Eructophagy: macrophages use autophagic machinery to burp out parts of their meal.

The phagolysosome is an antimicrobial and degradative organelle that plays key roles in macrophage-mediated inflammatory and homeostatic functions. Whereas mature phagolysosomes are known to sequester and degrade their contents into basic nutrients, they were not previously assigned an active role in amplifying inflammation. We have described a novel macrophage process in which partially digested immunostimulatory PAMPs are released extracellularly from the mature phagolysosome via discrete events we term eructophagy. Eructophagy is induced by proinflammatory stimuli, negatively regulated by IL4 and MTOR, and is dependent on key autophagy proteins, including fusion machinery of degradative and secretory autophagy. We propose that macrophages use eructophagy to release processed PAMPs/DAMPs to amplify local inflammation.

Multiplexed Phagosomal Assays for the Detection and Quantification of Bidirectional Exchange Between the Phagolysosomal Lumen and Extracellular Space.

The phagolysosome is an antimicrobial and degradative organelle that plays a key role in macrophage-mediated inflammation and homeostasis. Before being presented to the adaptive immune system, phagocytosed proteins must first be processed into immunostimulatory antigens. Until recently, little attention has been given to how other processed PAMPs and DAMPs can stimulate an immune response if they are sequestered in the phagolysosome. Eructophagy is a newly described process in macrophages that releases partially digested immunostimulatory PAMPs and DAMPs extracellularly from the mature phagolysosome to activate vicinal leukocytes. This chapter outlines approaches to observe and quantify eructophagy by simultaneously measuring several phagosomal parameters of individual phagosomes. These methods use specifically designed experimental particles capable of conjugating to multiple reporter/reference fluors in combination with real-time automated fluorescent microscopy. Through the use of high-content image analysis software, each phagosomal parameter can be evaluated quantitatively or semiquantitatively during post-analysis.

Macrophages disseminate pathogen associated molecular patterns through the direct extracellular release of the soluble content of their phagolysosomes.

Recognition of pathogen-or-damage-associated molecular patterns is critical to inflammation. However, most pathogen-or-damage-associated molecular patterns exist within intact microbes/cells and are typically part of non-diffusible, stable macromolecules that are not optimally immunostimulatory or available for immune detection. Partial digestion of microbes/cells following phagocytosis potentially generates new diffusible pathogen-or-damage-associated molecular patterns, however, our current understanding of phagosomal biology would have these molecules sequestered and destroyed within phagolysosomes. Here, we show the controlled release of partially-digested, soluble material from phagolysosomes of macrophages through transient, iterative fusion-fission events between mature phagolysosomes and the plasma membrane, a process we term eructophagy. Eructophagy is most active in proinflammatory macrophages and further induced by toll like receptor engagement. Eructophagy is mediated by genes encoding proteins required for autophagy and can activate vicinal cells by release of phagolysosomally-processed, partially-digested pathogen associated molecular patterns. We propose that eructophagy allows macrophages to amplify local inflammation through the processing and dissemination of pathogen-or-damage-associated molecular patterns.

Better Together: Current Insights Into Phagosome-Lysosome Fusion.

Following phagocytosis, the nascent phagosome undergoes maturation to become a phagolysosome with an acidic, hydrolytic, and often oxidative lumen that can efficiently kill and digest engulfed microbes, cells, and debris. The fusion of phagosomes with lysosomes is a principal driver of phagosomal maturation and is targeted by several adapted intracellular pathogens. Impairment of this process has significant consequences for microbial infection, tissue inflammation, the onset of adaptive immunity, and disease. Given the importance of phagosome-lysosome fusion to phagocyte function and the many virulence factors that target it, it is unsurprising that multiple molecular pathways have evolved to mediate this essential process. While the full range of these pathways has yet to be fully characterized, several pathways involving proteins such as members of the Rab GTPases, tethering factors and SNAREs have been identified. Here, we summarize the current state of knowledge to clarify the ambiguities in the field and construct a more comprehensive phagolysosome formation model. Lastly, we discuss how other cellular pathways help support phagolysosome biogenesis and, consequently, phagocyte function.