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

Abstract We feature a multi-parametric approach based on an imaging flow cytometry platform for examining phagocyte antimicrobial responses against the gram-negative bacterium Aeromonas veronii . This pathogen is known to induce strong inflammatory responses across a broad range of animal species, including humans. We examined the contribution of A . veronii to the induction of early phagocyte inflammatory processes in RAW 264.7 murine macrophages in vitro . We found that A . veronii , both in live or heat-killed forms, induced similar levels of macrophage activation based on NF-κB translocation. Although these macrophages maintained high levels of viability following heat-killed or live challenges with A . veronii , we identified inhibition of macrophage proliferation as early as 1 h post in vitro challenge. The characterization of phagocytic responses showed a time-dependent increase in phagocytosis upon A . veronii challenge, which was paired with a robust induction of intracellular respiratory burst responses. Interestingly, despite the overall increase in the production of reactive oxygen species (ROS) among RAW 264.7 macrophages, we found a significant reduction in the production of ROS among the macrophage subset that had bound A . veronii . Phagocytic uptake of the pathogen further decreased ROS production levels, even beyond those of unstimulated controls. Overall, this multi-parametric imaging flow cytometry-based approach allowed for segregation of unique phagocyte sub-populations and examination of their downstream antimicrobial responses, and should contribute to improved understanding of phagocyte responses against Aeromonas and other pathogens.