Investigating the Mediating Fusion Machinery and Biological Role of a Novel Late-Stage Phagocytic Event

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This dissertation investigates eructophagy, a newly discovered process in which macrophages release soluble contents of the phagolysosome (PL) through fusion with the plasma membrane (PM). This study aims to determine the molecular machinery involved in membrane fusion during eructophagy and its role in antigen presentation. Macrophages play a crucial role in combating infections and maintaining homeostasis by phagocytosing invading pathogens and cellular debris into the phagolysosome (PL). It was believed that the contents of the PL that were not employed for antigen presentation were completely digested and recycled as primary building blocks, while indigestible material were stored in lysosomes. Therefore, how macrophages make immunostimulatory molecules, such as nucleic acids, polysaccharides, or lipids, available for immune detection is not well understood. Recently, eructophagy emerged as a novel process that allows macrophages to expel soluble components of the PL into the external environment, potentially serving as a form of intercellular communication to disseminates otherwise unavailable bioactive molecules during inflammation. The molecular machinery underlying membrane fusion during eructophagy has not been elucidated. To address this, we used a lentiviral short hairpin RNA knockdown library of conditionally immortalized monocytes to target genes involved in fusion, fission, and trafficking events to examine their potential role in eructophagy. A novel microscopy-based assay that reports eructophagy events as flashes of fluorescence was used, and it was found that eructophagy is dependent on genes involved in degradative and secretory autophagy. This led to further exploration of the role of autophagosomes in eructophagy. Vesicular blebs protruding from PLs were observed before eructophagy and vanished after resolution. Interfering with autophagosome-PL or autophagosome-PM fusion modulated the incidences of PL-associated blebs, thereby suggesting that these structures are autophagosomes. Immunofluorescence demonstrated that blebs colocalize with markers of the mature autophagosome ATG5 and LC3B. Furthermore, using supernatant transfer and triple cell coculture experiments, we demonstrate that eructophagy is used by macrophages to transfer antigens to vicinal APCs to enhance antigen presentation to T cells. Collectively, this dissertation reveals a novel mechanism of macrophage-mediated antigen transfer and suggests a putative model in which blebs are mature autophagosomes bridging the PL to the PM to facilitate eructophagy.
Macrophage, Phagosome, Phagolysosome, Membrane fusion, Antigen presentation, Autophagy, Eructophagy
Nguyen, J. A. (2023). Investigating the mediating fusion machinery and biological role of a novel late-stage phagocytic event (Doctoral thesis, University of Calgary, Calgary, Canada). Retrieved from