Quantifying neutrophil extracellular trap release in a combined infection-inflammation NET-array device

Area of Science:

• Immunology
• Biomedical Engineering
• Cell Biology

Background:

• Neutrophil extracellular traps (NETs) are implicated in pathologies like COVID-19, with elevated levels indicating coagulopathy and immunothrombosis risks.
• Current methods for NET quantification use bulk measurements in simplified settings, lacking single-cell resolution and microenvironment context.
• Understanding NET release dynamics is crucial for developing targeted immunotherapies.

Purpose of the Study:

• To develop and validate a novel NET-array device for single-cell resolution quantification of NET release.
• To investigate the impact of infection (Pseudomonas aeruginosa) and inflammatory mediators on NET release in varied microenvironments.
• To analyze the spatiotemporal dynamics and characteristics of NET release.

Main Methods:

• Fabrication of a microfluidic NET-array device with wide chambers and constricted loops.
• Culture of primary human neutrophils with Pseudomonas aeruginosa PAO1 and inflammatory mediators (TNF-α, IL-6, LTB4).
• Time-lapse imaging and computer-vision-based image processing for automated quantification of individual NETs and their characteristics.

Main Results:

• The NET-array device enabled single-cell resolution imaging and quantification of NET release.
• Combined infection with Pseudomonas aeruginosa and inflammatory mediators (TNF-α, IL-6) significantly increased NET release compared to infection alone.
• NET release was reduced in constricted microenvironments within the device, and variable NET sizes were observed.
• Temporal dynamics and relative areas of NETs were quantified, revealing distinct release patterns under different conditions.

Conclusions:

• The novel NET-array device provides a powerful tool for studying NET release dynamics at single-cell resolution in defined microenvironments.
• This system enhances our understanding of how infection and inflammation interact to modulate NETosis.
• The device holds potential for high-throughput screening of immunotherapies targeting neutrophil-driven inflammation.