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Sub-cellular Biochemistry 2023Viruses are infectious entities that make use of the replication machinery of their hosts to produce more progenies, causing disease and sometimes death. To counter...
Viruses are infectious entities that make use of the replication machinery of their hosts to produce more progenies, causing disease and sometimes death. To counter viral infection, metazoan hosts are equipped with various defense mechanisms, from the rapid-evoking innate immune responses to the most advanced adaptive immune responses. Previous research demonstrated that cells in fruit flies and mice infected with Drosophila C virus and influenza, respectively, undergo apoptosis, which triggers the engulfment of apoptotic virus-infected cells by phagocytes. This process involves the recognition of eat-me signals on the surface of virus-infected cells by receptors of specialized phagocytes, such as macrophages and neutrophils in mice and hemocytes in fruit flies, to facilitate the phagocytic elimination of virus-infected cells. Inhibition of phagocytosis led to severe pathologies and death in both species, indicating that apoptosis-dependent phagocytosis of virus-infected cells is a conserved antiviral mechanism in multicellular organisms. Indeed, our understanding of the mechanisms underlying apoptosis-dependent phagocytosis of virus-infected cells has shed a new perspective on how hosts defend themselves against viral infection. This chapter explores the mechanisms of this process and its potential for developing new treatments for viral diseases.
Topics: Animals; Mice; Phagocytosis; Phagocytes; Immunity, Innate; Virus Diseases; Apoptosis; Antiviral Agents
PubMed: 38159224
DOI: 10.1007/978-3-031-40086-5_3 -
Current Opinion in Cell Biology Oct 2020Phagocytosis is a widespread and evolutionarily conserved process with diverse biological functions, ranging from engulfment of invading microbes during infection to... (Review)
Review
Phagocytosis is a widespread and evolutionarily conserved process with diverse biological functions, ranging from engulfment of invading microbes during infection to clearance of apoptotic debris in tissue homeostasis. Along with differences in biochemical composition, phagocytic targets greatly differ in physical attributes, such as size, shape, and rigidity, which are now recognized as important regulators of this process. Force exertion at the cell-target interface and cellular mechanical changes during phagocytosis are emerging as crucial factors underlying sensing of such target properties. With technological developments, mechanical aspects of phagocytosis are increasingly accessible experimentally, revealing remarkable organizational complexity of force exertion. An increasingly high-resolution picture is emerging of how target physical cues and cellular mechanical properties jointly govern important steps throughout phagocytic engulfment.
Topics: Animals; Biophysical Phenomena; Mechanotransduction, Cellular; Phagocytes; Phagocytosis
PubMed: 32698097
DOI: 10.1016/j.ceb.2020.05.011 -
Immunity Feb 2023β-glucosylceramide (β-GlcCer) accumulates in Gaucher disease, but how β-GlcCer, a Mincle ligand, causes characteristic neuroinflammation and neuronopathy is poorly...
β-glucosylceramide (β-GlcCer) accumulates in Gaucher disease, but how β-GlcCer, a Mincle ligand, causes characteristic neuroinflammation and neuronopathy is poorly understood. In this issue of Immunity, Shimizu et al. reveal that Mincle-dependent activation of microglia led to phagocytosis of neurons and neurologic symptoms.
Topics: Lectins; Microglia; Neurons; Phagocytosis; Selectins
PubMed: 36792566
DOI: 10.1016/j.immuni.2023.01.020 -
Frontiers in Immunology 2023Microglia are an integral part of central nervous system, but our understanding of microglial biology is limited due to the challenges in obtaining and culturing primary...
BACKGROUND
Microglia are an integral part of central nervous system, but our understanding of microglial biology is limited due to the challenges in obtaining and culturing primary human microglia. HMC3 is an important cell line for studying human microglia because it is readily accessible and straightforward to maintain in standard laboratories. Although HMC3 is widely used for microglial research, a robust genetic method has not been described. Here, we report a CRISPR genome editing platform, by the electroporation of Cas9 ribonucleoproteins (Cas9 RNP) and synthetic DNA repair templates, to enable rapid and precise genetic modifications of HMC3. For proof-of-concept demonstrations, we targeted the genes implicated in the regulation of amyloid beta (Aβ) and glioblastoma phagocytosis in microglia. We showed that CRISPR genome editing could enhance the phagocytic activities of HMC3.
