Candida albicans Targets a Lipid Raft/Dectin-1 Platform to Enter Human Monocytes and Induce Antigen Specific T Cell Responses

Abstract

Several pathogens have been described to enter host cells via cholesterol-enriched membrane lipid raft microdomains. We found that disruption of lipid rafts by the cholesterol-extracting agent methyl-β-cyclodextrin or by the cholesterol-binding antifungal drug Amphotericin B strongly impairs the uptake of the fungal pathogen Candida albicans by human monocytes, suggesting a role of raft microdomains in the phagocytosis of the fungus. Time lapse confocal imaging indicated that Dectin-1, the C-type lectin receptor that recognizes Candida albicans cell wall-associated β-glucan, is recruited to lipid rafts upon Candida albicans uptake by monocytes, supporting the notion that lipid rafts act as an entry platform. Interestingly disruption of lipid raft integrity and interference with fungus uptake do not alter cytokine production by monocytes in response to Candida albicans but drastically dampen fungus specific T cell response. In conclusion, these data suggest that monocyte lipid rafts play a crucial role in the innate and adaptive immune responses to Candida albicans in humans and highlight a new and unexpected immunomodulatory function of the antifungal drug Amphotericin B.

Integrin-based diffusion barrier separates membrane domains enabling the formation of microbiostatic frustrated phagosomes

“…We proceeded to probe the receptors whose signaling could potentiate the formation of the actin cuff. Because C-type lectin signaling contributes importantly to C. albicans phagocytosis ( de Turris et al, 2015 ; Tafesse et al, 2015 ; Xu et al, 2009 ), we analyzed whether Dectin1 accumulated in the membrane at sites where cuffs were evident. Remarkably, while Dectin1 was clearly concentrated in patches elsewhere along the frustrated phagocytic cup, it was poorly detectable by immunostaining near the actin cuff (ratio cuff: cup 0.60 ± 0.04; n = 30 p <0.0001; Figure 2A and inset).…”

C-type lectin-like receptor 2 (CLEC-2)-dependent DC migration is controlled by tetraspanin CD37

“…Furthermore, Dectin-1 has been proposed to be present in lipid rafts (Xu et al, 2009;De Turris et al, 2015) or tetraspanin (CD63, CD37) microdomains (Mantegazza et al, 2004;Meyer-Wentrup et al, 2007;Yan et al, 2014) on myeloid cells.…”

Sterol targeting drugs reveal life cycle stage-specific differences in trypanosome lipid rafts

“…C-Laurdan imaging was performed with and without treatment with sterol-specific drugs MBCD and Amphotericin B (Amp B), which respectively target cholesterol 50 – 52 and ergosterol 53 . Both MBCD and Amp B have been used extensively in previous investigations of lipid rafts 51 , 52 , 54 – 57 .…”

“…Lipid rafts were isolated using a detergent-free method 54 . 8 × 10 8 cells of either 2913 procyclic or Single Marker T. brucei , with or without a 30-min treatment with 20 mM MBCD or 20 μg/mL Amp B, were pelleted by centrifugation at 1000 × g, washed twice with PBSG (PBS with 2.3 g/L of glucose) and resuspended in 1 mL of base buffer (20 mM Tris-HCl, pH 7.8, 250 mM sucrose) supplemented with 1 mM CaCl 2 , 1 mM MgCl 2 , and protease inhibitor.…”

“…Cells were lysed by passage through a 22 gauge × 3 inch needle and centrifuged at 1000 × g. The supernatant was collected and the pellet was lysed again. The two supernatant fractions were combined and subjected to Optiprep TM gradient (0–25%) ultracentrifugation as described previously 54 at 52,000 × g using a SW41Ti rotor in a Beckman ultracentrifuge for 90 min. 800 μL fractions were analyzed by SDS-PAGE and western blotting with calflagin-specific mouse serum (1:1500), mt-hsp70-specific mouse serum (1:1000) or PFR2-specific rabbit serum (1:1000).…”