Activation of the innate immune receptor Dectin-1 upon formation of a 'phagocytic synapse'

Abstract

Innate immune cells must be able to distinguish between direct binding to microbes and detection of components shed from the surface of microbes located at a distance. Dectin-1 (also known as CLEC7A) is a pattern-recognition receptor expressed by myeloid phagocytes (macrophages, dendritic cells and neutrophils) that detects β-glucans in fungal cell walls and triggers direct cellular antimicrobial activity, including phagocytosis and production of reactive oxygen species (ROS). In contrast to inflammatory responses stimulated upon detection of soluble ligands by other pattern-recognition receptors, such as Toll-like receptors (TLRs), these responses are only useful when a cell comes into direct contact with a microbe and must not be spuriously activated by soluble stimuli. In this study we show that, despite its ability to bind both soluble and particulate β-glucan polymers, Dectin-1 signalling is only activated by particulate β-glucans, which cluster the receptor in synapse-like structures from which regulatory tyrosine phosphatases CD45 and CD148 (also known as PTPRC and PTPRJ, respectively) are excluded (Supplementary Fig. 1). The 'phagocytic synapse' now provides a model mechanism by which innate immune receptors can distinguish direct microbial contact from detection of microbes at a distance, thereby initiating direct cellular antimicrobial responses only when they are required.

Figure 1. Particulate, but not soluble, β-glucans induce Dectin-1 signalling

a Size (molecular weight or diameter) of β-glucan preparations used in this study. b-e Bone marrow-derived macrophages (bmM; b-d IFN-γ-primed overnight) were stimulated with 50 μg/ml β-glucans. b TNF-α production (24 h) was assessed by ELISA; data are means plus standard deviations of triplicate culture (*** p<0.001, n.s. not significant). c ROS production was assessed by luminol-ECL; datapoints are means of triplicate culture. d Syk activation (10 min) was assessed by intracellular flow cytometry. e p38 MAP kinase activation at the indicated times was assessed by immunoblotting. All data are representative of at least 3 independent experiments.

Figure 2. Immobilised β-glucans induce Dectin-1 signalling

a Soluble β-glucan binding (50 μg/ml, 10 min) to wild type and Dectin-1 -/- bone marrow-derived DC (bmDC) was assessed by flow cytometry. b, c IFN-γ-primed bmM were stimulated with soluble β-glucans (50 μg/ml) or β-glucans immobilised on either tissue culture plates (b – plate-coated) or 0.8 μm polystyrene latex beads (c – beads). ROS production was measured by luminol-ECL; datapoints are means of triplicate culture. d, e TNF-α production (24 h) by bmDC exposed to soluble/particulate (50 μg/ml), plate-immobilised or bead-coated soluble β-glucans was assessed by ELISA; data are expressed as means plus standard deviations of triplicate culture (*** p<0.001, n.s. not significant). All data are representative of at least 3 independent experiments.

Figure 3. CD45 and CD148 regulate Dectin-1 signalling

Responses of wild type and CD45/CD148-deficient bmM (b IFN-γ-primed) were examined. a Internalization of FITC-labelled zymosan particles (20 μg/ml, 10 min) was assessed by flow cytometry. b ROS production was measured by luminol-ECL; datapoints are means of triplicate culture. c TNF-α production (50 μg/ml WGP, 24 h) was assessed by ELISA; data are expressed as means plus standard deviations of triplicate culture (* p<0.01, *** p<0.001, n.s. not significant). d Syk activation (pSyk; green) by AlexaFluor647-labelled WGP (20 μg/ml, 1 min; blue) was assessed by confocal microscopy. e Inactive Lyn (pY507) levels following zymosan/WGP stimulation (50 μg/ml, 10 min) were assessed by immunoblotting. All data are representative of 3 independent experiments.

Figure 4. CD45 and CD148 phosphatases are excluded from the β-glucan particle contact site

a-c Confocal microscopy of SBPc-tagged Dectin-1-expressing RAW264.7 macrophages stimulated with zymosan (20 μg/ml, 1 min) and stained for CD45 (red) and SBPc tag (Dectin-1; green), phospho-tyrosine (pTyr; green) or active Syk (pSyk; green). z-stacks were analyzed to visualise the indicated particle contact sites (left panels) in cross-section (center panels). 3-dimensional isosurface models of the indicated contact sites were generated using ImageJ and ImageSurfer (right panels). d Resident peritoneal macrophages stimulated with WGP (20 μg/ml, 1 min) were stained (left panel) for CD45 (red) and active Syk (pSyk; green). e Dectin-1-expressing RAW264.7 macrophages stimulated with WGP (20 μg/ml, 1 min) were stained (left panel) for CD148 (red) and active Syk (pSyk; green). Isosurface models (d, e right panels) are of the indicated particle contact sites. f bmDC were added to tissue culture plates pre-coated with HMW soluble β-glucan (cHMW) or LPS (cLPS) and/or anti-CD45 or control IgG; some bmDC were stimulated with WGP (50 μg/ml). TNF-α production (24 h) was assessed by ELISA; data are means plus standard deviations of triplicate culture (*** p<0.001). All data are representative of at least 3 independent experiments.