The architecture of neutrophil extracellular traps investigated by atomic force microscopy

Abstract

Neutrophils are immune cells that engage in a suicidal pathway leading to the release of partially decondensed chromatin, or neutrophil extracellular traps (NETs). NETs behave as a double edged sword; they can bind to pathogens thereby ensnaring them and limiting their spread during infection; however, they may bind to host circulating materials and trigger thrombotic events, and are associated with autoimmune disorders. Despite the fundamental role of NETs as part of an immune system response, there is currently a very poor understanding of how their nanoscale properties are reflected in their macroscopic impact. In this work, using a combination of fluorescence and atomic force microscopy, we show that NETs appear as a branching filament network that results in a substantially organized porous structure with openings with 0.03 ± 0.04 μm(2) on average and thus in the size range of small pathogens. Topological profiles typically up to 3 ± 1 nm in height are compatible with a "beads on a string" model of nucleosome chromatin. Typical branch lengths of 153 ± 103 nm appearing as rigid rods and height profiles of naked DNA in NETs of 1.2 ± 0.5 nm are indicative of extensive DNA supercoiling throughout NETs. The presence of DNA duplexes could also be inferred from force spectroscopy and the occurrence of force plateaus that ranged from ∼65 pN to 300 pN. Proteolytic digestion of NETs resulted in widespread disassembly of the network structure and considerable loss of mechanical properties. Our results suggest that the underlying structure of NETs is considerably organized and that part of its protein content plays an important role in maintaining its mesh architecture. We anticipate that NETs may work as microscopic mechanical sieves with elastic properties that stem from their DNA-protein composition, which is able to segregate particles also as a result of their size. Such a behavior may explain their participation in capturing pathogens and their association with thrombosis.

Networks that stop the flow: A fresh look at fibrin and neutrophil extracellular traps

“…While many studies point to the primary importance of DNAses in terms of clearing NETs, a recent atomic force microscopy (AFM)-focused structural study suggests that proteins are pivotal in determining the stability of NETs and that proteases could be effective in dismantling them [128]. While the authors used trypsin to demonstrate this phenomenon in their in vitro setting, they speculate that NET-borne proteases, such as NE or proteinase-3 might carry out a similar function in vivo, thereby contributing to a more dispersed NET structure or even the gradual clearance thereof.…”

“…According to SEM and AFM studies, NETs form thinner threads (15-25 nm) with globular domains of 30-50 nm diameter, containing NET-bound proteins and enzymes [3]. While this thinner network generally has smaller pores than that of fibrin, in some regions, aggregation of strands creates thicker threads (up to 100 nm [3]) and openings that could fit bacterial cells [128]. Based on these data, and SEM images of composite NET-fibrin clots [48], it is tempting to speculate that fibrin and NETs complete each other: if bacteria slip through the pores of one network, they might still get caught up in another.…”

Extracellular Traps: An Ancient Weapon of Multiple Kingdoms

“…Studies that have attempted to quantify ETs via software programming [166,167] defined NETs from human neutrophils as changes in DNA/chromatin staining pattern [168]. ETs have been described at around 153 nm in length as a bead-on-a-string model [169]. Stimuli vary from a range of bacterial, viral, or parasitic pathogens, endogenous molecules to chemical compounds or trauma.…”

Neutrophil extracellular trap formation correlates with improved overall survival in ovarian cancer

“…Neutrophils exert their functions through phagocytosis, degranulation or release of neutrophil extracellular traps (NETs) [19], a physiologic way of cell death called NETosis [17,18,20]. NETs are DNA structures associated with proteins such as histones and others with antibacterial activity, including elastase (ENOA), myeloperoxidase (MPO), cathepsin G (CTG) and lactoferrin (LTF) [20,21]. Under normal conditions, after successful clearance of an infection, a resolution of inflammation is initiatiated.…”