Molecular mechanisms of NET formation and degradation revealed by intravital imaging in the liver vasculature

Abstract

Neutrophil extracellular traps (NETs) composed of DNA decorated with histones and proteases trap and kill bacteria but also injure host tissue. Here we show that during a bloodstream infection with methicillin-resistant Staphylococcus aureus, the majority of bacteria are sequestered immediately by hepatic Kupffer cells, resulting in transient increases in liver enzymes, focal ischaemic areas and a robust neutrophil infiltration into the liver. The neutrophils release NETs into the liver vasculature, which remain anchored to the vascular wall via von Willebrand factor and reveal significant neutrophil elastase (NE) proteolytic activity. Importantly, DNase although very effective at DNA removal, and somewhat effective at inhibiting NE proteolytic activity, fails to remove the majority of histones from the vessel wall and only partly reduces injury. By contrast, inhibition of NET production as modelled by PAD4-deficiency, or prevention of NET formation and proteolytic activity as modelled in NE−/− mice prevent collateral host tissue damage.

Biology, role and therapeutic potential of circulating histones in acute inflammatory disorders

“…Breakdown of DNA in NETs with DNAse only partially ameliorates NET‐related toxicity, as it does not affect the histone component 125. Concentrations below 10 μg/mL seem to have a signalling function and can induce calcium transients in cells 126.…”

“…The addition of histones and DNA also increases fibrin fibre thickness, clot stability and delayed clot lysis142 as well as reducing anti‐thrombin‐mediated thrombin inactivation and plasmin activity 143. In in vivo systems, the interplay between von Willebrand factor (vWF), platelets and neutrophils anchors neutrophils to otherwise healthy vessel walls and permits NETing neutrophils to initiate clot formation,125, 144 with propagation that may occur or be enhanced by the mechanisms described above. Deficiencies in degradation of vWF produce clinical microangiopathies (eg, thrombotic thrombocytopenic purpura), the severity of which is also closely correlated with circulating NET components in humans 145…”

Priming of neutrophils toward NETosis promotes tumor growth

“…27 The NETting process could also have a predominant role in the generation of a beneficial extracellular matrix by delivering proteinases and could also favor the release of inflammatory and angiogenic factors. For example, NETs have been shown to contain and activate matrix metalloproteinases, 28 preserve the enzymatic activity of elastase 29 and recruit platelets, 30 important cells for angiogenesis. 31 Neutrophils were described to suppress the cytotoxic CD8 C antitumoral response, which is associated with the generation of ROS.…”

Characterization of Filifactor alocis and its immune evasion strategies employed against human neutrophils.

“…First, neutrophil elastase (NE), a neutrophil-specific serine protease, functions in histone degradation, promotion of chromatin decondensation in the neutrophil nucleus, degradation of the nuclear envelope, antimicrobial activities within the neutrophil phagosome (170,376,377). Studies performed with both NE and MPO knockout mice show failed induction of NETs and an increased susceptibility to infection (170).…”

“…Several studies have highlighted the importance of NET component neutrophil elastase (NE) to the effective formation and function of NETs (170,376,377). Therefore, using a kit to detect extracellular release of NE, we sought to determine if its presence was found with nonopsonized and opsonized F. alocis challenge of neutrophils.…”