Phospholipase Cγ2 Is Critical for Dectin-1-mediated Ca²⁺ Flux and Cytokine Production in Dendritic Cells^*

Abstract

Dectin-1 is a C-type lectin that recognizes β-glucan in the cell walls of fungi and plays an important role in anti-fungal immunity. It signals via tyrosine kinase Syk and adaptor protein Card9 to activate NF-κB leading to proinflammatory cytokine production in dendritic cells (DCs). Other than this, not much else is known of the mechanism of Dectin-1 signaling. We demonstrate here that stimulation of DCs with zymosan triggers an intracellular Ca²⁺ flux that can be attenuated by a blocking anti-Dectin-1 antibody or by pre-treatment of cells with the phospholipase C (PLC) γ-inhibitor U73122, suggesting that Dectin-1 signals via a PLCγ pathway to induce Ca²⁺ flux in DCs. Interestingly, treatment of DCs with particulate curdlan, which specifically engages Dectin-1, results in the phosphorylation of both PLCγ1 and PLCγ2. However, we show that PLCγ2 is the critical enzyme for Dectin-1 signaling in DCs. PLCγ2-deficient DCs have drastic impairment of Ca²⁺ signaling and are defective in their secretion of interleukin 2 (IL-2), IL-6, IL-10, IL-12, IL-23, and tumor necrosis factor α. PLCγ2-deficient DCs also exhibit impaired activation of ERK and JNK MAPKs and AP-1 and NFAT transcription factors in response to Dectin-1 stimulation. In addition, PLCγ2-deficient DCs are also impaired in their activation of NF-κB upon Dectin-1 engagement due to defective assembly of the Card9-Bcl10-Malt1 complex and impaired IKKα/β activation and IκBα degradation. Thus, our data indicate that pattern recognition receptors such as Dectin-1 could elicit Ca²⁺ signaling and that PLCγ2 is a critical player in the Dectin-1 signal transduction pathway.