Microscopy-Based Tracking and Quantification Methods to Study Phagosome Resolution.

Phagosome resolution is a newly defined, terminal stage in the process of phagocytosis. During this phase, phagolysosomes are fragmented into smaller vesicles, which we called phagosome-derived vesicles (PDVs). PDVs gradually accumulate within macrophages, while the phagosomes diminish in size until the organelles are no longer detectable. Although PDVs share the same maturation markers as phagolysosomes, they are heterogeneous in size and very dynamic, which makes PDVs difficult to track. Thus, to analyze PDV populations in cells, we developed methods to differentiate PDVs from the phagosomes in which they were derived and further assess their characteristics. In this chapter, we describe two microscopy-based methods that can be used to quantify different aspects of phagosome resolution: volumetric analysis of phagosome shrinkage and PDV accumulation and co-occurrence analysis of various membrane markers with PDVs.

Aluminum hydroxide adjuvant diverts the uptake and trafficking of genetically detoxified pertussis toxin to lysosomes in macrophages.

Aluminum salts have been successfully utilized as adjuvants to enhance the immunogenicity of vaccine antigens since the 1930s. However, the cellular mechanisms behind the immune adjuvanticity effect of these materials in antigen-presenting cells are poorly understood. In this study, we investigated the uptake and trafficking of aluminum oxy-hydroxide (AlOOH), in RAW 264.7 murine and U-937 human macrophages-like cells. Furthermore, we determined the impact that the adsorption to AlOOH particulates has on the trafficking of a Bordetella pertussis vaccine candidate, the genetically detoxified pertussis toxin (gdPT). Our results indicate that macrophages internalize AlOOH by constitutive macropinocytosis assisted by the filopodial protrusions that capture the adjuvant particles. Moreover, we show that AlOOH has the capacity to nonspecifically adsorb IgG, engaging opsonic phagocytosis, which is a feature that may allow for more effective capture and uptake of adjuvant particles by antigen-presenting cells (APCs) at the site of vaccine administration. We found that AlOOH traffics to endolysosomal compartments that hold degradative properties. Importantly, while we show that gdPT escapes degradative endolysosomes and traffics toward the retrograde pathway, as reported for the wild-type pertussis toxin, the adsorption to AlOOH diverts gdPT to traffic to the adjuvant's lysosome-type compartments, which may be key for MHC-II-driven antigen presentation and activation of CD4+ T cell. Thus, our findings establish a direct link between antigen adsorption to AlOOH and the intracellular trafficking of antigens within antigen-presenting cells and bring to light a new potential mechanism for aluminum adjuvancy. Moreover, the in-vitro single-cell approach described herein provides a general framework and tools for understanding critical attributes of other vaccine formulations.

Phagosome resolution regenerates lysosomes and maintains the degradative capacity in phagocytes.

Phagocytes engulf unwanted particles into phagosomes that then fuse with lysosomes to degrade the enclosed particles. Ultimately, phagosomes must be recycled to help recover membrane resources that were consumed during phagocytosis and phagosome maturation, a process referred to as "phagosome resolution." Little is known about phagosome resolution, which may proceed through exocytosis or membrane fission. Here, we show that bacteria-containing phagolysosomes in macrophages undergo fragmentation through vesicle budding, tubulation, and constriction. Phagosome fragmentation requires cargo degradation, the actin and microtubule cytoskeletons, and clathrin. We provide evidence that lysosome reformation occurs during phagosome resolution since the majority of phagosome-derived vesicles displayed lysosomal properties. Importantly, we show that clathrin-dependent phagosome resolution is important to maintain the degradative capacity of macrophages challenged with two waves of phagocytosis. Overall, our work suggests that phagosome resolution contributes to lysosome recovery and to maintaining the degradative power of macrophages to handle multiple waves of phagocytosis.

Phagocytosis: what's on the menu? 1.

Phagocytosis is an evolutionarily conserved process. In Protozoa, phagocytosis fulfills a feeding mechanism, while in Metazoa, phagocytosis diversified to play multiple organismal roles, including immune defence, tissue homeostasis, and remodeling. Accordingly, phagocytes display a high level of plasticity in their capacity to recognize, engulf, and process targets that differ in composition and morphology. Here, we review how phagocytosis adapts to its multiple roles and discuss in particular the effect of target morphology in phagocytic uptake and phagosome maturation.