Microfluidic Assay Measures Increased Neutrophil Extracellular Traps Circulating in Blood after Burn Injuries

Masayuki Otawara,Felix Ellett,Yong-Ming Yu,Daniel Irimia,Maedeh Roushan,Xiao Wang

doi:10.1038/s41598-018-34952-0

Masayuki Otawara, Felix Ellett + Show 4 more

Open Access

https://doi.org/10.1038/s41598-018-34952-0

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Abstract

Cell-free DNA (cf-DNA) concentration in human plasma is often increased after burn and trauma injuries. Two major sources of cf-DNA are the parenchymal cells damaged by the injury and various circulating cells indirectly altered by the response to injury. The cf-DNA originating from neutrophils, also known as circulating neutrophil extracellular traps (cNETs), is of notable interest because cNETs have been associated with pathological processes in other conditions, including cancer, autoimmunity, etc. Both intact chromatin and oligonucleotides, which are the by-product of cf-DNA degradation, are assumed to contribute to the cf-DNA in patients. However, traditional assays for cf-DNA quantification do not distinguish between cNETs and cf-DNA of other origins and do not differentiate between intact chromatin and oligonucleotides. Here we measure the amount of intact cNETs in the circulation, using a microfluidic device that mechanically traps chromatin fibers directly from blood and an immunofluorescence protocol that detects neutrophil-specific proteins associated with chromatin. In a rat model of burn injury, we determined that the chromatin fibers in the circulation after injury originate exclusively from neutrophils and are cNETs. We found that the concentration of cNETs surges the first day after injury and then decreases slowly over several days. In a secondary sepsis model, which involved a burn injury followed by cecal-ligation-puncture, we measured additional increases in cNETs in the days after sepsis was induced. These results validate a microfluidic assay for the quantification of cNETs and will facilitate fruther studies probing the contribution of cNETs to complications after burns and sepsis.

Highlights

Neutrophil elastase (NE) is identified by immunofluorescence staining and is co-localized with the chromatin fibers trapped on chip, validating their neutrophil origin
Using our new microfluidic technique and immunostaining for neutrophilic elastase (NE) we show that the captured chromatin from blood is circulating neutrophil extracellular traps (cNETs)
As the samples flow through devices, chromatin fibers are captured in the post array (Fig. 1D)

Summary

Introduction

Neutrophil elastase (NE) is identified by immunofluorescence staining (green) and is co-localized with the chromatin fibers trapped on chip, validating their neutrophil origin. A mechanical mechanism has been proposed, when the trapping of cNETs inside in vitro capillary plexuses perturbs the distribution of flowing red blood cells, and may be responsible for localized tissue hypoxia and damage[21]. For this mechanism, the distinction between circulating chromatin fibers and oligonucleotides is critically important. Using our new microfluidic technique and immunostaining for neutrophilic elastase (NE) we show that the captured chromatin from blood is cNETs. we quantify the amount of cNETs and show that cNETs increase rapidly after burns and decrease slowly in the days after the injury. Secondary sepsis induced by cecal ligation puncture at day 9 post burn results in additional, progressive increase in the concentration of cNETs for several days

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