The activitiy of proteases play a crucial role in inflammatory processes as they contribute to white blood cell infiltration and the gradual destruction of the extracellular lung matrix in chronic inflammation. The activity levels of certain proteases such as neutrophil elastase (NE), cathepsin S and the matrix metalloproteases 9 and 12 (MMP-9, MMP-12) might be useful indicators for the progression of diseases such as cystic fibrosis or chronic obstructive pulmonary disease (COPD). We recently introduced the small molecule FRET protease reporters NEmo (for NE) and LaRee (for MMP-12) which enable the monitoring of protease activity at the single cell level by means of fluorescence microscopy. However, analysis of the corresponding imaging data and translating the results into easily interpretable measures for clinicians, have been very time-consuming and challenging.

Introduction: The epithelial sodium channel (ENaC) plays a crucial role in sodium transport across epithelial membranes and in the homeostasis of airway surface liquid (ASL) hydration. Proteolytic cleavage of the ENaC γ-subunit by channel-activating proteases (CAPs) increases the opening probability of the channel and thereby enhances sodium transport. Tarran and co-workers have shown that CAP expression is elevated in primary bronchial epithelial cultures of CF patients compared to tissue from healthy subjects (Tarran et al., 2006). However, the impact of CAPs on ENaC activity in cystic fibrosis lung disease is unknown.

Introduction: Neutrophilic airway inflammation is a hallmark of cystic fibrosis (CF) and previous studies identified free neutrophil elastase (NE) in bronchoalveolar lavage fluid and sputum as a key risk factor of early bronchiectasis and decline in lung function. However, the relevance of membrane-associated NE activity in the pathophysiology of CF lung disease is not well understood. In this study, we used a ratiometric Foerster resonance energy transfer (FRET) reporter (NEmo-2) to determine NE activity on the surface of sputum neutrophils and correlated NE levels with measurements of pulmonary function.

Background: Increased neutrophil elastase (NE) activity in bronchoalveolar lavage (BAL) fluid in newborns with cystic fibrosis (CF) has been shown to correlate with lung damage in juvenile age. Mice with airway- specific overexpression of the epithelial Na+ channel (βENaC-Tg) phenocopy CF lung disease with mucus obstruction, chronic inflammation and emphysema. Genetic deletion of NE in βENaC-Tg mice reduced airway inflammation and emphysema, indicating that NE may serve as a therapeutic target.

Proteases such as neutrophil elastase (NE) and matrix metalloprotease 12 (MMP-12) are key factors in inflammatory processes and contribute the gradual destruction of extracellular lung matrix in chronic inflammation. We now hypothesize that activity levels of inflammation-relevant proteases may be useful indicators for the onset and progression of obstructive lung diseases such as cystic fibrosis or COPD. With the recently published small molecule FRET protease reporters NEmo and LaRee, for detection of NE and MMP-12 activity, respectively, it is possible to monitor protease activity at the single cell level.