Intravital Confocal Microscopy of Dermal Innate Immune Responses to Flea-Transmitted Yersinia pestis

  • Jeffrey G. ShannonEmail author
  • B. Joseph Hinnebusch
Part of the Methods in Molecular Biology book series (MIMB, volume 2010)


The technique known as intravital microscopy (IVM), when used in conjunction with transgenic mice expressing fluorescent proteins in various cell populations, is a powerful tool with the potential to provide new insights into host-pathogen interactions in infectious disease pathogenesis in vivo. Yersinia pestis, the causative agent of plague, is typically deposited in a host’s skin during feeding of an infected flea. IVM has been used to characterize the innate immune response to Y. pestis in the skin and identify differences between the responses to needle-inoculated and flea-transmitted bacteria that would have been difficult, if not impossible, to detect by other means. Here we describe techniques used to image the neutrophil response to flea-transmitted Y. pestis in the dermis of live mice using conventional confocal microscopy.

Key words

Intravital microscopy Confocal microscopy Skin Yersinia pestis Xenopsylla cheopis Flea 



We thank Clayton Jarrett, Chris Bosio, Dave Bland, Dustin Van Hofwegen, Adelaide Miarinjara, and Ashley Schwarzer for their careful review of the manuscript, David Dorward and Vinod Nair for assistance with confocal microscopy, and Anita Mora for assistance with graphic arts. This research was supported by the Intramural Research Program of the National Institutes of Health, National Institute of Allergy and Infectious Diseases.


  1. 1.
    Jain R, Weninger W (2013) Shedding light on cutaneous innate immune responses: the intravital microscopy approach. Immunol Cell Biol 91(4):263–270. Scholar
  2. 2.
    Faust N, Varas F, Kelly LM, Heck S, Graf T (2000) Insertion of enhanced green fluorescent protein into the lysozyme gene creates mice with green fluorescent granulocytes and macrophages. Blood 96(2):719–726PubMedGoogle Scholar
  3. 3.
    Burnett SH, Kershen EJ, Zhang J, Zeng L, Straley SC, Kaplan AM, Cohen DA (2004) Conditional macrophage ablation in transgenic mice expressing a Fas-based suicide gene. J Leukoc Biol 75(4):612–623. Scholar
  4. 4.
    Jung S, Aliberti J, Graemmel P, Sunshine MJ, Kreutzberg GW, Sher A, Littman DR (2000) Analysis of fractalkine receptor CX(3)CR1 function by targeted deletion and green fluorescent protein reporter gene insertion. Mol Cell Biol 20(11):4106–4114CrossRefGoogle Scholar
  5. 5.
    Lindquist RL, Shakhar G, Dudziak D, Wardemann H, Eisenreich T, Dustin ML, Nussenzweig MC (2004) Visualizing dendritic cell networks in vivo. Nat Immunol 5(12):1243–1250. Scholar
  6. 6.
    Unutmaz D, Xiang W, Sunshine MJ, Campbell J, Butcher E, Littman DR (2000) The primate lentiviral receptor Bonzo/STRL33 is coordinately regulated with CCR5 and its expression pattern is conserved between human and mouse. J Immunol 165(6):3284–3292CrossRefGoogle Scholar
  7. 7.
    Benson RA, Brewer JM, Garside P (2017) Visualizing and tracking T cell motility in vivo. Methods Mol Biol 1591:27–41. Scholar
  8. 8.
    McArdle S, Mikulski Z, Ley K (2016) Live cell imaging to understand monocyte, macrophage, and dendritic cell function in atherosclerosis. J Exp Med 213(7):1117–1131. Scholar
  9. 9.
    Shannon JG, Bosio CF, Hinnebusch BJ (2015) Dermal neutrophil, macrophage and dendritic cell responses to Yersinia pestis transmitted by fleas. PLoS Pathog 11(3):e1004734. Scholar
  10. 10.
    Shannon JG, Hasenkrug AM, Dorward DW, Nair V, Carmody AB, Hinnebusch BJ (2013) Yersinia pestis subverts the dermal neutrophil response in a mouse model of bubonic plague. MBio 4(5):e00170–e00113. Scholar
  11. 11.
    Wade SE, Georgi JR (1988) Survival and reproduction of artificially fed cat fleas, Ctenocephalides felis Bouché (Siphonaptera: Pulicidae). J Med Entomol 25:186–190CrossRefGoogle Scholar
  12. 12.
    Bland DM, Brown LD, Jarrett CO, Hinnebusch BJ, Macaluso KR (2017) Methods in flea research. Biodefense and emerging infections research resources repository.
  13. 13.
    Hinnebusch BJ, Perry RD, Schwan TG (1996) Role of the Yersinia pestis hemin storage (hms) locus in the transmission of plague by fleas. Science 273(5273):367–370CrossRefGoogle Scholar
  14. 14.
    Bosio CF, Viall AK, Jarrett CO, Gardner D, Rood MP, Hinnebusch BJ (2014) Evaluation of the murine immune response to Xenopsylla cheopis flea saliva and its effect on transmission of Yersinia pestis. PLoS Negl Trop Dis 8(9):e3196. Scholar

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

Authors and Affiliations

  1. 1.Laboratory of Bacteriology, Rocky Mountain LaboratoriesNIAID, NIHHamiltonUSA

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