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The Journal of Pathology Jun 2010Autophagy is a fundamental and phylogenetically conserved self-degradation process that is characterized by the formation of double-layered vesicles (autophagosomes)... (Review)
Review
Autophagy is a fundamental and phylogenetically conserved self-degradation process that is characterized by the formation of double-layered vesicles (autophagosomes) around intracellular cargo for delivery to lysosomes and proteolytic degradation. The increasing significance attached to autophagy in development and disease in higher eukaryotes has placed greater importance on the validation of reliable, meaningful and quantitative assays to monitor autophagy in live cells and in vivo in the animal. To date, the detection of processed LC3B-II by western blot or fluorescence studies, together with electron microscopy for autophagosome formation, have been the mainstays for autophagy detection. However, LC3 expression levels can vary markedly between different cell types and in response to different stresses, and there is also concern that over-expression of tagged versions of LC3 to facilitate imaging and detection of autophagy interferes with the process itself. In addition, the realization that it is not sufficient to monitor static levels of autophagy but to measure 'autophagic flux' has driven the development of new or modified approaches to detecting autophagy. Here, we present a critical overview of current methodologies to measure autophagy in cells and in animals.
Topics: Animals; Autophagy; Biomarkers; Flow Cytometry; Humans; Lysosomes; Microscopy, Electron; Microscopy, Fluorescence; Microtubule-Associated Proteins; Phagosomes
PubMed: 20225337
DOI: 10.1002/path.2694 -
Frontiers in Immunology 2021The rapid and efficient phagocytic clearance of apoptotic cells, termed efferocytosis, is a critical mechanism in the maintenance of tissue homeostasis. Removal of... (Review)
Review
The rapid and efficient phagocytic clearance of apoptotic cells, termed efferocytosis, is a critical mechanism in the maintenance of tissue homeostasis. Removal of apoptotic cells through efferocytosis prevents secondary necrosis and the resultant inflammation caused by the release of intracellular contents. The importance of efferocytosis in homeostasis is underscored by the large number of inflammatory and autoimmune disorders, including atherosclerosis and systemic lupus erythematosus, that are characterized by defective apoptotic cell clearance. Although mechanistically similar to the phagocytic clearance of pathogens, efferocytosis differs from phagocytosis in that it is immunologically silent and induces a tissue repair response. Efferocytes face unique challenges resulting from the internalization of apoptotic cells, including degradation of the apoptotic cell, dealing with the extra metabolic load imposed by the processing of apoptotic cell contents, and the coordination of an anti-inflammatory, pro-tissue repair response. This review will discuss recent advances in our understanding of the cellular response to apoptotic cell uptake, including trafficking of apoptotic cell cargo and antigen presentation, signaling and transcriptional events initiated by efferocytosis, the coordination of an anti-inflammatory response and tissue repair, unique cellular metabolic responses and the role of efferocytosis in host defense. A better understanding of how efferocytic cells respond to apoptotic cell uptake will be critical in unraveling the complex connections between apoptotic cell removal and inflammation resolution and maintenance of tissue homeostasis.
Topics: Antigen Presentation; Apoptosis; Gene Expression Regulation; Homeostasis; Humans; Inflammation; Phagocytes; Phagocytosis; Phagosomes; Signal Transduction
PubMed: 33959122
DOI: 10.3389/fimmu.2021.631714 -
Immunity May 2005Phagocytosis requires receptor-mediated recognition of particles, usually in the guise of infectious agents and apoptotic cells. Phagosomes fuse with lysosomes to... (Review)
Review
Phagocytosis requires receptor-mediated recognition of particles, usually in the guise of infectious agents and apoptotic cells. Phagosomes fuse with lysosomes to generate phagolysosomes, which play a key role in enzymatic digestion of the internalized contents into component parts. Recent findings indicate that a simple paradigm of a single cognate receptor interaction that guides the phagosome to phagolysosome formation belies the complexity of combinatorial receptor recognition and diversity of phagosome function. In fact, phagosomes are comprised of hundreds of proteins that play a key role in deciphering the contents of the phagosome and in defining host response. In this review we discuss how the challenge of recognizing diverse molecular patterns is met by combinatorial interactions between phagocytic receptors. Furthermore, these combinations are dynamic and both sculpt the balance between a proinflammatory or anti-inflammatory response and direct phagosome diversity. We also indicate an important role for genetically tractable model organisms in defining key components of this evolutionarily conserved process.
Topics: Adaptation, Physiological; Animals; Antigen Presentation; Apoptosis; Autophagy; Humans; Immunity, Innate; Models, Immunological; Opsonin Proteins; Phagocytes; Phagocytosis; Phagosomes; Receptors, Cell Surface; Signal Transduction
PubMed: 15894272
DOI: 10.1016/j.immuni.2005.05.002 -
Methods (San Diego, Calif.) Mar 2015Xenophagy is an autophagic phenomenon that specifically involves pathogens and other non-host entities. Although the understanding of the relationship between...
