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New method catches cystic fibrosis inflammatory enzyme in the act

A combination of two techniques offers a clinically feasible way to measure inflammatory response in chronic lung diseases

by Jyoti Madhusoodanan, special to C&EN
March 19, 2019


People with chronic lung diseases such as cystic fibrosis are caught in a vicious circle: infections cause inflammation and immune cells rush into airways, but in the process of killing infectious bacteria, some enzymes remain bound to the surface of the immune cells that secreted them, so they cause further tissue damage and inflammation. A new method allows researchers to track this inflammatory enzyme activity in patient cells. The method, which relies on a combination of fluorescence-based approaches, could help monitor how patients respond to therapy (ACS Cent. Sci. 2019, DOI: 10.1021/acscentsci.8b00933).

Credit: ACS Cent. Sci.
In healthy neutrophil immune cells (left), the cathepsin G proteases do not cleave a fluorescent probe, causing it to glow yellow. In cells with cystic fibrosis (right), active cathepsin G cleaves the fluorescent peptide tag on the neutrophil surface, yielding a change in the fluorescence wavelength to produce a purple glow.

Carsten Schultz of Oregon Health and Science University and his colleagues developed the technique to measure the activity of cathepsin G, one of several proteases secreted by immune cells known as neutrophils. Although previous studies have examined other enzymes, Schultz wanted to see if cathepsin G was one of the enzymes still stuck on the cell surface and therefore a marker of inflammation. Previous studies analyzed clinical samples using flow cytometry, a cell-sorting technique, to test for the presence of related proteases, or confocal microscopy to measure the enzymes’ activity. But flow cytometry alone doesn’t reveal whether a surface protein is active—and causing inflammation—and microscopy requires several hours to process a single sample, making it unfeasible for routine clinical uses.

In the new study, the team tracked cathepsin G with a method for detecting peptide interactions, Förster resonance energy transfer (FRET), in which one fluorescent probe transfers energy to another when the two are close enough to do so, causing a glow at a particular wavelength. The team chose a peptide that cathepsin G would cleave, added a lipid tag so it would target the cell membrane, and tacked on two light sensitive molecules nestled closely together. When membrane-bound cathepsin G was active and digested the peptide, the two fluorescent parts split, changing the emitted signal.

To make use of the technique, the researchers first used flow cytometry to isolate cathepsin G-secreting neutrophils from other cells in sputum samples from people with and without cystic fibrosis. They then carried out the FRET tests on the neutrophils and found that the cells of people with cystic fibrosis showed approximately three times as much cathepsin G activity than control cells.

In conditions such as cystic fibrosis where inflammation already exists, such an assay “could help monitor response to a drug,” Schultz says, whereas in other lung conditions such as chronic obstructive pulmonary disease, the test “could help to pick up early inflammatory signals” to catch the condition early and track disease.

Combining flow cytometry with a fluorescence-based assay of enzyme activity is a viable approach for clinical use, says Matteo Guerra, graduate student at the University of Heidelberg and study co-author. Because the method assays common inflammatory markers, it can be adapted to other conditions such as arthritis, he says.

The results with clinical samples are comparable to other existing methods such as confocal microscopy, says Matthew Bogyo of Stanford University, who studies enzyme activity in biological systems and was not involved with the study. The strength of the study is that it could be used with clinical samples, allowing quicker diagnosis of inflammation than microscopy does.



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