Dectin-1 is a fungal pattern recognition receptor that binds to β-glucans and triggers cytokine production by facilitating interaction with TLR2 or by directly activating spleen tyrosine kinase (Syk). To assess the possible role of Dectin-1 in the innate response to mycobacteria, we used an in vitro system in which IL-12p40 production is measured in splenic dendritic cells (SpDC) following exposure to live Mycobacterium tuberculosis bacilli. Treatment of SpDC with laminarin or glucan phosphate, two molecules known to block Dectin-1-dependent activity, led to a reduction in M. tuberculosis-induced IL-12p40 as well as IL-12p70 production. Moreover, SpDC from Dectin-1 -/- chimeric mice displayed reduced IL-12p40 production in response to mycobacteria when compared with Dectin-sufficient DC. Laminarin treatment also inhibited mycobacterial-induced IL-12p40 production in DC from TLR2 -/- mice, arguing that Dectin-1 functions independently of TLR2 signaling in this system. Importantly, a Dectin-1 fusion protein was found to directly bind to live mycobacteria in a laminarin-inhibitable manner indicating the presence of ligands for the receptor in the bacterium and laminarin pretreatment resulted in reduced association of mycobacteria to SpDC. In additional experiments, mycobacterial stimulation was shown to be associated with increased phosphorylation of Syk and this response was inhibited by laminarin. Furthermore, pharmacologic inhibition of Syk reduced the M. tuberculosis-induced IL-12p40 response. Together, these findings support a role for Dectin-1 in promoting M. tuberculosis-induced IL-12p40 production by DC in which the receptor augments bacterial-host cell interaction and enhances the subsequent cytokine response through an unknown mechanism involving Syk signaling.

TLRs play a critical role in the induction of innate and adaptive immunity. However, TLRs have also been reported to mediate the pathophysiology of organ damage following ischemia/reperfusion (I/R) injury. We have reported that TLR4-/_ mice show decreased myocardial injury following I/R; however, the protective mechanisms have not been elucidated. We examined the role of the PI3K/Akt signaling pathway in TLR4-/- cardioprotection following I/R injury. TLR4_/_ and age-matched wild-type (WT) mice were subjected to myocardial ischemia for 45 min, followed by reperfusion for 4 h. Pharmacologic inhibitors of PI3K (wortmannin or LY294002) were administered 1 h before myocardial I/R. Myocardial infarct size/area at risk was reduced by 51.2% in TLR4-/- vs WT mice. Cardiac myocyte apoptosis was also increased in WT vs TLR4_/_ mice following I/R. Pharmacologic blockade of PI3K abrogated myocardial protection in TLR4_/_ mice following I/R. Specifically, heart infarct size/area at risk was increased by 98% in wortmannin and 101% in LY294002-treated TLR4-/- mice, when compared with control TLR4_/_ mice. These data indicate that protection against myocardial I/R injury in TLR4-/~ mice is mediated through a PI3K/Akt-dependent mechanism. The mechanisms by which PI3K/Akt are increased in the TLR4_/_ myocardium may involve increased phosphorylation/ inactivation of myocardial phosphatase and tensin homolog deleted on chromosome 10 as well as increased phosphorylation/inactivation of myocardial glycogen synthase kinase-3β. These data implicate innate immune signaling pathways in the pathology of acute myocardial I/R injury. These data also suggest that modulation of TLR4/PI3K/Akt-dependent signaling pathways may be a viable strategy for reducing myocardial I/R injury.

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The fungal pathogen Candida albicans has a multilayered cell wall composed of an outer layer of proteins glycosylated with N- or O-linked mannosyl residues and an inner skeletal layer of β-glucans and chitin. We demonstrate that cytokine production by human mononuclear cells or murine macrophages was markedly reduced when stimulated by C. albicans mutants defective in mannosylation. Recognition of mannosyl residues was mediated by mannose receptor binding to N-linked mannosyl residues and by TLR4 binding to O-linked mannosyl residues. Residual cytokine production was mediated by recognition of β-glucan by the dectin-1/TLR2 receptor complex. C. albicans mutants with a cell wall defective in mannosyl residues were less virulent in experimental disseminated candidiasis and elicited reduced cytokine production in vivo. We concluded that recognition of C. albicans by monocytes/macrophages is mediated by 3 recognition systems of differing importance, each of which senses specific layers of the C. albicans cell wall.