Xenophagy is an autophagic phenomenon that specifically involves pathogens and other non-host entities. Although the understanding of the relationship between autophagosomes and invading organisms has grown significantly in the past decade, the exact steps to confirm xenophagy has been not been thoroughly defined. Here we describe a methodical approach to confirming autophagy, its interaction with bacterial invasion, as well as the specific type of autophagic formation (i.e. autophagosome, autolysosome, phagolysosome). Further, we argue that xenophagy is not limited to pathogen interaction with autophagosome, but also non-microbial entities such as iron.
Topics: Autophagy; Brucella; Host-Pathogen Interactions; Humans; Infections; Lysosomes; Molecular Biology; Phagosomes
PubMed: 25497060
DOI: 10.1016/j.ymeth.2014.12.005 -
Nature Reviews. Microbiology Feb 2014Autophagy is a cellular process that targets proteins, lipids and organelles to lysosomes for degradation, but it has also been shown to combat infection with various... (Review)
Review
Autophagy is a cellular process that targets proteins, lipids and organelles to lysosomes for degradation, but it has also been shown to combat infection with various pathogenic bacteria. In turn, bacteria have developed diverse strategies to avoid autophagy by interfering with autophagy signalling or the autophagy machinery and, in some cases, they even exploit autophagy for their growth. In this Review, we discuss canonical and non-canonical autophagy pathways and our current knowledge of antibacterial autophagy, with a focus on the interplay between bacterial factors and autophagy components.
Topics: Autophagy; Bacteria; Host-Pathogen Interactions; Lysosomes; Phagocytosis; Phagosomes
PubMed: 24384599
DOI: 10.1038/nrmicro3160 -
Cell Research Jan 2014
Topics: Animals; Autophagy; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Humans; Phagosomes
PubMed: 24384820
DOI: 10.1038/cr.2014.1 -
The European Respiratory Journal Mar 2021Sarcoidosis and tuberculosis are granulomatous pulmonary diseases characterised by heightened immune reactivity to antigens. We hypothesised that an unsupervised...
INTRODUCTION
Sarcoidosis and tuberculosis are granulomatous pulmonary diseases characterised by heightened immune reactivity to antigens. We hypothesised that an unsupervised analysis comparing the molecular characteristics of granulomas formed in response to antigens in patients with sarcoidosis or latent tuberculosis infection (LTBI) would provide novel insights into the pathogenesis of sarcoidosis.
METHODS
A genomic analysis identified differentially expressed genes in granuloma-like cell aggregates formed by sarcoidosis (n=12) or LTBI patients (n=5) in an established human granuloma model wherein peripheral blood mononuclear cells were exposed to antigens (beads coated with purified protein derivative) and cultured for 7 days. Pathway analysis of differentially expressed genes identified canonical pathways, most notably antigen processing and presentation phagolysosomes, as a prominent pathway in sarcoidosis granuloma formation. The phagolysosomal pathway promoted mechanistic target of rapamycin complex 1 (mTORc1)/STAT3 signal transduction. Thus, granuloma formation and related immune mediators were evaluated in the absence or presence of various pre-treatments known to prevent phagolysosome formation (chloroquine) or phagosome acidification (bafilomycin A1) or directly inhibit mTORc1 activation (rapamycin).
RESULTS
In keeping with genomic analyses indicating enhanced phagolysosomal activation and predicted mTORc1 signalling, it was determined that sarcoidosis granuloma formation and related inflammatory mediator release was dependent upon phagolysosome assembly and acidification and mTORc1/S6/STAT3 signal transduction.
CONCLUSIONS
Sarcoidosis granulomas exhibit enhanced and sustained intracellular antigen processing and presentation capacities, and related phagolysosome assembly and acidification are required to support mTORc1 signalling to promote sarcoidosis granuloma formation.
Topics: Granuloma; Humans; Leukocytes, Mononuclear; Phagosomes; Sarcoidosis; Signal Transduction; TOR Serine-Threonine Kinases
PubMed: 32943400
DOI: 10.1183/13993003.02695-2020 -
Nature Cell Biology Oct 2019Phosphoinositides have a pivotal role in the maturation of nascent phagosomes into microbicidal phagolysosomes. Following degradation of their contents, mature...
Phosphoinositides have a pivotal role in the maturation of nascent phagosomes into microbicidal phagolysosomes. Following degradation of their contents, mature phagolysosomes undergo resolution, a process that remains largely uninvestigated. Here we studied the role of phosphoinositides in phagolysosome resolution. Phosphatidylinositol-4-phosphate (PtdIns(4)P), which is abundant in maturing phagolysosomes, was depleted as they tubulated and resorbed. Depletion was caused, in part, by transfer of phagolysosomal PtdIns(4)P to the endoplasmic reticulum, a process mediated by oxysterol-binding protein-related protein 1L (ORP1L), a RAB7 effector. ORP1L formed discrete tethers between the phagolysosome and the endoplasmic reticulum, resulting in distinct regions with alternating PtdIns(4)P depletion and enrichment. Tubules emerged from PtdIns(4)P-rich regions, where ADP-ribosylation factor-like protein 8B (ARL8B) and SifA- and kinesin-interacting protein/pleckstrin homology domain-containing family M member 2 (SKIP/PLEKHM2) accumulated. SKIP binds preferentially to monophosphorylated phosphoinositides, of which PtdIns(4)P is most abundant in phagolysosomes, contributing to their tubulation. Accordingly, premature hydrolysis of PtdIns(4)P impaired SKIP recruitment and phagosome resolution. Thus, resolution involves phosphoinositides and tethering of phagolysosomes to the endoplasmic reticulum.
