Advertisement

Human Neutrophil Isolation and Degranulation Responses to Yersinia pestis Infection

  • Kara R. Eichelberger
  • William E. GoldmanEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2010)

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.

Key words

Neutrophil Yersinia pestis Degranulation Granule exocytosis Neutrophil isolation Plague Cell culture Flow cytometry 

References

  1. 1.
    Kolaczkowska E, Kubes P (2013) Neutrophil recruitment and function in health and inflammation. Nat Rev Immunol 13:159–175CrossRefGoogle Scholar
  2. 2.
    Lacy P (2006) Mechanisms of degranulation in neutrophils. Allergy Asthma Clin Immunol 2:98–108CrossRefGoogle Scholar
  3. 3.
    Sengeløv H, Follin P, Kjeldsen L, Lollike K, Dahlgren C, Borregaard N (1995) Mobilization of granules and secretory vesicles during in vivo exudation of human neutrophils. J Immunol 154:4157–4165PubMedGoogle Scholar
  4. 4.
    Soehnlein O, Lindbom L (2009) Neutrophil-derived azurocidin alarms the immune system. J Leukoc Biol 85:344–351CrossRefGoogle Scholar
  5. 5.
    Pereira HA, Shafer WM, Pohl J, Martin LE, Spitznagel JK (1990) CAP37, a human neutrophil-derived chemotactic factor with monocyte specific activity. J Clin Invest 85:1468–1476CrossRefGoogle Scholar
  6. 6.
    Sahoo M, Del Barrio L, Miller MA, Re F (2014) Neutrophil elastase causes tissue damage that decreases host tolerance to lung infection with Burkholderia species. PLoS Pathog 10:e1004327CrossRefGoogle Scholar
  7. 7.
    Winterbourn CC, Kettle AJ, Hampton MB (2016) Reactive oxygen species and neutrophil function. Annu Rev Biochem 85:765–792CrossRefGoogle Scholar
  8. 8.
    Perry RD, Fetherston JD (1997) Yersinia pestis--etiologic agent of plague. Clin Microbiol Rev 10:35–66CrossRefGoogle Scholar
  9. 9.
    Viboud GI, Bliska JB (2005) Yersinia outer proteins: role in modulation of host cell signaling responses and pathogenesis. Annu Rev Microbiol 59:69–89CrossRefGoogle Scholar
  10. 10.
    Pechous RD, Sivaraman V, Price PA, Stasulli NM, Goldman WE (2013) Early host cell targets of Yersinia pestis during primary pneumonic plague. PLoS Pathog 9:e1003679CrossRefGoogle Scholar
  11. 11.
    Marketon MM, DePaolo RW, DeBord KL, Jabri B, Schneewind O (2005) Plague bacteria target immune cells during infection. Science 309:1739–1741CrossRefGoogle Scholar
  12. 12.
    Spinner JL, Cundiff JA, Kobayashi SD (2008) Yersinia pestis type III secretion system-dependent inhibition of human polymorphonuclear leukocyte function. Infect Immun 76:3754–3760CrossRefGoogle Scholar
  13. 13.
    Stasulli NM, Eichelberger KR, Price PA, Pechous RD, Montgomery SA, Parker JS, Goldman WE (2015) Spatially distinct neutrophil responses within the inflammatory lesions of pneumonic plague. MBio 6:e01530–e01515CrossRefGoogle Scholar
  14. 14.
    Spinner JL, Hasenkrug AM, Shannon JG, Kobayashi SD, Hinnebusch BJ (2016) Role of the Yersinia YopJ protein in suppressing interleukin-8 secretion by human polymorphonuclear leukocytes. Microbes Infect 18:21–29CrossRefGoogle Scholar
  15. 15.
    Gonzalez RJ, Lane MC, Wagner NJ, Weening EH, Miller VL (2015) Dissemination of a highly virulent pathogen: tracking the early events that define infection. PLoS Pathog 11:e1004587CrossRefGoogle Scholar
  16. 16.
    Bosio CF, Jarrett CO, Gardner D, Hinnebusch BJ (2012) Kinetics of innate immune response to Yersinia pestis after intradermal infection in a mouse model. Infect Immun 80:4034–4045CrossRefGoogle Scholar
  17. 17.
    Lathem WW, Crosby SD, Miller VL, Goldman WE (2005) Progression of primary pneumonic plague: a mouse model of infection, pathology, and bacterial transcriptional activity. Proc Natl Acad Sci U S A 102:17786–17791CrossRefGoogle Scholar
  18. 18.
    Bubeck SS, Cantwell AM, Dube PH (2007) Delayed inflammatory response to primary pneumonic plague occurs in both outbred and inbred mice. Infect Immun 75:697–705CrossRefGoogle Scholar
  19. 19.
    Pechous RD, Broberg CA, Stasulli NM, Miller VL, Goldman WE (2015) In vivo transcriptional profiling of Yersinia pestis reveals a novel bacterial mediator of pulmonary inflammation. MBio 6:e02302–e14–11CrossRefGoogle Scholar
  20. 20.
    Amulic B, Cazalet C, Hayes GL, Metzler KD, Zychlinsky A (2012) Neutrophil function: from mechanisms to disease. Annu Rev Immunol 30:459–489CrossRefGoogle Scholar
  21. 21.
    Kuhns DB, Long Priel DA, Chu J, Zarember KA (2015) Isolation and functional analysis of human neutrophils. Curr Protoc Immunol 111:7.23.1–7.2316CrossRefGoogle Scholar
  22. 22.
    Michiels T, Wattiau P, Brasseur R, Ruysschaert JM, Cornelis G (1990) Secretion of Yop proteins by Yersiniae. Infect Immun 58:2840–2849PubMedPubMedCentralGoogle Scholar
  23. 23.
    Freitas M, Porto G, Lima JLFC, Fernandes E (2008) Isolation and activation of human neutrophils in vitro. The importance of the anticoagulant used during blood collection. Clin Biochem 41:570–575CrossRefGoogle Scholar
  24. 24.
    Cowland JB, Borregaard N (2016) Granulopoiesis and granules of human neutrophils. Immunol Rev 273:11–28CrossRefGoogle Scholar

Copyright information

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

Authors and Affiliations

  1. 1.Department of Microbiology and ImmunologyUniversity of North Carolina at Chapel HillChapel HillUSA

Personalised recommendations