Abstract
Neutrophils are the first line of defense at the site of an infection. They encounter and kill microbes intracellularly upon phagocytosis or extracellularly by degranulation of antimicrobial proteins and the release of Neutrophil Extracellular Traps (NETs). NETs were shown to ensnare and kill microbes. However, their complete protein composition and the antimicrobial mechanism are not well understood. Using a proteomic approach, we identified 24 NET-associated proteins. Quantitative analysis of these proteins and high resolution electron microscopy showed that NETs consist of modified nucleosomes and a stringent selection of other proteins. In contrast to previous results, we found several NET proteins that are cytoplasmic in unstimulated neutrophils. We demonstrated that of those proteins, the antimicrobial heterodimer calprotectin is released in NETs as the major antifungal component. Absence of calprotectin in NETs resulted in complete loss of antifungal activity in vitro. Analysis of three different Candida albicans in vivo infection models indicated that NET formation is a hitherto unrecognized route of calprotectin release. By comparing wild-type and calprotectin-deficient animals we found that calprotectin is crucial for the clearance of infection. Taken together, the present investigations confirmed the antifungal activity of calprotectin in vitro and, moreover, demonstrated that it contributes to effective host defense against C. albicans in vivo. We showed for the first time that a proportion of calprotectin is bound to NETs in vitro and in vivo.
Highlights
Neutrophils are an essential component of the innate immune response since neutropenia or impairment of neutrophil function results in microbial infections that are often fatal [1]
We found that calprotectin is the major antifungal Neutrophil Extracellular Traps (NETs)-bound protein
Calprotectin was known to be antimicrobial but here we demonstrate that NET formation is a novel release mechanism for this cytoplasmic protein
Summary
Neutrophils are an essential component of the innate immune response since neutropenia or impairment of neutrophil function results in microbial infections that are often fatal [1]. Neutrophil Extracellular Traps (NETs) are released during a novel form of cell death that requires ROS produced by the NADPH-oxidase complex [6]. During this process, the nucleus decondenses and intracellular membranes disintegrate allowing the mixing of nuclear and cytoplasmic components. Whereas bacteria [3] and parasites [14] probably are killed by histones in NETs, in a previous study we found that purified histones did affect Candida albicans in vitro only poorly [13] It remains to be determined whether histones or other antifungal effectors in NETs kill or inhibit fungi. This seems to be of importance since previous reports have demonstrated that histones and histone peptides kill different fungal species such as Cryptococcus neoformans and Candida tropicalis [15,16,17]
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