Human Neutrophil Isolation and Degranulation Responses to Yersinia pestis Infection

Methods Mol Biol. 2019;2010:197-209. doi: 10.1007/978-1-4939-9541-7_14.

Abstract

Neutrophils are the primary immune cell recruited to the site of bacterial infection, where they can rapidly deploy vesicles filled with various pro-inflammatory and anti-microbial proteins. This degranulation process, combined with oxidative and nitrosative mechanisms, is a major part of the initial host response to kill microorganisms. Neutrophils are one of the main cell types that interact with Yersinia pestis during infection, which is often lethal in the absence of prompt antibiotic treatment. Intradermal inoculation of Y. pestis results in bubonic plague, and inhalation of aerosolized droplets containing Y. pestis results in pneumonic plague. Although neutrophils are recruited to the site of inoculation during both bubonic and pneumonic plague, the neutrophils fail to clear Y. pestis, and, during pneumonic plague, contribute to the development of severe pneumonia. Subverting neutrophil responses is critical to the development of fulminant disease, yet the mechanisms by which Y. pestis impairs neutrophils are poorly understood. Cell culture models are important tools for studying Y. pestis interactions with immune cells. We describe a cell culture model for the infection of human neutrophils with Y. pestis. Neutrophils are isolated from human peripheral blood at high purity and subsequently infected with Y. pestis. We specifically focus on the application of this in vitro infection assay to the analysis of neutrophil degranulation responses.

Keywords: Cell culture; Degranulation; Flow cytometry; Granule exocytosis; Neutrophil; Neutrophil isolation; Plague; Yersinia pestis.

MeSH terms

  • Cell Degranulation
  • Cell Separation / methods
  • Flow Cytometry / methods
  • Humans
  • Lung / immunology
  • Lung / microbiology
  • Neutrophil Infiltration
  • Neutrophils / immunology*
  • Neutrophils / microbiology
  • Neutrophils / physiology
  • Plague / immunology*
  • Plague / microbiology
  • Pneumonia / immunology
  • Pneumonia / microbiology
  • Yersinia pestis / immunology*