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Collaborative induction of inflammatory responses by dectin-1 and Toll-like receptor 2.

Gantner BN, Simmons RM, Canavera SJ, Akira S, Underhill DM - J. Exp. Med. (2003)

Bottom Line: Toll-like receptors (TLRs) mediate recognition of a wide range of microbial products including lipopolysaccharides, lipoproteins, flagellin, and bacterial DNA, and signaling through TLRs leads to the production of inflammatory mediators.In this report we have examined how dectin-1, a lectin family receptor for beta-glucans, collaborates with TLRs in recognizing microbes.The receptor is recruited to phagosomes containing zymosan particles but not to phagosomes containing immunoglobulin G-opsonized particles.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, University of Washington, Seattle, 98103, USA.

ABSTRACT
Toll-like receptors (TLRs) mediate recognition of a wide range of microbial products including lipopolysaccharides, lipoproteins, flagellin, and bacterial DNA, and signaling through TLRs leads to the production of inflammatory mediators. In addition to TLRs, many other surface receptors have been proposed to participate in innate immunity and microbial recognition, and signaling through some of these receptors is likely to cooperate with TLR signaling in defining inflammatory responses. In this report we have examined how dectin-1, a lectin family receptor for beta-glucans, collaborates with TLRs in recognizing microbes. Dectin-1, which is expressed at low levels on macrophages and high levels on dendritic cells, contains an immunoreceptor tyrosine-based activation motif-like signaling motif that is tyrosine phosphorylated upon activation. The receptor is recruited to phagosomes containing zymosan particles but not to phagosomes containing immunoglobulin G-opsonized particles. Dectin-1 expression enhances TLR-mediated activation of nuclear factor kappa B by beta-glucan-containing particles, and in macrophages and dendritic cells dectin-1 and TLRs are synergistic in mediating production of cytokines such as interleukin 12 and tumor necrosis factor alpha. Additionally, dectin-1 triggers production of reactive oxygen species, an inflammatory response that is primed by TLR activation. The data demonstrate that collaborative recognition of distinct microbial components by different classes of innate immune receptors is crucial in orchestrating inflammatory responses.

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Dectin-1 and TLR2 collaborate in zymosan recognition by HEK 293 cells. (a and b) Activation of NF-κB (ELAM luciferase) was measured in HEK 293 cells transiently transfected with the indicated expression vectors and stimulated with 100 μg/ml zymosan or 100 ng/ml PAM3CSK4 lipopeptide for 4 h. (c) Activation of NF-κB was measured in HEK 293 cells transfected with expression vectors for TLR2 (together with either CD14 or dectin-1) and stimulated with 100 μg/ml zymosan in the indicated concentrations of laminarin.
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fig2: Dectin-1 and TLR2 collaborate in zymosan recognition by HEK 293 cells. (a and b) Activation of NF-κB (ELAM luciferase) was measured in HEK 293 cells transiently transfected with the indicated expression vectors and stimulated with 100 μg/ml zymosan or 100 ng/ml PAM3CSK4 lipopeptide for 4 h. (c) Activation of NF-κB was measured in HEK 293 cells transfected with expression vectors for TLR2 (together with either CD14 or dectin-1) and stimulated with 100 μg/ml zymosan in the indicated concentrations of laminarin.

Mentions: It is not clear whether in the presence of TLR-mediated inflammatory signaling additional receptors contribute to modulation of gene expression. Therefore, we explored whether recognition by a phagocytic receptor for zymosan (dectin-1) could contribute to TLR-mediated signaling. As we and others have previously observed, HEK 293 cells (that do not normally express TLR2 or respond to TLR2 stimuli) transiently transfected with TLR2 and an NF-κB luciferase reporter plasmid respond strongly to the soluble TLR2 stimulus PAM3CSK4 lipopeptide. However, the cells respond poorly to zymosan, suggesting that additional recognition molecules are required for efficient TLR2–mediated detection of zymosan (Fig. 2 a). Coexpression of dectin-1 (that is not endogenously expressed by HEK 293 cells; reference 16) enhanced TLR2–mediated recognition of zymosan to levels comparable to stimulation by the soluble lipopeptide (Fig. 2 a). Although expression of dectin-1 alone in HEK 293 cells conferred the ability to phagocytose zymosan (unpublished data), it did not confer NF-κB activation by zymosan in this system (Fig. 2 a), suggesting that dectin-1 signaling alone is not sufficient to activate NF-κB and associated downstream inflammatory responses. Dectin-1–enhanced signaling was clearly dependent on TLR2 activation because expression of a signaling-deficient mutant of TLR2, TLR2-P681H (11), blocked NF-κB activation (Fig. 2 a). Dectin-1 signaling is required for enhanced TLR2 responses because mutation of dectin-1 either by truncation of the intracellular amino terminal 38 acids (Dectin-Δ38) or by substitution of tyrosine 15 (in the ITAM-like motif) to serine (DectinY15S) abrogated the enhancement (Fig. 2 b). Dectin-enhanced signaling was dependent on recognition of β-glucan on the particle because laminarin (a soluble β-glucan from the brown seaweed Laminaria digitata) completely blocked the enhancement (Fig. 2 c). Although we have observed dectin-1 and TLR2 enriched together on phagosomes containing zymosan, we have not detected direct physical interactions between the receptors, suggesting that cooperation occurs by parallel activation (unpublished data). We have previously observed that CD14 (a soluble or GPI-linked protein) can facilitate TLR2–mediated inflammatory responses to zymosan (11), so we compared the requirement for β-glucan recognition by CD14 and dectin-1. Laminarin had no effect on intracellular signaling by TLR2 coexpressed with CD14 (Fig. 2 c), demonstrating that neither CD14 nor TLR2 directly recognizes β-glucan. Taken together, these observations demonstrate that dectin-1 enhances TLR signaling in response to β-glucan–containing particulate stimuli.


Collaborative induction of inflammatory responses by dectin-1 and Toll-like receptor 2.

Gantner BN, Simmons RM, Canavera SJ, Akira S, Underhill DM - J. Exp. Med. (2003)

Dectin-1 and TLR2 collaborate in zymosan recognition by HEK 293 cells. (a and b) Activation of NF-κB (ELAM luciferase) was measured in HEK 293 cells transiently transfected with the indicated expression vectors and stimulated with 100 μg/ml zymosan or 100 ng/ml PAM3CSK4 lipopeptide for 4 h. (c) Activation of NF-κB was measured in HEK 293 cells transfected with expression vectors for TLR2 (together with either CD14 or dectin-1) and stimulated with 100 μg/ml zymosan in the indicated concentrations of laminarin.
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Related In: Results  -  Collection

