Quantifying Neutrophil Extracellular Trap Release and Citrullinated Histone H3 in an Infection-Inflammation NET-Array Microsystem

Submission 48

Tuesday-Talks 3-Selected talk-06

Presented by: Caroline N. Jones

Udaya Sree Datla ^{1, 2}, Bhaskar Vundurthy ⁴, Jessica S. Hook ⁵, Nidhi Menon ⁶, Tarik Shihabeddin ², David W. Schmidtke ^{1, 2, 3}, Jessica G. Moreland ⁵, Marko Z. Radic ⁷, Caroline N. Jones ^{1, 2, 3}

¹ Department of Surgery, University of Texas Southwestern medical center Dallas, Dallas, TX, USA.
² Department of Bioengineering, University of Texas at Dallas, Richardson, TX, USA.
³ Department of Biomedical Engineering, University of Texas Southwestern medical center Dallas, Dallas, TX, USA.
⁴ Robotics Institute, Carnegie Mellon University, Pittsburgh, PA, USA.
⁵ Department of Pediatrics, University of Texas Southwestern medical center Dallas, Dallas, TX, USA.
⁶ HueDx, Philadelphia, PA, USA.
⁷ Department of Microbiology, Immunology and Biochemistry, University of Tennessee Health Science Center, Memphis, TN, USA.

Introduction: Excessive release of neutrophil extracellular traps (NETs) has been linked to various human pathologies, notably COVID-19, where elevated levels are indicative of increased risks of coagulopathy and immunothrombosis. Traditional immunoassays often lack single-cell resolution and struggle to capture the complexities of microenvironments. Human microphysiological models (microsystems) enable quantification of single-cell dynamics and behavioromes, such as NETosis, within physiologically relevant microenvironments.

Aims: Our objective was to develop a NET-array microsystem capable of accurately quantifying human citrullinated histone H3-positive NET-release at a single-cell level within infection and inflammation-rich microenvironments.

Methodology: The NET-array microsystem, featuring open chambers and constricted loops, was designed to simulate infection and inflammation-rich microenvironments. Primary human neutrophils were exposed to Pseudomonas aeruginosa PAO1 and inflammatory cytokines, including tumor necrosis factor-α and interleukin-6. Time-lapse imaging captured the release of NETs, including citrullinated histone H3-positive NETs, while computer-vision-based image processing methods were developed to automate quantification.

Main Results: Our study unveiled a significant increase in NET release in response to Pseudomonas aeruginosa PAO1 when combined with inflammatory cytokines compared to infection alone. Notably, we measured increased citrullinated histone H3-positive NET-release to PAO1 when challenged with tumor necrosis factor-α and interleukin-6, assessed at the conclusion of the live–dead NETs assay. Additionally, confinement within loops of the device led to reduced NET release. Our NET-array device is currently deployed at the University of Texas at Southwestern Medical Center for high-throughput screening of novel immunotherapies aimed at fine-tuning NET release to mitigate pathological neutrophil-driven inflammation.