Steps Of Phagocytosis Research Articles

Santa-maria is a glial phagocytic receptor that acts with SIMU to recognize and engulf apoptotic neurons.

The elimination of superfluous neurons via apoptosis and subsequent glial phagocytosis is crucial for the development of the central nervous system (CNS). In Drosophila, two glial phagocytic receptors, six-microns-under (SIMU) and Draper, mediate the phagocytosis of apoptotic neurons during embryogenesis. However, in simu;draper double-mutant embryos, some apoptotic neurons are still engulfed by the glia, suggesting the involvement of additional receptors. Here, we discover the Drosophila CD36 homolog Santa-maria, a transmembrane receptor, which is specifically expressed in embryonic phagocytic glia and plays a major role in the recognition and engulfment steps of phagocytosis. Our data demonstrate that santa-maria genetically interacts with simu and draper, while the protein product binds apoptotic cells and physically interacts with the SIMU protein. Moreover, we reveal that triple knockout of genes for all three glial phagocytic receptors (i.e., simu, draper, and santa-maria) causes partial lethality, thus illuminating their role in development, particularly in the developing nervous system.

The different roles of V-ATPase a subunits in phagocytosis/endocytosis and autophagy

ABSTRACT Microglia are specialized macrophages responsible for the clearance of dead neurons and pathogens by phagocytosis and degradation. The degradation requires phagosome maturation and acidification provided by the vesicular- or vacuolar-type H+-translocating adenosine triphosphatase (V-ATPase), which is composed of the cytoplasmic V1 domain and the membrane-embedded Vo domain. The V-ATPase a subunit, an integral part of the Vo domain, has four isoforms in mammals. The functions of different isoforms on phagosome maturation in different cells/species remain controversial. Here we show that mutations of both the V-ATPase Atp6v0a1 and Tcirg1b/Atp6v0a3 subunits lead to the accumulation of phagosomes in zebrafish microglia. However, their mechanisms are different. The V-ATPase Atp6v0a1 subunit is mainly distributed in early and late phagosomes. Defects of this subunit lead to a defective transition from early phagosomes to late phagosomes. In contrast, The V-ATPase Tcirg1b/Atp6v0a3 subunit is primarily located on lysosomes and regulates late phagosome-lysosomal fusion. Defective Tcirg1b/Atp6v0a3, but not Atp6v0a1 subunit leads to reduced acidification and impaired macroautophagy/autophagy in microglia. We further showed that ATP6V0A1/a1 and TCIRG1/a3 subunits in mouse macrophages preferentially located in endosomes and lysosomes, respectively. Blocking these subunits disrupted early-to-late endosome transition and endosome-to-lysosome fusion, respectively. Taken together, our results highlight the essential and conserved roles played by different V-ATPase subunits in multiple steps of phagocytosis and endocytosis across various species. Abbrevations: Apoe: apolipoprotein E; ANXA5/annexin V: annexin A5; ATP6V0A1/a1: ATPase H+-transporting V0 subunit a1; ATP6V0A2/a2: ATPase H+-transporting V0 subunit a2; ATP6V0A4/a4: ATPase H+-transporting V0 subunit a4; dpf: days post-fertilization; EEA1: early endosome antigen 1; HOPS: homotypic fusion and protein sorting; LAMP1: lysosomal associated membrane protein 1; Lcp1: lymphocyte cytosolic protein 1 (L-plastin); Map1lc3/Lc3: microtubule-associated protein 1 light chain 3; NR: neutral red; PBS: phosphate-buffered saline; PtdIns: phosphatidylinositol; PtdIns3P: phosphatidylinositol-3-phosphate; PtdIns(3,5)P2: phosphatidylinositol (3,5)-bisphosphate; RAB4: RAB4, member RAS oncogene family; RAB5: RAB5, member RAS oncogene family; RAB7: RAB7, member RAS oncogene family; TCIRG1/Atp6v0a3/a3: T cell immune regulator 1, ATPase H+-transporting V0 subunit a3; V-ATPase: vacuolar-type H+-translocating adenosine triphosphatase; Xla.Tubb2b/NBT: tubulin beta 2B class IIb.

