Fungal and Bacterial Killing by Neutrophils

Abstract

Neutrophils are professional phagocytes of the innate immune system that are essential to control bacterial and fungal infections. These cells engulf and kill invading microbes. Additionally, activated neutrophils are able to release neutrophil extracellular traps (NETs). These fibers consist of chromatin decorated with antimicrobial proteins to trap and kill microbes. Appropriate quantitative methods are required to understand the nature of interactions of neutrophils with pathogens. Here we present assays to measure killing mediated by phagocytosis, by NETs, by a combination of both, and by granular extract. As examples, we use Candida albicans for fungal and Shigella flexneri for bacterial pathogens.

Mannheimia haemolyticaand Its Leukotoxin Cause Neutrophil Extracellular Trap Formation by Bovine Neutrophils

“…Other investigators have shown that NETs are capable of trapping and killing extracellular pathogens (12,27,28,31,34,52,64). Using fluorescein-labeled M. haemolytica cells, we found that bacterial cells were trapped, in a time-dependent manner, by NETs (Fig.…”

IntracellularShigellaremodels its LPS to dampen the innate immune recognition and evade inflammasome activation

“…Human neutrophils were isolated from blood obtained from the blood bank in a protocol approved by the ethics committee of the Charité Hospital (Berlin, Germany). Neutrophils were purified by Histopaque/Percoll (68). After isolation, neutrophils were resuspended in Hanks balanced salt solution (Gibco) containing cations supplemented with 5% FCS and seeded at 7.5 × 10 4 neutrophils per well in a 96-well plate and allowed to rest for 30 min.…”

Afa/Dr Diffusely Adhering Escherichia coli Strain C1845 Induces Neutrophil Extracellular Traps That Kill Bacteria and Damage Human Enterocyte-Like Cells

“…4). Bacterial killing dependent on netosis, considered as the DNase-sensitive mechanism on activated cells, was calculated as previously reported (24) from CFU values obtained with PMA-stimulated cells treated and not treated with DNase. Netosis accounted for 18.0% Ϯ 4.1% of WT C1845 bacterial killing by PLB-985 cells (Fig.…”

“…The percentage of bacterial killing by netosis was determined as previously reported (24), considering the number of bacteria recovered after challenge of PMA-stimulated cells versus the number recovered after challenge of PMA-DNase-treated cells, as follows: [(df ϫ CFU with DNase Ϫ df ϫ CFU with no DNase)/df ϫ CFU DNase] ϫ 100, where df is the dilution factor and CFU represents numbers of CFU.…”