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Detection of Neutrophil Extracellular Traps in Urine

  • Yanbao Yu
  • Keehwan Kwon
  • Rembert PieperEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2021)

Abstract

Neutrophils are important mediators of the antimicrobial defense during urinary tract infections (UTIs). When activated at the site of infection, these innate immune cells phagocytose and neutralize an invading pathogen. Another neutrophil defense strategy is the release of effectors, such as antimicrobial peptides and proteins stored in neutrophil granules and reactive oxygen species. Their release can be facilitated by cellular signals that trigger chromatic decondensation and the disruption of nuclear membranes, followed by granule and plasma membrane disintegration, DNA release into the extracellular milieu, and neutrophil cell death. Neutrophil extracellular traps (NETs) form. If microbial pathogens are the cause of neutrophil infiltration, they are entrapped in the network of DNA fibers that characterize NETs and are exposed to antimicrobial granule effectors and histones that bind to the extracellular DNA fibers. Here, we describe nonmicroscopic methods applied to clinical (urine sediment) samples to identify and characterize NETs associated with UTI. A stepwise extraction procedure using PBS, deoxyribonuclease I digestion and SDS-based solubilization is described. This is followed by native gel analysis to visualize protein–DNA macromolecular assemblies and proteomic analysis to identify signature proteins and their quantities in NETs. Microbes observed to be entrapped in NETs in the process of the innate immune response to the infection are Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Staphylococcus aureus, and Enterococcus faecalis.

Key words

Neutrophil extracellular traps Neutrophils Urinary tract infection Urine Inflammation Proteomics Proteus mirabilis 

Notes

Acknowledgments

This work was supported in part by the grant NIH-1R01GM103598 (National Institutes of Health, National Institute of General Medical Sciences).

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.The J. Craig Venter InstituteRockvilleUSA

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