Transcriptomics reveal a unique phago-mixotrophic response to low nutrient concentrations in the prasinophyte Pterosperma cristatum.

Constitutive mixoplankton-plastid-bearing microbial eukaryotes capable of both phototrophy and phagotrophy-are ubiquitous in marine ecosystems and facilitate carbon transfer to higher trophic levels within aquatic food webs, which supports enhanced sinking carbon flux. However, the regulation of the relative contribution of photosynthesis and prey consumption remains poorly characterized. We investigated the transcriptional dynamics behind this phenotypic plasticity in the prasinophyte green alga Pterosperma cristatum. Based on what is known of other mixoplankton species that cannot grow without photosynthesis (obligate phototrophs), we hypothesized that P. cristatum uses phagotrophy to circumvent the restrictions imposed on photosynthesis by nutrient depletion, to obtain nutrients from ingested prey, and to maintain photosynthetic carbon fixation. We observed an increase in feeding as a response to nutrient depletion, coinciding with an upregulation of expression for genes involved in essential steps of phagocytosis including prey recognition, adhesion and engulfment, transport and maturation of food vacuoles, and digestion. Unexpectedly, genes involved in the photosynthetic electron transfer chain, pigment biosynthesis, and carbon fixation were downregulated as feeding increased, implying an abatement of photosynthesis. Contrary to our original hypothesis, our results therefore suggest that depletion of inorganic nutrients triggered an alteration of trophic behavior from photosynthesis to phagotrophy in P. cristatum. While this behavior distinguishes P. cristatum from other groups of constitutive mixoplankton, its physiological response aligns with recent discoveries from natural microbial communities. These findings indicate that mixoplankton communities in nutrient-limited oceans can regulate photosynthesis against bacterivory based on nutrient availability.

Detection and Quantification of Cryptococcus Uptake by Phagocytic Cells Using Imaging Flow Cytometry.

Cryptococcus neoformans, the predominant etiological agent of cryptococcosis, is an encapsulated fungal pathogen found ubiquitously in the environment that causes pneumonia and life-threatening infections of the central nervous system. Following inhalation of yeasts or desiccated basidiospores into the lung alveoli, resident pulmonary phagocytic cells aid in the identification and eradication of Cryptococcus yeast through their arsenal of pattern recognition receptors (PRRs). PRRs recognize conserved pathogen-associated molecular patterns (PAMPs), such as branched mannans, β-glucans, and chitins that are the major components of the fungal cell wall. However, the key receptors/ligand interactions required for cryptococcal recognition and eventual fungal clearance have yet to be elucidated. Here we present an imaging flow cytometer (IFC) method that offers a novel quantitative cellular imaging and population statistics tool to accurately measure phagocytosis of fungal cells. It has the capacity to measure two distinct steps of phagocytosis: association/attachment and internalization in a high-throughput and quantitative manner that is difficult to achieve with other technologies. Results from these IFC studies allow for the potential to identify PRRs required for recognition, uptake, and subsequent activation of cytokine production, as well as other effector cell responses required for fungal clearance.

Mechanism of resistance to phagocytosis and pulmonary persistence in mucoid Pseudomonas aeruginosa.

Pseudomonas aeruginosa is known for its ability to form biofilms, which are dependent on the production of exopolysaccharides. During chronic colonization of the airway and biofilm formation, P. aeruginosa converts to a mucoid phenotype, indicating production of the exopolysaccharide alginate. The mucoid phenotype promotes resistance to phagocytic killing, but the mechanism has not been established. To better understand the mechanism of phagocytic evasion conferred by alginate production, Human (THP-1) and murine (MH-S) macrophage cell lines were used to determine the effects of alginate production on macrophage binding, signaling and phagocytosis. Phagocytosis assays using mucoid clinical isolate FRD1 and its non-mucoid algD mutant showed that alginate production inhibited opsonic and non-opsonic phagocytosis, but exogenous alginate was not protective. Alginate caused a decrease in binding to murine macrophages. Blocking antibodies to CD11b and CD14 showed that these receptors were important for phagocytosis and were blocked by alginate. Furthermore, alginate production decreased the activation of signaling pathways required for phagocytosis. Mucoid and non-mucoid bacteria induced similar levels of MIP-2 from murine macrophages. This study demonstrated for the first time that alginate on the bacterial surface inhibits receptor-ligand interactions important for phagocytosis. Our data suggest that there is a selection for alginate conversion that blocks the earliest steps in phagocytosis, leading to persistence during chronic pulmonary infections.