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fig2: Dectin-1 and TLR2 collaborate in zymosan recognition by HEK 293 cells. (a and b) Activation of NF-κB (ELAM luciferase) was measured in HEK 293 cells transiently transfected with the indicated expression vectors and stimulated with 100 μg/ml zymosan or 100 ng/ml PAM3CSK4 lipopeptide for 4 h. (c) Activation of NF-κB was measured in HEK 293 cells transfected with expression vectors for TLR2 (together with either CD14 or dectin-1) and stimulated with 100 μg/ml zymosan in the indicated concentrations of laminarin.
Mentions: It is not clear whether in the presence of TLR-mediated inflammatory signaling additional receptors contribute to modulation of gene expression. Therefore, we explored whether recognition by a phagocytic receptor for zymosan (dectin-1) could contribute to TLR-mediated signaling. As we and others have previously observed, HEK 293 cells (that do not normally express TLR2 or respond to TLR2 stimuli) transiently transfected with TLR2 and an NF-κB luciferase reporter plasmid respond strongly to the soluble TLR2 stimulus PAM3CSK4 lipopeptide. However, the cells respond poorly to zymosan, suggesting that additional recognition molecules are required for efficient TLR2–mediated detection of zymosan (Fig. 2 a). Coexpression of dectin-1 (that is not endogenously expressed by HEK 293 cells; reference 16) enhanced TLR2–mediated recognition of zymosan to levels comparable to stimulation by the soluble lipopeptide (Fig. 2 a). Although expression of dectin-1 alone in HEK 293 cells conferred the ability to phagocytose zymosan (unpublished data), it did not confer NF-κB activation by zymosan in this system (Fig. 2 a), suggesting that dectin-1 signaling alone is not sufficient to activate NF-κB and associated downstream inflammatory responses. Dectin-1–enhanced signaling was clearly dependent on TLR2 activation because expression of a signaling-deficient mutant of TLR2, TLR2-P681H (11), blocked NF-κB activation (Fig. 2 a). Dectin-1 signaling is required for enhanced TLR2 responses because mutation of dectin-1 either by truncation of the intracellular amino terminal 38 acids (Dectin-Δ38) or by substitution of tyrosine 15 (in the ITAM-like motif) to serine (DectinY15S) abrogated the enhancement (Fig. 2 b). Dectin-enhanced signaling was dependent on recognition of β-glucan on the particle because laminarin (a soluble β-glucan from the brown seaweed Laminaria digitata) completely blocked the enhancement (Fig. 2 c). Although we have observed dectin-1 and TLR2 enriched together on phagosomes containing zymosan, we have not detected direct physical interactions between the receptors, suggesting that cooperation occurs by parallel activation (unpublished data). We have previously observed that CD14 (a soluble or GPI-linked protein) can facilitate TLR2–mediated inflammatory responses to zymosan (11), so we compared the requirement for β-glucan recognition by CD14 and dectin-1. Laminarin had no effect on intracellular signaling by TLR2 coexpressed with CD14 (Fig. 2 c), demonstrating that neither CD14 nor TLR2 directly recognizes β-glucan. Taken together, these observations demonstrate that dectin-1 enhances TLR signaling in response to β-glucan–containing particulate stimuli.

Bottom Line: Toll-like receptors (TLRs) mediate recognition of a wide range of microbial products including lipopolysaccharides, lipoproteins, flagellin, and bacterial DNA, and signaling through TLRs leads to the production of inflammatory mediators.In this report we have examined how dectin-1, a lectin family receptor for beta-glucans, collaborates with TLRs in recognizing microbes.The receptor is recruited to phagosomes containing zymosan particles but not to phagosomes containing immunoglobulin G-opsonized particles.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology, University of Washington, Seattle, 98103, USA.

ABSTRACT
Toll-like receptors (TLRs) mediate recognition of a wide range of microbial products including lipopolysaccharides, lipoproteins, flagellin, and bacterial DNA, and signaling through TLRs leads to the production of inflammatory mediators. In addition to TLRs, many other surface receptors have been proposed to participate in innate immunity and microbial recognition, and signaling through some of these receptors is likely to cooperate with TLR signaling in defining inflammatory responses. In this report we have examined how dectin-1, a lectin family receptor for beta-glucans, collaborates with TLRs in recognizing microbes. Dectin-1, which is expressed at low levels on macrophages and high levels on dendritic cells, contains an immunoreceptor tyrosine-based activation motif-like signaling motif that is tyrosine phosphorylated upon activation. The receptor is recruited to phagosomes containing zymosan particles but not to phagosomes containing immunoglobulin G-opsonized particles. Dectin-1 expression enhances TLR-mediated activation of nuclear factor kappa B by beta-glucan-containing particles, and in macrophages and dendritic cells dectin-1 and TLRs are synergistic in mediating production of cytokines such as interleukin 12 and tumor necrosis factor alpha. Additionally, dectin-1 triggers production of reactive oxygen species, an inflammatory response that is primed by TLR activation. The data demonstrate that collaborative recognition of distinct microbial components by different classes of innate immune receptors is crucial in orchestrating inflammatory responses.

Show MeSH
Related in: MedlinePlus