Dectin-1 participates in β-glucan- or PMA-induced neutrophil extracellular trap formation during antifungal defense.

Abstract

Objectives: The formation of neutrophil extracellular traps (NETs) plays a crucial role in neutrophil-mediated defense against fungal infections and has become a hot topic of immunological research. This study aimed to investigate whether high expression of Dectin-1, a key pattern recognition receptor, contributes to NET formation in response to fungal pathogens.

Methods: Human neutrophils were isolated and characterized, then stimulated with cell wall β-glucan to induce NET formation. Phorbol 12-myristate 13-acetate (PMA), a diacylglycerol mimetic, was used as a positive control. Dectin-1 antibody was used to determine the functional significance of Dectin-1 in the formation of NETs. NET formation was detected by Sytox Green staining, myeloperoxidase (MPO) and neutrophil elastase (NE) immunofluorescence staining, and western blot analysis. The relative kits, 2',7'-dichlorodihydrofluorescein diacetate staining and MitoSOX Red staining were used to determine the mechanism of Dectin-1 induced NET formation.

Results: Dectin-1 was overexpressed in β-glucan- and PMA-treated neutrophils. Dectin-1 deficiency reduced NET formation, accompanied by decreased Sytox Green fluorescence, lower levels of dsDNA content, and decreased expression of NE, MPO and citrullinated histone H3 (H3Cit). Dectin-1-mediated NET formation was dependent on reactive oxygen species (ROS) produced by NADPH oxidase (NOX), NOX2 protein and mitochondrial superoxide. Moreover, up-regulated Dectin-1 expression activated the extracellular regulated protein kinases (ERK) and p38 MAPK pathways, which were critical for the induction of NETs.

Conclusion: Dectin-1 promotes NET formation in neutrophils stimulated by β-glucan or PMA through activation of the ERK and p38 signaling pathways, which might contribute to defense against fungal pathogens.