Neutrophil Extracellular Traps Contain Calprotectin, a Cytosolic Protein Complex Involved in Host Defense against Candida albicans

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. Neutrophils are phagocytes that disarm and kill microbes by engulfing them. Less well characterized than their phagocytic killing mechanisms is how neutrophils cope with microbes that are too large to be internalized. Notably, neutrophils may also kill or inhibit extracellularly by releasing Neutrophil Extracellular Traps (NETs). NETs are fibers made of chromatin (histones and DNA) decorated with antimicrobial proteins. NETs ensnare and kill microbes, such as bacteria, fungi and parasites. We wanted to find out if and how NETs control pathogenic fungi that can form large filaments such as Candida albicans. We purified all NET-bound proteins and identified 24 of them. We found that calprotectin is the major antifungal NET-bound protein. Calprotectin was known to be antimicrobial but here we demonstrate that NET formation is a novel release mechanism for this cytoplasmic protein. The NET matrix comes in close contact with the fungi and the high local concentration of calprotectin in the NETs supports the antifungal activity. Furthermore, in mice calprotectin is essential for an efficient antifungal response to Candida albicans in skin, lung and systemic infections. In tissue sections from these animals we detected NETs and NET-associated calprotectin. Thus, our study gives more insights into mechanisms how the immune system copes with fungal pathogens.