Protease FRET Reporters Targeting Neutrophil Extracellular Traps

Abstract

Neutrophil extracellular traps (NETs) consist of DNA released by terminally stimulated neutrophils. They fine-tune inflammation, kill pathogens, activate macrophages, contribute to airway mucus obstruction in cystic fibrosis, and facilitate tumor metastasis after dormancy. Neutrophil proteases such as elastase (NE) and cathepsin G (CG) attach to NETs and contribute to the diverse immune outcome. However, because of the lack of suitable tools, little spatiotemporal information on protease activities on NETs is available in a pathophysiological context to date. Here, we present H-NE and H-CG, two FRET-based reporters armed with a DNA minor groove binder, which monitor DNA-bound NE and CG activity, respectively. The probes revealed that only NE maintains its catalytic ability when localized to DNA. Further, we demonstrated elevated protease activity within the extracellular DNA of sputum from cystic fibrosis patients. Finally, H-NE showed NE activity at single-cell and free DNA resolution within mouse lung slices, a difficult to achieve task with available substrate-based reporters.

Proteases, Mucus, and Mucosal Immunity in Chronic Lung Disease

“…Induction of NET formation and subsequent increases in extracellular DNA may contribute to increased mucus viscosity. NETs also provide a protective lattice around proteases preventing access and inhibition by their natural inhibitors [ 91 , 92 ]. Bacterial species in the airway use mucolytic proteases to promote colonisation by inhibiting entrapment in the mucus layer and to gain access to the airway epithelium.…”

Neutrophil extracellular traps in chronic lung disease: implications for pathogenesis and therapy

“…NETs and inflammation in chronic lung disease CF NETs have been extensively studied in CF. CF sputum has been shown to be heavily composed of NET and NET-related proteins, including NE, MPO, calprotectin and resistin [40][41][42][43][44][45]. NE, which has long been recognised as a biomarker of disease severity in CF and is believed to contribute to disease progression, retains its catalytic ability when bound to DNA and has been shown to have elevated protease activity within the extracellular DNA (eDNA) of sputum from CF patients [42].…”

“…CF sputum has been shown to be heavily composed of NET and NET-related proteins, including NE, MPO, calprotectin and resistin [40][41][42][43][44][45]. NE, which has long been recognised as a biomarker of disease severity in CF and is believed to contribute to disease progression, retains its catalytic ability when bound to DNA and has been shown to have elevated protease activity within the extracellular DNA (eDNA) of sputum from CF patients [42]. Additional NET-associated markers such as resistin and MPO are associated with worse disease outcomes and reduction in lung function in CF [41,46].…”

Significance of Mast Cell Formed Extracellular Traps in Microbial Defense

“…• A serine protease expressed in primary granules [95] • In humans, NE translocates from azurophilic granules to the nucleus upon formation of NET where it cleaves histones and contributes to chromatin decondensation by partially degrading specific histones [96] • Neutrophils of NE −/− mice produce NETs when stimulated by PMA [97] • Maintains its catalytic ability after being localized to DNA [98] • It is suggested that NE blocking would largely abrogate the protease activity associated with NETs [99] Myeloperoxidase • Synergies with NE in decondensation of chromatin during NETosis [96] • A granule component of neutrophil that possesses antiviral activity [100] Cathepsin G • Cleaves the pro-IL-1α precursor and produces more IL-1α through which it activates endothelial cells [101] • Plays a role in platelet activation, platelet aggregation, and dense granule secretion [102,103] Leukocyte proteinase 3…”