Abstract
Abstract Abundant NETosis has been observed in many human pathologies, including in COVID-19 patients. Excessive neutrophil extracellular trap (NET) production is known to lead to unfavorable coagulopathy and immunothrombosis in these patients. Traditional immunoassays employed to quantify NETosis focus on bulk measurements of released chromatin in simplified microenvironments. In this study, we fabricated a novel NET-array device to quantify NETs with single-cell resolution in the presence bacteria and inflammatory cytokines. The device was engineered to have open chambers and constricted channels to measure NETosis at varying degrees of mechanical confinement. The microsystem enabled us to take time-lapse images of individual primary human neutrophils releasing NETs in clinically relevant microenvironments. We then developed a computer-vision-based image processing method to automate the quantification of individual NETs. We showed a significant increase in NET release to Pseudomonas aeruginosaPAO1 infection when challenged with the inflammatory cytokines Tumor Necrosis Factor-α and Interleukin-6 (but not Leukotriene B 4), compared to the infection alone. We confirmed reduced NET release in confined channels in the microsystem. We quantified the temporal dynamics and differences in the areas of NETs released, showing a high percentage of large NETs with Pseudomonas-inflammation mediator treatment. Overall, our NET-array device serves as a proof-of-concept model to further our understanding of the spatiotemporal dynamics of NET release to infection in a defined microenvironment. Our NET-array device is now being used for high-throughput and cost-effective screening of novel immunotherapies on human neutrophils. NIH NIGMS (R35GM133610-01)
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