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From the plasma membrane to tangled DNA webs: a roadway to track, investigate and employ spatially localized neutrophil elastase and cathepsin G activities

Guerra, Matteo

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Airways muco-obstruction and irreversible neutrophil-driven inflammation cause bronchiectasis in lung diseases such as cystic fibrosis (CF) and chronic obstructive pulmonary diseases (COPD). To enter the airway lumen, neutrophils secrete their proteases, namely cathepsin G (CG), neutrophil elastase (NE), proteinase 3 (PR3) and neutrophil serine protease 4 (NSP4). The released neutrophil serine proteases (NSPs) contribute directly and indirectly to the innate immunity. Released NSPs’ action is usually counteracted by endogenous antiproteases. However, the delicate balance between these two components is broken in chronic inflammation. Strikingly, NSPs greedily associate to the surface of the secreting neutrophil, to the myriad of extracellular vesicles filling the airway fluid and to the tangled DNA webs made of neutrophil extracellular traps (NETs). When fastened to such structures, NSPs seem to be less accessible to antiproteases and their persisting activity damages the connective tissue. As a result, more proinflammatory stimuli are released and the outcome is a vicious cycle leading to non-resolving airway neutrophilia. In order to expand our palette of fluorescent tools and to propose an alternative drug target and inflammatory biomarker, we developed of a new series of Förster resonance energy transfer (FRET)-based reporters, which revealed high cathepsin G activity in CF and COPD airways. Also, we were inspired by the demand of novel advanced diagnostic technologies to examine sputum samples in a hospital environment. Therefore, we established a new assay based on the combination of spatially localized FRET probes and flow cytometry. This combination was shown to be a valuable diagnostic technique applicable in a basic and translational biomedical context. The simplicity and throughput of the new method opened the doors to two novel biomedically relevant projects. First, to identify new inflammatory markers, we investigated the discriminants and common traits of inflammation in CF and COPD airways. We carried out a comprehensive characterization of sputum samples via analysis of protease activities, cytokines and antiprotease levels. We found that COPD airways appear to be characterized by less severe inflammation featuring elevated but not uttermost marker levels, compared to CF airways. As a key marker, high membrane-bound protease activity was the most significant indicator for COPD, suggesting this trait as a highly relevant early-inflammation biomarker. Second, we wondered if in addition to the neutrophil surface, CF- and COPD-derived exosomes carry active NE and how to measure such activity at a single nanoparticle level. Therefore, we adapted our cytometric assay to monitor protease activity on human sputum particles as small as 100 nm in diameter. We showed that CF exosomes acquired NE at their surface in the inflamed airways and exported it to surrounding cells. Finally, we synthesized small-molecule probes designed to attach to DNA with the help of a DNA minor groove binder (Hoechst). The respective reporters were able to detect and quantify NE and CG activity on NETs, making them valuable tools to study the eclectic effect these enzymes have when embedded in DNA webs. Our reporters revealed that DNA-bound NE retained its catalytical activity. When applied to 5 μm mouse lung slices, the probe allowed to both distinguish single cell nuclei and to quantify cell-specific NE activity within the section. In conclusion, the activity of enzymes like CG and NE can now be studied with unprecedented spatial resolution. Furthermore, this work brings a flow cytometric assay into biomedical research which, in combination with an expanding palette of FRET-based tools, bears the potential to allow for rapid and detailed diagnosis and treatment evaluation for lung disease patients, ideally at the early stage of the disease.

Item Type: Dissertation
Supervisor: Schultz, Dr. Carsten
Place of Publication: Heidelberg
Date of thesis defense: 30 June 2020
Date Deposited: 30 Sep 2020 12:43
Date: 2020
Faculties / Institutes: The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
Service facilities > European Molecular Biology Laboratory (EMBL)
Subjects: 500 Natural sciences and mathematics
570 Life sciences
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