METHODS
We performed CRISPR gene knockout (KO) in HMC3 by the electroporation of pre-assembled Cas9 RNP. Co-introduction of DNA repair templates allowed site-specific knock-in (KI) of an epitope tag, a synthetic promoter and a fluorescent reporter gene. The editing efficiencies were determined genotypically by DNA sequencing and phenotypically by immunofluorescent staining and flow cytometry. The gene-edited HMC3 cells were examined by fluorescent Aβ and glioblastoma phagocytosis assays.
RESULTS
Our platform enabled robust single (>90%) and double (>70%) KO without detectable off-target editing by high throughput DNA sequencing. We also inserted a synthetic SFFV promoter to efficiently upregulate the expression of endogenous and genes associated with microglial phagocytosis. The CRISPR-edited HMC3 showed stable phenotypes and enhanced phagocytosis of fluorescence-labeled Aβ1-42 peptides. Confocal microscopy further confirmed the localization of Aβ aggregates in the acidified lysosomes. HMC3 mutants also changed the phagocytic characteristic toward apoptotic glioblastoma cells.
CONCLUSION
CRISPR genome editing by Cas9 RNP electroporation is a robust approach to genetically modify HMC3 for functional studies such as the interrogation of Aβ and tumor phagocytosis, and is readily adoptable to investigate other aspects of microglial biology.
Topics: Humans; Gene Editing; CRISPR-Cas Systems; Microglia; Glioblastoma; Amyloid beta-Peptides; Phagocytosis
PubMed: 37483607
DOI: 10.3389/fimmu.2023.1169725 -
Methods in Molecular Biology (Clifton,... 2023Many plasma membrane receptors and ligands form nanoscale clusters on the plasma membrane surface. However, methods for directly and precisely manipulating nanoscale...
Many plasma membrane receptors and ligands form nanoscale clusters on the plasma membrane surface. However, methods for directly and precisely manipulating nanoscale protein localization are limited, making understanding the effects of this clustering difficult. DNA origami allows precise control over nanoscale protein localization with high fidelity and adaptability. Here, we describe how we have used this technique to study how nanoscale protein clustering affects phagocytosis. We provide protocols for conjugating DNA origami structures to supported lipid bilayer-coated beads to assay phagocytosis and planar glass coverslips for TIRF microscopy. The core aspects of this protocol can be translated to study other immune signaling pathways and should enable the implementation of previously inaccessible investigations.
Topics: Phagocytosis; Cell Membrane; DNA; Lipid Bilayers; Signal Transduction
PubMed: 37106190
DOI: 10.1007/978-1-0716-3135-5_19 -
Circulation Research Jan 2024
Topics: Humans; Efferocytosis; Macrophages; Phagocytosis; Inflammation
PubMed: 38236949
DOI: 10.1161/CIRCRESAHA.123.324011 -
Frontiers in Immunology 2021Mammalian phagocytes can phagocytose (i.e. eat) other mammalian cells in the body if they display certain signals, and this phagocytosis plays fundamental roles in... (Review)
Review
Mammalian phagocytes can phagocytose (i.e. eat) other mammalian cells in the body if they display certain signals, and this phagocytosis plays fundamental roles in development, cell turnover, tissue homeostasis and disease prevention. To phagocytose the correct cells, phagocytes must discriminate which cells to eat using a 'phagocytic code' - a set of over 50 known phagocytic signals determining whether a cell is eaten or not - comprising find-me signals, eat-me signals, don't-eat-me signals and opsonins. Most opsonins require binding to eat-me signals - for example, the opsonins galectin-3, calreticulin and C1q bind asialoglycan eat-me signals on target cells - to induce phagocytosis. Some proteins act as 'self-opsonins', while others are 'negative opsonins' or 'phagocyte suppressants', inhibiting phagocytosis. We review known phagocytic signals here, both established and novel, and how they integrate to regulate phagocytosis of several mammalian targets - including excess cells in development, senescent and aged cells, infected cells, cancer cells, dead or dying cells, cell debris and neuronal synapses. Understanding the phagocytic code, and how it goes wrong, may enable novel therapies for multiple pathologies with too much or too little phagocytosis, such as: infectious disease, cancer, neurodegeneration, psychiatric disease, cardiovascular disease, ageing and auto-immune disease.
