Filamentous Bacteria as Targets to Study Phagocytosis.

Filamentous targets are internalized via phagocytic cups that last for several minutes before closing to form a phagosome. This characteristic offers the possibility to study key events in phagocytosis with greater spatial and temporal resolution than is possible to achieve using spherical particles, for which the transition from a phagocytic cup to an enclosed phagosome occurs within a few seconds after particle attachment. In this chapter, we provide methodologies to prepare filamentous bacteria and describe how they can be used as targets to study different aspects of phagocytosis.

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.