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 β-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 κB by β-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 α. 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.

The ability of Candida albicans to rapidly and reversibly switch between yeast and filamentous morphologies is crucial to pathogenicity, and it is thought that the filamentous morphology provides some advantage during interaction with the mammalian immune system. Dectin-1 is a receptor that binds b-glucans and is important for macrophage phagocytosis of fungi. The receptor also collaborates with Toll-like receptors for inflammatory activation of phagocytes by fungi. We show that yeast cell wall bglucan is largely shielded from Dectin-1 by outer wall components. However, the normal mechanisms of yeast budding and cell separation create permanent scars which expose sufficient b-glucan to trigger antimicrobial responses through Dectin-1, including phagocytosis and activation of reactive oxygen production. During filamentous growth, no cell separation or subsequent b-glucan exposure occurs, and the pathogen fails to activate Dectin-1. The data demonstrate a mechanism by which C. albicans shape alone directly contributes to the method by which phagocytes recognize the fungus.

Dectin-1 is a lectin receptor for ␤-glucan that is important for innate macrophage recognition of fungi and contributes to phagocytosis, reactive oxygen production, and induction of inflammatory cytokines. The mechanisms by which Dectin-1 mediates intracellular signaling are just beginning to be defined. Spleen tyrosine kinase (Syk) is a protein tyrosine kinase that is critical for adaptive immune responses where it mediates signaling through B-cell receptors, T-cell receptors, and Fc receptors. Here we report that Dectin-1 activates Syk in macrophages and is important for Dectin-1-stimulated reactive oxygen production, but not for phagocytosis. Syk activation is restricted to a subpopulation of macrophages that is in equilibrium with cells that cannot activate the pathway. The proportion of macrophages using this signaling pathway can be modulated by cytokine treatment. Thus, Dectin-1 signaling reveals dynamic macrophage heterogeneity in inflammatory activation potential. (Blood. 2005;106:2543-2550)

A recent report also implicated Dectin-1 in macrophage activation by mycobacteria (9). Together these reports demonstrate that Dectin-1 collaborates with TLRs in the induction of proinflammatory cytokines. We have previously reported collaboration between Dectin-1 and TLR2 in the activation of NF-B (2). Additionally, a recent report has suggested that Dectin-1 can directly activate NF-B in dendritic cells via the signaling adaptor molecule CARD9 (10). Recognition of zymosan and live C. albicans yeast by Dectin-1 also triggers phagocytosis and the production of reactive oxygen species (ROS) 3 by mechanisms independent of TLR signaling (1-3, 11). Thus, Dectin-1 is a key coordinator of macrophage/dendritic cell antimicrobial responses.Dectin-1 has an ITAM-like motif in its intracellular tail and mutation of this motif results in loss of its signaling activity (1-3, 11, 12). ITAM-based signaling is classically associated with lymphocyte Ag receptors (TCR and BCR) ligation, resulting in activation of Src and Syk kinases. We recently demonstrated that Dectin-1 ligation triggers Src-Syk activation in macrophages and that Syk signaling is required for ROS induction (11). Rogers et al. also observed zymosan-stimulated Syk activation in their recent investigation of Dectin-1 signaling in dendritic cells (3). Dectin-1 ligation triggered Syk-dependent induction of IL-2 and IL-10.We now report that recognition of zymosan and live C. albicans yeast by Dectin-1 triggers activation of NFAT in macrophages and dendritic cells. The NFAT family of transcription factors comprises 4 closely related members with overlapping functions (NFATc1 through NFATc4) and the distantly related NFAT5, which is important for cellular responses to hypertonic stress (reviewed in Ref. 13). The role of NFAT activation has been defined most comprehensively in T cells: thymocyte development, T cell differentiation, T cell activation, and anergy are all regulated by NFAT (reviewed in Ref. 13). In T cells, NFAT proteins are normally inactive and highly phosphorylated, and TCR stimulation triggers dephosphorylation by the calcium-dependent phosphatase calcineurin. Dephosphorylated NFAT proteins enter the nucleus where they form strong collaborative complexes on DNA with a variety of transcription factors to integrate signaling pathways. In addition, NFAT transcription factors also regulate cell differentiation outside the immune system, with established roles in processes such as cardiac valve development, skeletal muscle differentiation, and osteoclastogenesis (reviewed in Ref. 14).A role for NFAT activation in the innate antimicrobial response has not previously been demonstrated. In this study we show that Dectin-1-mediated NFAT activation in macrophages and dendritic cells regulates the induction of early growth response (Egr) family transcription factors Egr2 and Egr3, as well as key inflammatory mediators cyclooxygenase-2 (COX-2), IL-2, IL-10, and IL-12 p70.