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

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.