Topics: ADP-Ribosylation Factors; Animals; CRISPR-Cas Systems; Endoplasmic Reticulum; Gene Editing; Gene Expression Regulation; Humans; Mice; Monocytes; Phagocytosis; Phagosomes; Phosphatidylinositol Phosphates; Primary Cell Culture; Proteolysis; RAW 264.7 Cells; RNA, Small Interfering; Receptors, Steroid; Signal Transduction; Vesicular Transport Proteins; rab GTP-Binding Proteins; rab7 GTP-Binding Proteins
PubMed: 31570833
DOI: 10.1038/s41556-019-0394-2 -
Frontiers in Immunology 2023Bacterial infections still impose a significant burden on humanity, even though antimicrobial agents have long since been developed. In addition to individual severe... (Review)
Review
Bacterial infections still impose a significant burden on humanity, even though antimicrobial agents have long since been developed. In addition to individual severe infections, the f fatality rate of sepsis remains high, and the threat of antimicrobial-resistant bacteria grows with time, putting us at inferiority. Although tremendous resources have been devoted to the development of antimicrobial agents, we have yet to recover from the lost ground we have been driven into. Looking back at the evolution of treatment for cancer, which, like infectious diseases, has the similarity that host immunity eliminates the lesion, the development of drugs to eliminate the tumor itself has shifted from a single-minded focus on drug development to the establishment of a treatment strategy in which the de-suppression of host immunity is another pillar of treatment. In infectious diseases, on the other hand, the development of therapies that strengthen and support the immune system has only just begun. Among innate immunity, the first line of defense that bacteria encounter after invading the host, the molecular mechanisms of the phagolysosome pathway, which begins with phagocytosis to fusion with lysosome, have been elucidated in detail. Bacteria have a large number of strategies to escape and survive the pathway. Although the full picture is still unfathomable, the molecular mechanisms have been elucidated for some of them, providing sufficient clues for intervention. In this article, we review the host defense mechanisms and bacterial evasion mechanisms and discuss the possibility of host-directed therapy for bacterial infection by intervening in the phagolysosome pathway.
Topics: Humans; Bacterial Infections; Immunity, Innate; Anti-Infective Agents; Bacteria; Phagosomes; Communicable Diseases
PubMed: 37841276
DOI: 10.3389/fimmu.2023.1227467 -
Molecular & Cellular Proteomics : MCP Jan 2022The lysosome represents a central degradative compartment of eukaryote cells, yet little is known about the biogenesis and function of this organelle in parasitic...
The lysosome represents a central degradative compartment of eukaryote cells, yet little is known about the biogenesis and function of this organelle in parasitic protists. Whereas the mannose 6-phosphate (M6P)-dependent system is dominant for lysosomal targeting in metazoans, oligosaccharide-independent sorting has been reported in other eukaryotes. In this study, we investigated the phagolysosomal proteome of the human parasite Trichomonas vaginalis, its protein targeting and the involvement of lysosomes in hydrolase secretion. The organelles were purified using Percoll and OptiPrep gradient centrifugation and a novel purification protocol based on the phagocytosis of lactoferrin-covered magnetic nanoparticles. The analysis resulted in a lysosomal proteome of 462 proteins, which were sorted into 21 classes. Hydrolases represented the largest functional class and included proteases, lipases, phosphatases, and glycosidases. Identification of a large set of proteins involved in vesicular trafficking (80) and turnover of actin cytoskeleton rearrangement (29) indicate a dynamic phagolysosomal compartment. Several cysteine proteases such as TvCP2 were previously shown to be secreted. Our experiments showed that secretion of TvCP2 was strongly inhibited by chloroquine, which increases intralysosomal pH, thus indicating that TvCP2 secretion occurs through lysosomes rather than the classical secretory pathway. Unexpectedly, we identified divergent homologues of the M6P receptor TvMPR in the phagolysosomal proteome, although T. vaginalis lacks enzymes for M6P formation. To test whether oligosaccharides are involved in lysosomal targeting, we selected the lysosome-resident cysteine protease CLCP, which possesses two glycosylation sites. Mutation of any of the sites redirected CLCP to the secretory pathway. Similarly, the introduction of glycosylation sites to secreted β-amylase redirected this protein to lysosomes. Thus, unlike other parasitic protists, T. vaginalis seems to utilize glycosylation as a recognition marker for lysosomal hydrolases. Our findings provide the first insight into the complexity of T. vaginalis phagolysosomes, their biogenesis, and role in the unconventional secretion of cysteine peptidases.
Topics: Cysteine; Cysteine Proteases; Humans; Lysosomes; Peptide Hydrolases; Phagosomes; Proteomics; Trichomonas vaginalis
PubMed: 34763061
DOI: 10.1016/j.mcpro.2021.100174