Topics: Animals; Calreticulin; Cellular Senescence; Humans; Intercellular Adhesion Molecule-3; Opsonin Proteins; Phagocytosis; Phosphatidylserines; Polysaccharides; Signal Transduction; Synapses
PubMed: 34177884
DOI: 10.3389/fimmu.2021.629979 -
Critical Reviews in Oncology/hematology Jul 2023Erythrocytes are the most abundant type of cells in the blood and have a relatively simple structure when mature; they have a long life-span in the circulatory system.... (Review)
Review
Erythrocytes are the most abundant type of cells in the blood and have a relatively simple structure when mature; they have a long life-span in the circulatory system. The primary function of erythrocytes is as oxygen carriers; however, they also play an important role in the immune system. Erythrocytes recognize and adhere to antigens and promote phagocytosis. The abnormal morphology and function of erythrocytes are also involved in the pathological processes of some diseases. Owing to the large number and immune properties of erythrocytes, their immune functions should not be ignored. Currently, research on immunity is focused on immune cells other than erythrocytes. However, research on the immune function of erythrocytes and the development of erythrocyte-mediated applications is of great significance. Therefore, we aimed to review the relevant literature and summarize the immune functions of erythrocytes.
Topics: Humans; Erythrocytes; Immune System; Phagocytosis; Oxygen
PubMed: 37236411
DOI: 10.1016/j.critrevonc.2023.104039 -
F1000Research 2020Despite their comparatively low abundance in biological membranes, phosphoinositides are key to the regulation of a diverse array of signaling pathways and direct... (Review)
Review
Despite their comparatively low abundance in biological membranes, phosphoinositides are key to the regulation of a diverse array of signaling pathways and direct membrane traffic. The role of phosphoinositides in the initiation and progression of endocytic pathways has been studied in considerable depth. Recent advances have revealed that distinct phosphoinositide species feature prominently in clathrin-dependent and -independent endocytosis as well as in phagocytosis and macropinocytosis. Moreover, a variety of intracellular and cell-associated pathogens have developed strategies to commandeer host cell phosphoinositide metabolism to gain entry and/or metabolic advantage, thereby promoting their survival and proliferation. Here, we briefly survey the current knowledge on the involvement of phosphoinositides in endocytosis, phagocytosis, and macropinocytosis and highlight several examples of molecular mimicry employed by pathogens to either "hitch a ride" on endocytic pathways endogenous to the host or create an entry path of their own.
Topics: Clathrin; Endocytosis; Phagocytosis; Phosphatidylinositols; Protein Transport
PubMed: 32494357
DOI: 10.12688/f1000research.22393.1 -
Genes Jan 2023is the enteric protozoan parasite responsible for amebiasis. Trophozoites of ingest human cells in the intestine and other organs, which is the hallmark of its... (Review)
Review
is the enteric protozoan parasite responsible for amebiasis. Trophozoites of ingest human cells in the intestine and other organs, which is the hallmark of its pathogenesis. Phagocytosis and trogocytosis are pivotal biological functions for its virulence and also contribute to the proliferation of nutrient uptake from the environment. We previously elucidated the role of a variety of proteins associated with phagocytosis and trogocytosis, including Rab small GTPases, Rab effectors, including retromer, phosphoinositide-binding proteins, lysosomal hydrolase receptors, protein kinases, and cytoskeletal proteins. However, a number of proteins involved in phagocytosis and trogocytosis remain to be identified, and mechanistic details of their involvement must be elucidated at the molecular level. To date, a number of studies in which a repertoire of proteins associated with phagosomes and potentially involved in phagocytosis have been conducted. In this review, we revisited all phagosome proteome studies we previously conducted in order to reiterate information on the proteome of phagosomes. We demonstrated the core set of constitutive phagosomal proteins and also the set of phagosomal proteins recruited only transiently or in condition-dependent fashions. The catalogs of phagosome proteomes resulting from such analyses can be a useful source of information for future mechanistic studies as well as for confirming or excluding a possibility of whether a protein of interest in various investigations is likely or is potentially involved in phagocytosis and phagosome biogenesis.
Topics: Humans; Entamoeba histolytica; Proteome; Proteomics; Phagocytosis; Phagosomes; rab GTP-Binding Proteins
PubMed: 36833306
DOI: 10.3390/genes14020379