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.

Release of Active Peptidyl Arginine Deiminases by Neutrophils Can Explain Production of Extracellular Citrullinated Autoantigens in Rheumatoid Arthritis Synovial Fluid

“…Consistent with the findings from a study by Urban et al 18, only a small number of proteins, comprising mainly granular and nuclear proteins, could be confirmed to be present in NETs, as compared to the wide range of proteins released into the supernatant during NETosis (see Supplementary Table 1, available on the Arthritis & Rheumatology web site at http://onlinelibrary.wiley.com/doi/10.1002/art.39313/abstract). Consistent with the findings from Western blot analysis, PAD2 and PAD4 were detected in both the supernatant and the DNase I–treated NET fraction of neutrophils from 7 patients.…”

“…Supernatants of either stimulated or unstimulated neutrophils were harvested, and the wells were washed 3 times to remove unbound proteins, as described previously 18. Cell‐associated NETs were solubilized with 10 units/ml DNase I for 20 minutes in 500 μl RPMI medium and protease inhibitor cocktail at a dilution of 1:200 (P8340; Sigma‐Aldrich).…”

Elevated plasma neutrophil elastase concentration is associated with disease activity in patients with thrombotic thrombocytopenic purpura

“…NETs are implicated in immune defense [20], sepsis [21] and autoimmunity [22]. NET is formed by a DNA-histones scaffold that contains granule proteins such as elastase and myeloperoxidase, as well as antimicrobial peptides [23]. Results obtained in PMNE knock-out mice showed that these animals do not form NETs in a pulmonary model of bacterial infection [24].…”

Human neutrophils produce extracellular traps against Paracoccidioides brasiliensis

“…An increasing number of bacteria, fungi, viruses and protozoan parasites have been shown to induce NETs (Brinkmann et al, 2004;Guimarães-Costa et al, 2009;Urban et al, 2009;Saitoh et al, 2012;Jenne et al, 2013) but to date only three fungal species (Candida albicans, Aspergillus spp. and Cryptococcus gattii) have been reported to activate their production (Urban et al, 2006;Bruns et al, 2010;Springer et al, 2010;Bianchi et al, 2011).…”