Fluorescent dyes light up immune cells’ DNA traps

Like Spider Man shooting webs at enemies, immune cells called neutrophils sometimes fling out a sticky mesh of DNA and proteins as a massive snare. These structures, known as neutrophil extracellular traps (NETs), catch pathogens and trigger inflammatory responses, implicating them in autoimmune diseases and severe immune responses such as those observed in COVID-19. But NETs are also difficult to image amongst the sea of other DNA and proteins outside of cells. Now, a new method to track the activity of DNA-bound enzymes could help researchers better understand the role of NETs in immunity (J. Am. Chem. Soc. 2020, DOI: 10.1021/jacs.0c08130).

Researchers would like to visualize the activity of enzymes attached to NETs to better understand their role in chewing up microbes and promoting inflammation. But such enzymes could be released by immune cells when they die, so it’s difficult to know whether the enzymes are both active and attached to a NET. “As a community, we still struggle to detect NETs properly,” says immunologist Paul Kubes of the University of Calgary, who was not part of the study.

Carsten Schultz of Oregon Health and Science University and colleagues designed a more precise way to study the antimicrobial actions of NETs. They made a reporter molecule with two parts: a dye that binds to DNA and a pair of fluorescent dyes linked by a small peptide that can get snipped by the enzyme elastase, which commonly attaches to NETs. The paired fluorescent dyes quench each other’s fluorescence when they’re close together, and the dye that binds to DNA reveals when the reporter has latched onto a potential NET. When they added the molecule to a solution containing DNA with elastase bound to it, the enzyme cut the peptide, causing a spike in fluorescence from the now separated, unquenched dyes.

The team then tested the reporter molecule on neutrophils triggered to release NETs. The researchers found a two-fold increase in fluorescence over 2 h, indicating the presence of active, DNA-bound elastase that had snipped the peptide. The method could image NETs in isolated immune cells, tissue samples from mice, and sputum samples from people with cystic fibrosis, a condition in which inflammatory NETs can damage lung tissue.

“We could associate enzyme activity with these large blobs of DNA in sputum samples from cystic fibrosis patients for the first time,” says Matteo Guerra of the University of Heidelberg and the European Molecular Biology Laboratory, who is first author on the study.

The team also tested a version of the reporter molecule that has a peptide cleaved by another common NET-associated enzyme called cathepsin G and found that this enzyme is inactive when bound to DNA. In 2018, another group found that NET-bound elastase, but not cathepsin, “woke” dormant cancer cells (Science 2018, DOI: 10.1126/science.aao4227); the new results suggest that the two enzymes are regulated in different ways when bound to NETs.

Reporter molecules that can track enzymes in NETs are likely to be “very useful,” Kubes says, especially in studies of potential drugs that could block NET activity where it’s harmful, as in cancer or autoimmune conditions.