Activation of the Innate Immune Receptor Dectin-1 by Clustering

Abstract

Dectin-1 is a pattern-recognition receptor that plays a key role in anti-fungal immunity. It specifically recognizes beta-glucans, polymers of glucose found on the fungal cell wall. Upon ligand binding, dectin-1 triggers signaling cascades that lead to various cellular responses, including phagocytosis and the release of pro-inflammatory mediators, which all culminate in the generation of an effective immune response and eradication of the pathogen. The molecular mechanisms underlying dectin-1 activation and signal transduction are not fully understood. The aim of this study is to elucidate such mechanisms. We propose that upon binding of fungal particles, dectin-1 receptors cluster and assemble into multimeric complexes. In these clusters, dectin-1 becomes activated and promotes the recruitment and activation of Syk (spleen tyrosine kinase), which in turn triggers subsequent signaling cascades. To test our hypothesis, we stably expressed human dectin-1 in RAW 264.7 macrophages. Various levels of dectin-1 clustering were induced using antibody cross-linking or beta-glucans of different molecular weights. The corresponding stimulation of dectin-1 was detected by determining the activation of several downstream effectors (Src, Syk, NF-kB). We demonstrate that antibody cross-linking and larger ligands are able to induce more Syk phosphorylation than smaller ligands. To quantify the level of dectin-1 clustering, single molecule analysis was employed. Upon antibody cross-linking or ligand binding dectin-1 formed nanodomains of about 500 nm of diameter, which were identified as the nucleation site for intracellular signaling. Additionally, the phosphorylation and recruitment of Syk to regions of the plasma membrane rich in dectin-1 clusters was observed using confocal microscopy. Together, our results suggest that receptor clustering, is the mechanism by which dectin-1 is activated.