Convolutional Neural Networks-Based Image Analysis for the Detection and Quantification of Neutrophil Extracellular Traps.

Aneta Manda-Handzlik,Adrianna Cieloch,Edyta Heropolitanska-Pliszka,Urszula Demkow,Krzysztof Fiok

doi:10.3390/cells9020508

Abstract

Over a decade ago, the formation of neutrophil extracellular traps (NETs) was described as a novel mechanism employed by neutrophils to tackle infections. Currently applied methods for NETs release quantification are often limited by the use of unspecific dyes and technical difficulties. Therefore, we aimed to develop a fully automatic image processing method for the detection and quantification of NETs based on live imaging with the use of DNA-staining dyes. For this purpose, we adopted a recently proposed Convolutional Neural Network (CNN) model called Mask R-CNN. The adopted model detected objects with quality comparable to manual counting—Over 90% of detected cells were classified in the same manner as in manual labelling. Furthermore, the inhibitory effect of GW 311616A (neutrophil elastase inhibitor) on NETs release, observed microscopically, was confirmed with the use of the CNN model but not by extracellular DNA release measurement. We have demonstrated that a modern CNN model outperforms a widely used quantification method based on the measurement of DNA release and can be a valuable tool to quantitate the formation process of NETs.

Highlights

Neutrophils are the most abundant leukocytes in human blood, constituting the first line of defense against infecting pathogens
To create the neutrophil extracellular traps (NETs) dataset, neutrophils isolated from healthy blood donors were stimulated with
NETs formation could be consecutively observed as the loss of lobulated nuclear shape, rounding of the nucleus and loosening of the chromatin structure followed by the rupture of cell membrane and release of cloud-like NETs structures

Summary

Introduction

Neutrophils are the most abundant leukocytes in human blood, constituting the first line of defense against infecting pathogens. Cells 2020, 9, 508 composed of a DNA backbone ornamented with antimicrobial proteins, such as myeloperoxidase (MPO), neutrophil elastase (NE), cathepsin B, and histones Due to their physical properties and presence of high concentrations of lytic proteins, NETs are suggested to act as efficient traps for immobilizing and neutralizing pathogens. The current consensus states that NETs can be released either by the cells undergoing lytic cell death (in a process called NETosis) or without the destruction of plasma membranes [4] In the latter mechanism, activation of neutrophils results in the release of nuclear DNA after the fusion of DNA-containing vesicles with plasma membranes or the extrusion of mitochondrial DNA [5,6]. The results of these studies might contribute to the development of new strategies for managing conditions arising from improper NETs formation and/or degradation [8]

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Conclusion