Dectin-1 is a specific receptor for β-glucans and a major receptor for fungal particles on macrophages (Mφ). It is a type II membrane receptor that has a C-terminal, NK-like, C-type lectin-like domain separated from the cell membrane by a short stalk region and a cytoplasmic immunoreceptor tyrosine-based activation-like motif. We observed functional differences in dectin-1-dependent recognition of fungal particles by Mφ from different mouse strains. RT-PCR analysis revealed that mice have at least two splice forms of dectin-1, generated by differential usage of exon 3, encoding the full-length dectin-1A and a stalkless Mφ dectin-1B. Mφ from BALB/c mice and genetically related mice expressed both isoforms in similar amounts, whereas Mφ from C57BL/6 and related mice mainly expressed the smaller isoform. NIH-3T3 fibroblast and RAW264.7 macrophage cell lines stably expressing either isoform were able to bind and phagocytose zymosan at 37°C. However, binding by the smaller dectin-1B isoform was significantly affected at lower temperatures. These properties were shared by the equivalent human isoforms. The relative ability of each of the isoforms to induce TNF-α production in RAW264.7 Mφ was also found to be different. These results are the first evidence that dectin-1 isoforms are functionally distinct and indicate that differential isoform usage may represent a mechanism of regulating cellular responses to β-glucans.

Dectin-1 is the primary pattern recognition receptor for fungal glucans. Dectin-1 mediates the internalization and biological response to glucans. We examined the effect of i.v. or i.p. glucan phosphate (GP) administration on Dectin-1 membrane expression in murine peripheral blood leukocytes, splenocytes, bone marrow, and peritoneal cells from 3 h to 10 days after injection. Circulating leukocytes were also examined for uptake and internalization of glucans from the blood. Fluorescent-labeled GP was taken up from the systemic circulation by circulating peripheral leukocytes, splenocytes, and peritoneal cells. Following internalization, glucan colocalized with Dectin-1 in an intracellular vesicle. A single parenteral injection of GP resulted in a significant reduction (∼33-85%) in peripheral leukocyte membrane-associated Dectin-1 positivity that lasted for up to 7 days. The loss of leukocyte membrane-associated Dectin-1 after GP administration was primarily due to decreased levels of Dectin-1 on neutrophil and monocyte membranes with no significant changes in the percentage of neutrophils or monocytes circulating in the blood. Administration of control carbohydrate polymers, i.e., mannan or pullulan, which are not ligands for Dectin-1, did not decrease Dectin-1 leukocyte positivity, indicating that the effect on Dectin-1 is specific to glucans. In fact, mannan administration increased leukocyte Dectin-1 positivity, thus demonstrating a differential effect on leukocyte Dectin-1, compared with GP. We conclude that systemic administration of GP has a specific and prolonged effect on loss of leukocyte membrane Dectin-1 positivity. These data may have important implications for developing dosing regimens for immunomodulatory carbohydrates.

Nuclear factor-kappaB (NF-kappaB) is a key transcription factor that regulates the expression of genes involved in immune and inflammatory responses and in cell death and survival. This chapter describes in detail the method for measuring the NF-kappaB binding activity in the cultured human mast cell line HMC-1 using the electrophoretic mobility shift assay: the activation of NF-kappaB was illustrated by adding lipopolysaccharide to mast cells, nuclear proteins were isolated, and the NF-kappaB binding activity was determined. We also demonstrate the specificity of the NF-kappaB binding activity using a competition and supershift assay with specific antibodies that recognize NF-kappaB subunits p50 and p65. Lipopolysaccharide caused a rapid and significant increase in NF-kappaB binding activity.

Pattern-recognition receptors (PRRs) detect molecular signatures of microbes and initiate immune responses to infection. Prototypical PRRs such as Toll-like receptors (TLRs) signal via a conserved pathway to induce innate response genes. In contrast, the signaling pathways engaged by other classes of putative PRRs remain ill defined. Here, we demonstrate that the β-glucan receptor Dectin-1, a yeast binding C type lectin known to synergize with TLR2 to induce TNFα and IL-12, can also promote synthesis of IL-2 and IL-10 through phosphorylation of the membrane proximal tyrosine in the cytoplasmic domain and recruitment of Syk kinase. syk-/- dendritic cells (DCs) do not make IL-10 or IL-2 upon yeast stimulation but produce IL-12, indicating that the Dectin-1/Syk and Dectin-1/TLR2 pathways can operate independently. These results identify a novel signaling pathway involved in pattern recognition by C type lectins and suggest a potential role for Syk kinase in regulation of innate immunity.