Mechanochemistry as a Sustainable Method for the Preparation of Fluorescent Ugi BODIPY Adducts

AbstractMechanochemistry, that is, the use of mechanical energy produced by milling or grinding to perform chemical transformations, was used as a green strategy using a minimal amount of solvent for the preparation of highly valuable formyl functionalized BODIPY dyes. The electronic properties of the meso‐aryl group play a key role in the fluorescence response of the adducts resulting from the Ugi reaction. Such an understanding of the molecular structure‐fluorescence interplay explains the brightness of these dyes in the cellular media monitored by fluorescence imaging. In the present report, we provide flow cytometry and fluorescence microscopy evidence that the novel dyes efficiently stain peripheral blood mononuclear cells (PBMCs) and Candida albicans cells without disturbing cell integrity. Moreover, it is demonstrated that by staining C. albicans cells, we can monitor the early and late steps of phagocytosis, using human monocyte‐derived macrophages. The dye series presented herein represent a novel application for formyl functionalized BODIPYs, expanding their applications in monitoring biological processes.

Squeezing in a Meal: Myosin Functions in Phagocytosis.

Phagocytosis is a receptor-mediated, actin-dependent process of internalization of large extracellular particles, such as pathogens or apoptotic cells. Engulfment of phagocytic targets requires the activity of myosins, actin-dependent molecular motors, which perform a variety of functions at distinct steps during phagocytosis. By applying force to actin filaments, the plasma membrane, and intracellular proteins and organelles, myosins can generate contractility, directly regulate actin assembly to ensure proper phagocytic internalization, and translocate phagosomes or other cargo to appropriate cellular locations. Recent studies using engineered microenvironments and phagocytic targets have demonstrated how altering the actomyosin cytoskeleton affects phagocytic behavior. Here, we discuss how studies using genetic and biochemical manipulation of myosins, force measurement techniques, and live-cell imaging have advanced our understanding of how specific myosins function at individual steps of phagocytosis.

Advanced Analysis of Photoreceptor Outer Segment Phagocytosis by RPE Cells in Culture.

Retinal pigment epithelial (RPE) cells are among the most actively phagocytic cells in nature. Primary RPE and stable RPE cell lines provide experimental model systems that possess the same phagocytic machinery as RPE in situ. Upon experimental challenge with isolated photoreceptor outer segment fragments (POS), these cells promptly and efficiently recognize, bind, internalize, and digest POS. Here, we describe experimental procedures to isolate POS from porcine eyes and to feed POS to RPE cells in culture. Furthermore, we provide experimental protocols to synchronize the POS binding and engulfment steps of phagocytosis. Finally, we describe three different and complementary methods to quantify total POS uptake by RPE cells and to discriminate surface-bound from engulfed POS.

Autophagy mediates phosphatidylserine exposure and phagosome degradation during apoptosis through specific functions of GABARAP/LGG-1 and LC3/LGG-2

ABSTRACTPhagocytosis and macroautophagy/autophagy are 2 processes involved in lysosome-mediated clearance of extracellular and intracellular components, respectively. Recent studies have identified the recruitment of the autophagic protein LC3 during phagocytosis of apoptotic corpses in what is now called LC3-associated phagocytosis (LAP). LAP is a distinct process from autophagy but it relies on some members of the autophagy pathway to allow efficient degradation of the phagocytosed cargo. We investigated whether both LC3/LGG-2 and GABARAP/LGG-1 are involved in phagocytosis of apoptotic corpses during embryonic development of Caenorhabditis elegans. We discovered that both LGG-1 and LGG-2 are involved in the correct elimination of apoptotic corpses, but that they have different functions. lgg-1 and lgg-2 mutants present a delay in phagocytosis of apoptotic cells but genetic analyses indicate that LGG-1 and LGG-2 act upstream and downstream of the engulfment pathways, respectively. Moreover, LGG-1 and LGG-2 display different cellular localizations with enrichment in apoptotic corpses and phagocytic cells, respectively. For both LGG-1 and LGG-2, subcellular localization is vesicular and dependent on UNC-51/ULK1, BEC-1/BECN1 and the lipidation machinery, indicating that their functions during phagocytosis of apoptotic corpses mainly rely on autophagy. Finally, we show that LGG-1 is involved in the exposure of the ‘eat-me signal’ phosphatidylserine at the surface of the apoptotic cell to allow its recognition by the phagocytic cell, whereas LGG-2 is involved in later steps of phagocytosis to allow efficient cell corpse clearance by mediating the maturation/degradation of the phagosome.

