Exploring a Novel Late Stage Phagolysosomal Event in Macrophages
Date
2020-09-17
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Abstract
The work presented in this thesis characterizes a novel late-stage phagolysosomal event of macrophages wherein the mature phagolysosome transiently fuses with the cellular membrane and releases its soluble but not particulate matter, a process we termed eructophagy. Phagosomal maturation through fusion and fission with the endolysosomal system was believed to be unidirectional, resulting in the degradation of phagocytosed cargo to basic building blocks. Through the development of a live-cell imaging assay to simultaneously monitor multiple phagolysosomal lumenal biochemistries in real-time, we observed eructophagy as an iterative process that occurred in certain phagolysosomes within a population of macrophages. Eructophagy was additionally observed in macrophages and dendritic cells from mice and humans. Upon further examination of eructophagy, we found that interferon-gamma (IFN-?) activation of macrophages significantly increased eructophagy but interleukin-4 (IL-4) activation almost completely inhibited eructophagy. Furthermore, macrophages derived from mice conditionally deficient in key autophagic proteins had impaired rates of eructophagy, suggesting a role for canonical autophagic machinery in eructophagy. Employing an shRNA screen of cellular fusion machinery associated with lysosomes, autophagosomes, and the cell membrane in macrophages derived from conditionally-immortalized myeloid precursor cells, we have constructed a potential fusion model of eructophagy. Pathogen and Damage-associated molecular patterns (PAMPS and DAMPS) are molecules from exogenous and endogenous sources respectively, that can induce innate immunity. Despite their recognition and downstream signaling being well established, little is known about how they are generated from larger source material. Here, we propose that eructophagy is a method of PAMP and DAMP dissemination through the release of partially-digested material from the phagolysosome. We demonstrate that phagolysosomal-dependent processing of CpG DNA and N-formyl peptides conjugated to particles within phagolysosomes occurs before their extracellular release, and subsequent activation of vicinal cells. Taken together, we present eructophagy as a novel phagolysosomal process of pro-inflammatory macrophages that is modulated by canonical autophagic machinery and utilizes fusion proteins associated with secretory autophagy. We propose that eructophagy offers a solution to the paradigm of how PAMPs and DAMPs are generated from larger source material before their dissemination.
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Greene, C. J. (2020). Exploring a Novel Late Stage Phagolysosomal Event in Macrophages (Doctoral thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca.