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Cells can die as a consequence of being phagocytosed by other cells — a form of cell death that has been called phagotrophy, cell cannibalism, programmed cell removal and primary phagocytosis. However, these are all different manifestations of cell death by phagocytosis (termed ‘phagoptosis’ for short). The engulfed cells die as a result of cytotoxic oxidants, peptides and degradative enzymes within acidic phagolysosomes. Cell death by phagocytosis was discovered by Metchnikov in the 1880s, but was neglected until recently. It is now known to contribute to developmental cell death in nematodes, Drosophila and mammals, and is central to innate and adaptive immunity against pathogens. Cell death by phagocytosis mediates physiological turnover of erythrocytes and other leucocytes, making it the most abundant form of cell death in the mammalian body. Immunity against cancer is also partly mediated by macrophage phagocytosis of cancer cells, but cancer cells can also phagocytose host cells and other cancer cells in order to survive. Recent evidence indicates neurodegeneration and other neuropathologies can be mediated by microglial phagocytosis of stressed neurons. Thus, despite cell death by phagocytosis being poorly recognized, it is one of the oldest, commonest and most important forms of cell death.Phagocytosis-mediated cell death — also known as ‘phagoptosis’ — regulates developmental processes, cell turnover and immunity to infections and cancer. Here, Brown summarizes the key molecular interactions involved in cell death by phagocytosis and the relevance of this process for host health.

Neurons in the developing brain undergo extensive structural refinement as nascent circuits adopt their mature form. This physical transformation of neurons is facilitated by the engulfment and degradation of axonal branches and synapses by surrounding glial cells, including microglia and astrocytes. However, the small size of phagocytic organelles and the complex, highly ramified morphology of glia have made it difficult to define the contribution of these and other glial cell types to this crucial process. Here, we used large-scale, serial section transmission electron microscopy (TEM) with computational volume segmentation to reconstruct the complete 3D morphologies of distinct glial types in the mouse visual cortex, providing unprecedented resolution of their morphology and composition. Unexpectedly, we discovered that the fine processes of oligodendrocyte precursor cells (OPCs), a population of abundant, highly dynamic glial progenitors, frequently surrounded small branches of axons. Numerous phagosomes and phagolysosomes (PLs) containing fragments of axons and vesicular structures were present inside their processes, suggesting that OPCs engage in axon pruning. Single-nucleus RNA sequencing from the developing mouse cortex revealed that OPCs express key phagocytic genes at this stage, as well as neuronal transcripts, consistent with active axon engulfment. Although microglia are thought to be responsible for the majority of synaptic pruning and structural refinement, PLs were ten times more abundant in OPCs than in microglia at this stage, and these structures were markedly less abundant in newly generated oligodendrocytes, suggesting that OPCs contribute substantially to the refinement of neuronal circuits during cortical development.

The pathogenicity of quartz involves lysosomal alteration in alveolar macrophages. This event triggers the inflammatory cascade that may lead to quartz-induced silicosis and eventually lung cancer. Experiments with synthetic quartz crystals recently showed that quartz dust is cytotoxic only when the atomic order of the crystal surfaces is upset by fracturing. Cytotoxicity was not observed when quartz had as-grown, unfractured surfaces. These findings raised questions on the potential impact of quartz surfaces on the phagolysosomal membrane upon internalization of the particles by macrophages. To gain insights on the surface-induced cytotoxicity of quartz, as-grown and fractured quartz particles in respirable size differing only in surface properties related to fracturing were prepared and physico-chemically characterized. Synthetic quartz particles were compared to a well-known toxic commercial quartz dust. Membranolysis was assessed on red blood cells, and quartz uptake, cell viability and effects on lysosomes were assessed on human PMA-differentiated THP-1 macrophages, upon exposing cells to increasing concentrations of quartz particles (10–250 µg/ml). All quartz samples were internalized, but only fractured quartz elicited cytotoxicity and phagolysosomal alterations. These effects were blunted when uptake was suppressed by incubating macrophages with particles at 4 °C. Membranolysis, but not cytotoxicity, was quenched when fractured quartz was incubated with cells in protein-supplemented medium. We propose that, upon internalization, the phagolysosome environment rapidly removes serum proteins from the quartz surface, restoring quartz membranolytic activity in the phagolysosomes. Our findings indicate that the cytotoxic activity of fractured quartz is elicited by promoting phagolysosomal membrane alteration.

Induction of lipid-laden foamy macrophages is a cellular hallmark of tuberculosis (TB) disease, which involves the transformation of infected phagolysosomes from a site of killing into a nutrient-rich replicative niche. Here, we show that a terpenyl nucleoside shed from Mycobacterium tuberculosis, 1-tuberculosinyladenosine (1-TbAd), caused lysosomal maturation arrest and autophagy blockade, leading to lipid storage in M1 macrophages. Pure 1-TbAd, or infection with terpenyl nucleoside-producing M. tuberculosis, caused intralysosomal and peribacillary lipid storage patterns that matched both the molecules and subcellular locations known in foamy macrophages. Lipidomics showed that 1-TbAd induced storage of triacylglycerides and cholesterylesters and that 1-TbAd increased M. tuberculosis growth under conditions of restricted lipid access in macrophages. Furthermore, lipidomics identified 1-TbAd-induced lipid substrates that define Gaucher's disease, Wolman's disease, and other inborn lysosomal storage diseases. These data identify genetic and molecular causes of M. tuberculosis-induced lysosomal failure, leading to successful testing of an agonist of TRPML1 calcium channels that reverses lipid storage in cells. These data establish the host-directed cellular functions of an orphan effector molecule that promotes survival in macrophages, providing both an upstream cause and detailed picture of lysosome failure in foamy macrophages.

Macrophages are critical to innate immunity due to their ability to phagocytose bacteria. The macrophage phagolysosome is a highly acidic organelle with potent antimicrobial properties, yet remarkably, ingested Staphylococcus aureus replicates within this niche. Herein we demonstrate that S. aureus requires the GraXRS regulatory system for growth within this niche, while the SaeRS and AgrAC two-component regulatory systems and the α-phenol soluble modulins are dispensable. Importantly, we find that it is exposure to acidic pH that is required for optimal growth of S. aureus inside fully acidified macrophage phagolysosomes. Exposure of S. aureus to acidic pH evokes GraS signaling, which in turn elicits an adaptive response that endows the bacteria with increased resistance to antimicrobial effectors, such as antimicrobial peptides, encountered inside macrophage phagolysosomes. Notably, pH-dependent induction of antimicrobial peptide resistance in S. aureus requires the GraS sensor kinase. GraS and MprF, a member of the GraS regulon, play an important role for bacterial survival in the acute stages of systemic infection, where in murine models of infection, S. aureus resides within liver-resident Kupffer cells. We conclude that GraXRS represents a vital regulatory system that functions to allow S. aureus to evade killing, prior to commencement of replication, within host antibacterial immune cells. IMPORTANCE S. aureus can infect any site of the body, including the microbicidal phagolysosome of the macrophage. The ability of S. aureus to infect diverse niches necessitates that the bacteria be highly adaptable. Here we show that S. aureus responds to phagolysosome acidification to evoke changes in gene expression that enable the bacteria to resist phagolysosomal killing and to promote replication. Toxin production is dispensable for this response; however, the bacteria require the sensor kinase GraS, which transduces signals in response to acidic pH. GraS is necessary for phagolysosomal replication and survival of S. aureus in the acute stage of systemic infection. Disruption of this S. aureus adaptation would render S. aureus susceptible to phagocyte restriction. S. aureus can infect any site of the body, including the microbicidal phagolysosome of the macrophage. The ability of S. aureus to infect diverse niches necessitates that the bacteria be highly adaptable. Here we show that S. aureus responds to phagolysosome acidification to evoke changes in gene expression that enable the bacteria to resist phagolysosomal killing and to promote replication. Toxin production is dispensable for this response; however, the bacteria require the sensor kinase GraS, which transduces signals in response to acidic pH. GraS is necessary for phagolysosomal replication and survival of S. aureus in the acute stage of systemic infection. Disruption of this S. aureus adaptation would render S. aureus susceptible to phagocyte restriction.

Conidia of the airborne human-pathogenic fungus Aspergillus fumigatus are inhaled by humans. In the lung, they are phagocytosed by alveolar macrophages and intracellularly processed. In macrophages, however, conidia can interfere with the maturation of phagolysosomes to avoid their elimination. To investigate whether polymeric particles (PPs) can reach this intracellular pathogen in macrophages, we formulated dye-labeled PPs with a size allowing for their phagocytosis. PPs were efficiently taken up by RAW 264.7 macrophages and were found in phagolysosomes. When macrophages were infected with conidia prior to the addition of PPs, we found that they co-localized in the same phagolysosomes. Mechanistically, the fusion of phagolysosomes containing PPs with phagolysosomes containing conidia was observed. Increasing concentrations of PPs increased fusion events, resulting in 14% of phagolysosomes containing both conidia and PPs. We demonstrate that PPs can reach conidia-containing phagolysosomes, making these particles a promising carrier system for antimicrobial drugs to target intracellular pathogens.Key points• Polymer particles of a size larger than 500 nm are internalized by macrophages and localized in phagolysosomes.• These particles can be delivered to Aspergillus fumigatus conidia-containing phagolysosomes of macrophages.• Enhanced phagolysosome fusion by the use of vacuolin1 can increase particle delivery.

Macrophage-expressed gene 1 (MPEG1/Perforin-2) is a perforin-like protein that functions within the phagolysosome to damage engulfed microbes. MPEG1 is thought to form pores in target membranes, however, its mode of action remains unknown. We use cryo-Electron Microscopy (cryo-EM) to determine the 2.4 Å structure of a hexadecameric assembly of MPEG1 that displays the expected features of a soluble prepore complex. We further discover that MPEG1 prepore-like assemblies can be induced to perforate membranes through acidification, such as would occur within maturing phagolysosomes. We next solve the 3.6 Å cryo-EM structure of MPEG1 in complex with liposomes. These data reveal that a multi-vesicular body of 12 kDa (MVB12)-associated β-prism (MABP) domain binds membranes such that the pore-forming machinery of MPEG1 is oriented away from the bound membrane. This unexpected mechanism of membrane interaction suggests that MPEG1 remains bound to the phagolysosome membrane while simultaneously forming pores in engulfed bacterial targets. Macrophage-expressed gene 1 (MPEG1) functions within the phagolysosome to damage engulfed microbes, presumably via forming pores in target membranes. In order to provide insights into the mechanism of MPEG1 function and membrane binding, the authors present structures of hexadecameric MPEG1 prepores both in solution and in complex with liposomes.

Acidic pH arrests the growth of Mycobacterium tuberculosis in vitro (pH < 5.8) and is thought to significantly contribute to the ability of macrophages to control M. tuberculosis replication. However, this pathogen has been shown to survive and even slowly replicate within macrophage phagolysosomes (pH 4.5 to 5) [M. S. Gomes et al., Infect. Immun. 67, 3199–3206 (1999)] [S. Levitte et al., Cell Host Microbe 20, 250–258 (2016)]. Here, we demonstrate that M. tuberculosis can grow at acidic pH, as low as pH 4.5, in the presence of host-relevant lipids. We show that lack of phosphoenolpyruvate carboxykinase and isocitrate lyase, two enzymes necessary for lipid assimilation, is cidal to M. tuberculosis in the presence of oleic acid at acidic pH. Metabolomic analysis revealed that M. tuberculosis responds to acidic pH by altering its metabolism to preferentially assimilate lipids such as oleic acid over carbohydrates such as glycerol. We show that the activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is impaired in acid-exposed M. tuberculosis likely contributing to a reduction in glycolytic flux. The generation of endogenous reactive oxygen species at acidic pH is consistent with the inhibition of GAPDH, an enzyme well-known to be sensitive to oxidation. This work shows that M. tuberculosis alters its carbon diet in response to pH and provides a greater understanding of the physiology of this pathogen during acid stress.

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. The detection of conserved motifs by pattern recognition receptors is a crucial component of the innate detection of pathogens and danger signals via conserved pattern recognition receptors. Here the authors define a pathway that transfers partially digested material from the phagolysosomal pathway of macrophages to release at the plasma membrane which is associated with enhanced inflammatory potential, by a process they introduce as eructophagy.

Phagocytosis is a fundamental process of cells to capture and ingest foreign particles. Small unicellular organisms such as free-living amoeba use this process to acquire food. In pluricellular organisms, phagocytosis is a universal phenomenon that all cells are able to perform (including epithelial, endothelial, fibroblasts, etc.), but some specialized cells (such as neutrophils and macrophages) perform this very efficiently and were therefore named professional phagocytes by Rabinovitch. Cells use phagocytosis to capture and clear all particles larger than 0.5 µm, including pathogenic microorganisms and cellular debris. Phagocytosis involves a series of steps from recognition of the target particle, ingestion of it in a phagosome (phagocytic vacuole), maturation of this phagosome into a phagolysosome, to the final destruction of the ingested particle in the robust antimicrobial environment of the phagolysosome. For the most part, phagocytosis is an efficient process that eliminates invading pathogens and helps maintaining homeostasis. However, several pathogens have also evolved different strategies to prevent phagocytosis from proceeding in a normal way. These pathogens have a clear advantage to perpetuate the infection and continue their replication. Here, we present an overview of the phagocytic process with emphasis on the antimicrobial elements professional phagocytes use. We also summarize the current knowledge on the microbial strategies different pathogens use to prevent phagocytosis either at the level of ingestion, phagosome formation, and maturation, and even complete escape from phagosomes.