Abstract
Neutrophil extracellular traps (NETs) are believed to be essential in controlling several bacterial pathogens. Quantification of NETs in vitro is an important tool in studies aiming to clarify the biological and chemical factors contributing to NET production, stabilization and degradation. This estimation can be performed on the basis of fluorescent microscopy images using appropriate labelings. In this context, it is desirable to automate the analysis to eliminate both the tedious process of manual annotation and possible operator-specific biases. We propose a framework for the automated determination of NET content, based on visually annotated images which are used to train a supervised machine-learning method. We derive several methods in this framework. The best results are obtained by combining these into a single prediction. The overall Q(2) of the combined method is 93%. By having two experts label part of the image set, we were able to compare the performance of the algorithms to the human interoperator variability. We find that the two operators exhibited a very high correlation on their overall assessment of the NET coverage area in the images (R(2) is 97%), although there were consistent differences in labeling at pixel level (Q(2), which unlike R(2) does not correct for additive and multiplicative biases, was only 89%). Open source software (under the MIT license) is available at https://github.com/luispedro/Coelho2015_NetsDetermination for both reproducibility and application to new data.
Highlights
Neutrophils are important effectors of the innate immune system in mammals, constituting the first line of defense against many microbial pathogens
It has been shown that some inflammatory mediators, as well as a wide range of microbes including bacteria, fungi and protozoa, can stimulate neutrophils to undergo a distinctive form of cell death designated NETosis (Yipp and Kubes, 2013)
This process leads to the extracellular release of fibers of DNA associated with histones, granule proteins and peptides—neutrophil extracellular traps (NETs)
Summary
Neutrophils are important effectors of the innate immune system in mammals, constituting the first line of defense against many microbial pathogens. It has been shown that some inflammatory mediators, as well as a wide range of microbes including bacteria, fungi and protozoa, can stimulate neutrophils to undergo a distinctive form of cell death designated NETosis (Yipp and Kubes, 2013). This process leads to the extracellular release of fibers of DNA associated with histones, granule proteins and peptides—neutrophil extracellular traps (NETs). These are capable of entrapping microbial pathogens and mediate their extracellular killing. Since the first identification of NETs by Brinkmann et al (2004), this has become an area of active research, with many studies relying on the in vitro production and quantification of NETs as means to further understand the mechanisms involved in their formation, as well as their importance in pathogen clearance and in the development of multiple diseases (Buchanan et al, 2006; Kessenbrock et al, 2009; Marin-Esteban et al, 2012; Neumann et al, 2014; Wartha et al, 2007; Yost et al, 2009)
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