Muco-obstructive
lung diseases feature extensive bronchiectasis
due to the uncontrolled release of neutrophil serine proteases into
the airways. To assess if cathepsin G (CG) is a novel key player in
chronic lung inflammation, we developed membrane-bound (mSAM) and
soluble (sSAM) FRET reporters. The probes quantitatively revealed
elevated CG activity in samples from 46 patients. For future basic
science and personalized clinical applications, we developed a rapid,
highly informative, and easily translatable small-molecule FRET flow
cytometry assay for monitoring protease activity including cathepsin
G. We demonstrated that mSAM distinguished healthy from patient cells
by FRET-based flow cytometry with excellent correlation to confocal
microscopy data.
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.
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