Monitoring protease activity in human sputum samples by FRET-based probes via statistics-based fluorescence image analysis

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.

Here we introduce an updated version of FluoQ, a macro for the popular image analysis software ImageJ to rapidly quantify the imaging data. The macro provides statistical analysis of the imaging data and automatically outputs the results in multiple graphic and numeric displays and thereby facilitating rapid data interpretation, particularly for a homogenous cell population. For the analysis of sputum samples, a heterogenous cell mixture, a feature was added to FluoQ which allows to manually assign different regions of interest (ROIs) to distinct cell types.

Using R, we differentially analyzed the results of multiple experiments by cell type. We found the protease reporters NEmo2 and LaRee1 indicate different protease activities in different cell types. Having the protocol and image analysis for these assays established, we are currently developing new protease probes for cathepsin S and MMP-9 in order to better understand the protease involvement in disease mechanisms leading to structural lung damage.

*Presenting author