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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.


Comparison of BM-DC response to Man and Gal-LPS.A, two LPS used in this study are shown. Man-LPS from H. alvei PCM 1223 has a mannosylated repeating unit, whereas Gal-LPS from S. enterica O66 has a galactosylated repeat. B, HEK293 cells stably transfected with TLR4-MD2 were cultured in the presence of LPS. The TLR4 activation was monitored by measuring alkaline phosphatase activity using the substrate. C, mouse BM-DCs were stimulated with 1 μg/ml Man-LPS or 4 μg/ml Gal-LPS for 7 h. The amount of TNFα and IL-10 in the culture supernatant was analyzed by ELISA. 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 < 0.001.
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Figure 1: Comparison of BM-DC response to Man and Gal-LPS.A, two LPS used in this study are shown. Man-LPS from H. alvei PCM 1223 has a mannosylated repeating unit, whereas Gal-LPS from S. enterica O66 has a galactosylated repeat. B, HEK293 cells stably transfected with TLR4-MD2 were cultured in the presence of LPS. The TLR4 activation was monitored by measuring alkaline phosphatase activity using the substrate. C, mouse BM-DCs were stimulated with 1 μg/ml Man-LPS or 4 μg/ml Gal-LPS for 7 h. The amount of TNFα and IL-10 in the culture supernatant was analyzed by ELISA. 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 < 0.001.

Mentions: In this study, we investigated the contribution of the α-linked mannosylated O-antigen in the LPS activation of myeloid cells. We compared DC response and Dectin-2 binding to the mannosylated LPS (Man-LPS) from H. alvei PCM 1223 and E. coli O9a with the LPS from Salmonella enterica O66 or K. pneumonia O1, which has the galactosylated O-antigen (Gal-LPS) (Fig. 1A). We observed binding between Man-LPS and Dectin-2, which led to augmentation of TLR4 response in mouse DCs and human monocytes. These results demonstrate a novel role of mannosylated O-antigen in activation of TLR4 in myeloid cells.


Dectin-2 Recognizes Mannosylated O-antigens of Human Opportunistic Pathogens and Augments Lipopolysaccharide Activation of Myeloid Cells *
Comparison of BM-DC response to Man and Gal-LPS.A, two LPS used in this study are shown. Man-LPS from H. alvei PCM 1223 has a mannosylated repeating unit, whereas Gal-LPS from S. enterica O66 has a galactosylated repeat. B, HEK293 cells stably transfected with TLR4-MD2 were cultured in the presence of LPS. The TLR4 activation was monitored by measuring alkaline phosphatase activity using the substrate. C, mouse BM-DCs were stimulated with 1 μg/ml Man-LPS or 4 μg/ml Gal-LPS for 7 h. The amount of TNFα and IL-10 in the culture supernatant was analyzed by ELISA. 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 < 0.001.
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Related In: Results  -  Collection

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Figure 1: Comparison of BM-DC response to Man and Gal-LPS.A, two LPS used in this study are shown. Man-LPS from H. alvei PCM 1223 has a mannosylated repeating unit, whereas Gal-LPS from S. enterica O66 has a galactosylated repeat. B, HEK293 cells stably transfected with TLR4-MD2 were cultured in the presence of LPS. The TLR4 activation was monitored by measuring alkaline phosphatase activity using the substrate. C, mouse BM-DCs were stimulated with 1 μg/ml Man-LPS or 4 μg/ml Gal-LPS for 7 h. The amount of TNFα and IL-10 in the culture supernatant was analyzed by ELISA. 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 < 0.001.
Mentions: In this study, we investigated the contribution of the α-linked mannosylated O-antigen in the LPS activation of myeloid cells. We compared DC response and Dectin-2 binding to the mannosylated LPS (Man-LPS) from H. alvei PCM 1223 and E. coli O9a with the LPS from Salmonella enterica O66 or K. pneumonia O1, which has the galactosylated O-antigen (Gal-LPS) (Fig. 1A). We observed binding between Man-LPS and Dectin-2, which led to augmentation of TLR4 response in mouse DCs and human monocytes. These results demonstrate a novel role of mannosylated O-antigen in activation of TLR4 in myeloid cells.

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.