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Multi-parametric analysis of phagocyte antimicrobial responses using imaging flow cytometry.

Jeffrey J Havixbeck1, Michael E Wong1, Juan A More Bayona1

  • 1Department of Biological Sciences, University of Alberta, Edmonton, Alberta T6G 2P5, Canada.

Journal of Immunological Methods
|April 12, 2015
PubMed
Summary

Aeromonas veronii triggers macrophage activation and phagocytosis but inhibits proliferation. Interestingly, phagocytosis of A. veronii reduces reactive oxygen species (ROS) production in macrophages.

Keywords:
AeromonasImaging flow cytometryMacrophageMulti-parametric analysisPhagocytosis

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Area of Science:

  • Immunology
  • Microbiology
  • Cell Biology

Background:

  • Aeromonas veronii is a gram-negative bacterium known to cause significant inflammatory responses in various animal species, including humans.
  • Understanding phagocyte responses to A. veronii is crucial for developing effective treatments against this pathogen.

Purpose of the Study:

  • To investigate the early phagocyte inflammatory and antimicrobial responses to Aeromonas veronii in vitro.
  • To characterize the impact of A. veronii on macrophage activation, proliferation, phagocytosis, and respiratory burst.

Main Methods:

  • Utilized a multi-parametric imaging flow cytometry platform to analyze RAW 264.7 murine macrophages.
  • Assessed NF-κB translocation for macrophage activation, cell viability, proliferation, phagocytosis, and reactive oxygen species (ROS) production.
  • Examined responses to both live and heat-killed A. veronii.

Main Results:

  • A. veronii, live or heat-killed, induced comparable NF-κB translocation, indicating similar macrophage activation.
  • Macrophage proliferation was inhibited as early as 1 hour post-challenge, despite maintained cell viability.
  • Phagocytosis and intracellular respiratory burst increased over time, but ROS production was significantly reduced in macrophages that had bound or phagocytosed A. veronii.

Conclusions:

  • Multi-parametric imaging flow cytometry effectively segregates phagocyte subpopulations and their responses.
  • A. veronii elicits complex phagocyte responses, including reduced ROS production upon pathogen interaction.
  • This approach enhances the understanding of phagocyte defense mechanisms against Aeromonas and other pathogens.