incubation with the indicated inhibitors. The results are presented as the percent inhibition, which represents the ratio between uninhibited cells and those pretreated with inhibitors.
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pone-0098512-g007: Cell signaling molecules that possibly mediate the macrophage activation induced by ArtinM.Peritoneal macrophages were pretreated with the inhibitors PD98059 (p42/44 MAPK or ERK 1/2), SB202190 (p38 MAPK), SP600125 (c-Jun N-terminal kinase), and LY-294002 (PI3K), or with medium alone (absence of inhibitor) and then stimulated with ArtinM (39 nM). After 48 h, IL-12p40 and IL-10 levels in the culture supernatants were assessed by ELISA. (A) Cytokine production reported as pg/mL (mean ± SEM) and statistical comparison were performed using a one-way analysis of variance followed by Bonferroni's multiple comparison test. *p<0.05. (B) Inhibition of ArtinM induced cytokine production after pre-incubation with the indicated inhibitors. The results are presented as the percent inhibition, which represents the ratio between uninhibited cells and those pretreated with inhibitors.

Mentions: Because full activation of macrophages by ArtinM requires interaction with TLR2, we investigated the signaling pathways used to promote IL-12p40 and IL-10 production by assessing the effects of several pharmacological inhibitors (PD98059, SB202190, SP600125, and LY-294002) on the response to ArtinM stimulation. We evaluated IL-12p40 and IL-10 production by macrophages that were treated with inhibitors and then stimulated with ArtinM. IL-12p40 production induced by ArtinM was significantly inhibited by SB202190 and SP600125, indicating the involvement of p38 MAPK and JNK (Figure 7). In addition to these signaling molecules, PI3K was involved in IL-10 production, as indicated by the inhibitory effect of LY-294002 (Figure 7). Therefore, the signaling molecules activated in ArtinM-stimulated macrophages are partially specific for the production of different cytokines.


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)

Cell signaling molecules that possibly mediate the macrophage activation induced by ArtinM.Peritoneal macrophages were pretreated with the inhibitors PD98059 (p42/44 MAPK or ERK 1/2), SB202190 (p38 MAPK), SP600125 (c-Jun N-terminal kinase), and LY-294002 (PI3K), or with medium alone (absence of inhibitor) and then stimulated with ArtinM (39 nM). After 48 h, IL-12p40 and IL-10 levels in the culture supernatants were assessed by ELISA. (A) Cytokine production reported as pg/mL (mean ± SEM) and statistical comparison were performed using a one-way analysis of variance followed by Bonferroni's multiple comparison test. *p<0.05. (B) Inhibition of ArtinM induced cytokine production after pre-incubation with the indicated inhibitors. The results are presented as the percent inhibition, which represents the ratio between uninhibited cells and those pretreated with inhibitors.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0098512-g007: Cell signaling molecules that possibly mediate the macrophage activation induced by ArtinM.Peritoneal macrophages were pretreated with the inhibitors PD98059 (p42/44 MAPK or ERK 1/2), SB202190 (p38 MAPK), SP600125 (c-Jun N-terminal kinase), and LY-294002 (PI3K), or with medium alone (absence of inhibitor) and then stimulated with ArtinM (39 nM). After 48 h, IL-12p40 and IL-10 levels in the culture supernatants were assessed by ELISA. (A) Cytokine production reported as pg/mL (mean ± SEM) and statistical comparison were performed using a one-way analysis of variance followed by Bonferroni's multiple comparison test. *p<0.05. (B) Inhibition of ArtinM induced cytokine production after pre-incubation with the indicated inhibitors. The results are presented as the percent inhibition, which represents the ratio between uninhibited cells and those pretreated with inhibitors.
Mentions: Because full activation of macrophages by ArtinM requires interaction with TLR2, we investigated the signaling pathways used to promote IL-12p40 and IL-10 production by assessing the effects of several pharmacological inhibitors (PD98059, SB202190, SP600125, and LY-294002) on the response to ArtinM stimulation. We evaluated IL-12p40 and IL-10 production by macrophages that were treated with inhibitors and then stimulated with ArtinM. IL-12p40 production induced by ArtinM was significantly inhibited by SB202190 and SP600125, indicating the involvement of p38 MAPK and JNK (Figure 7). In addition to these signaling molecules, PI3K was involved in IL-10 production, as indicated by the inhibitory effect of LY-294002 (Figure 7). Therefore, the signaling molecules activated in ArtinM-stimulated macrophages are partially specific for the production of different cytokines.

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