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Dectin-2 Recognizes Mannosylated O-antigens of Human Opportunistic Pathogens and Augments Lipopolysaccharide Activation of Myeloid Cells *

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ABSTRACT

LPS consists of a relatively conserved region of lipid A and core oligosaccharide and a highly variable region of O-antigen polysaccharide. Whereas lipid A is known to bind to the Toll-like receptor 4 (TLR4)-myeloid differentiation factor 2 (MD2) complex, the role of the O-antigen remains unclear. Here we report a novel molecular interaction between dendritic cell-associated C-type lectin-2 (Dectin-2) and mannosylated O-antigen found in a human opportunistic pathogen, Hafnia alvei PCM 1223, which has a repeating unit of [-Man-α1,3-Man-α1,2-Man-α1,2-Man-α1,2-Man-α1,3-]. H. alvei LPS induced higher levels of TNFα and IL-10 from mouse bone marrow-derived dendritic cells (BM-DCs), when compared with Salmonella enterica O66 LPS, which has a repeat of [-Gal-α1,6-Gal-α1,4-[Glc-β1,3]GalNAc-α1,3-GalNAc-β1,3-]. In a cell-based reporter assay, Dectin-2 was shown to recognize H. alvei LPS. This binding was inhibited by mannosidase treatment of H. alvei LPS and by mutations in the carbohydrate-binding domain of Dectin-2, demonstrating that H. alvei LPS is a novel glycan ligand of Dectin-2. The enhanced cytokine production by H. alvei LPS was Dectin-2-dependent, because Dectin-2 knock-out BM-DCs failed to do so. This receptor cross-talk between Dectin-2 and TLR4 involved events including spleen tyrosine kinase (Syk) activation and receptor juxtaposition. Furthermore, another mannosylated LPS from Escherichia coli O9a also bound to Dectin-2 and augmented TLR4 activation of BM-DCs. Taken together, these data indicate that mannosylated O-antigens from several Gram-negative bacteria augment TLR4 responses through interaction with Dectin-2.

No MeSH data available.


Co-stimulation of BM-DCs with Gal-LPS and yeast mannan failed to enhance cytokine production. BM-DCs were stimulated with the indicated stimuli, and cytokine production was analyzed as described in the legend to Fig. 1C. Data are representative of three independent experiments with similar results. Error bars, S.D. Statistical analyses were performed by one-way ANOVA followed by Tukey's test. **, p < 0.01; ***, p < 0.001; n.s., not statistically significant.
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Figure 5: Co-stimulation of BM-DCs with Gal-LPS and yeast mannan failed to enhance cytokine production. BM-DCs were stimulated with the indicated stimuli, and cytokine production was analyzed as described in the legend to Fig. 1C. Data are representative of three independent experiments with similar results. Error bars, S.D. Statistical analyses were performed by one-way ANOVA followed by Tukey's test. **, p < 0.01; ***, p < 0.001; n.s., not statistically significant.

Mentions: Because Man-LPS has the binding epitopes for both Dectin-2 and TLR4, we hypothesized that receptor juxtaposition by Man-LPS is the mechanism underpinning the synergy. To test this hypothesis, BM-DCs were stimulated with Gal-LPS as a TLR4 ligand in the presence of yeast α-linked mannan, a known Dectin-2 ligand. As shown in Fig. 5, the addition of yeast mannan was not sufficient to enhance cytokine production, compared with the Gal-LPS, demonstrating that receptor juxtaposition is required to achieve synergy.


Dectin-2 Recognizes Mannosylated O-antigens of Human Opportunistic Pathogens and Augments Lipopolysaccharide Activation of Myeloid Cells *
Co-stimulation of BM-DCs with Gal-LPS and yeast mannan failed to enhance cytokine production. BM-DCs were stimulated with the indicated stimuli, and cytokine production was analyzed as described in the legend to Fig. 1C. Data are representative of three independent experiments with similar results. Error bars, S.D. Statistical analyses were performed by one-way ANOVA followed by Tukey's test. **, p < 0.01; ***, p < 0.001; n.s., not statistically significant.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5016159&req=5

Figure 5: Co-stimulation of BM-DCs with Gal-LPS and yeast mannan failed to enhance cytokine production. BM-DCs were stimulated with the indicated stimuli, and cytokine production was analyzed as described in the legend to Fig. 1C. Data are representative of three independent experiments with similar results. Error bars, S.D. Statistical analyses were performed by one-way ANOVA followed by Tukey's test. **, p < 0.01; ***, p < 0.001; n.s., not statistically significant.
Mentions: Because Man-LPS has the binding epitopes for both Dectin-2 and TLR4, we hypothesized that receptor juxtaposition by Man-LPS is the mechanism underpinning the synergy. To test this hypothesis, BM-DCs were stimulated with Gal-LPS as a TLR4 ligand in the presence of yeast α-linked mannan, a known Dectin-2 ligand. As shown in Fig. 5, the addition of yeast mannan was not sufficient to enhance cytokine production, compared with the Gal-LPS, demonstrating that receptor juxtaposition is required to achieve synergy.

View Article: PubMed Central - PubMed

ABSTRACT

LPS consists of a relatively conserved region of lipid A and core oligosaccharide and a highly variable region of O-antigen polysaccharide. Whereas lipid A is known to bind to the Toll-like receptor 4 (TLR4)-myeloid differentiation factor 2 (MD2) complex, the role of the O-antigen remains unclear. Here we report a novel molecular interaction between dendritic cell-associated C-type lectin-2 (Dectin-2) and mannosylated O-antigen found in a human opportunistic pathogen, Hafnia alvei PCM 1223, which has a repeating unit of [-Man-&alpha;1,3-Man-&alpha;1,2-Man-&alpha;1,2-Man-&alpha;1,2-Man-&alpha;1,3-]. H. alvei LPS induced higher levels of TNF&alpha; and IL-10 from mouse bone marrow-derived dendritic cells (BM-DCs), when compared with Salmonella enterica O66 LPS, which has a repeat of [-Gal-&alpha;1,6-Gal-&alpha;1,4-[Glc-&beta;1,3]GalNAc-&alpha;1,3-GalNAc-&beta;1,3-]. In a cell-based reporter assay, Dectin-2 was shown to recognize H. alvei LPS. This binding was inhibited by mannosidase treatment of H. alvei LPS and by mutations in the carbohydrate-binding domain of Dectin-2, demonstrating that H. alvei LPS is a novel glycan ligand of Dectin-2. The enhanced cytokine production by H. alvei LPS was Dectin-2-dependent, because Dectin-2 knock-out BM-DCs failed to do so. This receptor cross-talk between Dectin-2 and TLR4 involved events including spleen tyrosine kinase (Syk) activation and receptor juxtaposition. Furthermore, another mannosylated LPS from Escherichia coli O9a also bound to Dectin-2 and augmented TLR4 activation of BM-DCs. Taken together, these data indicate that mannosylated O-antigens from several Gram-negative bacteria augment TLR4 responses through interaction with Dectin-2.

No MeSH data available.