Flavonoids of Artocarpus heterophyllus Lam. heartwood inhibit the innate immune responses of human phagocytes.

To investigate the effects of flavonoids isolated from Artocarpus heterophyllus. heartwood on chemotaxis, phagocytosis, reactive oxygen species (ROS) production and myeloperoxidase (MPO) activity of human phagocytes. Chemotaxis was evaluated using a modified Boyden chamber and phagocytosis was determined by flowcytometer. Respiratory burst was investigated by luminol-based chemiluminescence assay while MPO activity was determined by colorimetric assay. Artocarpanone and artocarpin strongly inhibited all steps of phagocytosis. Artocarpanone and artocarpin showed strong chemotactic activity with IC50 values of 6.96 and 6.10 μm, respectively, which were lower than that of ibuprofen (7.37 μm). Artocarpanone was the most potent compound in inhibiting ROS production of polymorphonuclear leucocytes and monocytes with IC50 values comparable to those of aspirin. Artocarpin at 100 μg/ml inhibited phagocytosis of opsonized bacteria (28.3%). It also strongly inhibited MPO release with an IC50 value (23.3 μm) lower than that of indomethacin (69 μm). Structure-activity analysis indicated that the number of hydroxyl group, the presence of prenyl group and variation of C-2 and C-3 bonds might contribute towards their phagocytosis. Artocarpanone and artocarpin were able to suppress strongly the phagocytosis of human phagocytes at different steps and have potential to be developed into potent anti-inflammatory agents.

Modulation of Zinc Homeostasis in Acanthamoeba castellanii as a Possible Antifungal Strategy against Cryptococcus gattii.

Cryptococcus gattii is a basidiomycetous yeast that can be found in the environment and is one of the agents of cryptococcosis, a life-threatening disease. During its life cycle, cryptococcal cells take hold inside environmental predators such as amoebae. Despite their evolutionary distance, macrophages and amoebae share conserved similar steps of phagocytosis and microbial killing. To evaluate whether amoebae also share other antifungal strategies developed by macrophages, we investigated nutritional immunity against cryptococcal cells. We focused on zinc homeostasis modulation in Acanthamoeba castellanii infected with C. gattii. The intracellular proliferation rate (IPR) in amoebae was determined using C. gattii R265 and mutants for the ZIP1 gene, which displays defects of growth in zinc-limiting conditions. We detected a reduced IPR in cells lacking the ZIP1 gene compared to wild-type strains, suggesting that amoebae produce a low zinc environment to engulfed cells. Furthermore, flow cytometry analysis employing the zinc probe Zinpyr-1 confirmed the reduced concentration of zinc in cryptococcal-infected amoebae. qRT-PCR analysis of zinc transporter-coding genes suggests that zinc export by members of the ZnT family would be involved in the reduced intracellular zinc concentration. These results indicate that amoebae may use nutritional immunity to reduce fungal cell proliferation by reducing zinc availability for the pathogen.

Comprehensive proteome analysis of lysosomes reveals the diverse function of macrophages in immune responses.

Phagocytosis and autophagy in macrophages have been shown to be essential to both innate and adaptive immunity. Lysosomes are the main catabolic subcellular organelles responsible for degradation and recycling of both extracellular and intracellular material, which are the final steps in phagocytosis and autophagy. However, the molecular mechanisms underlying lysosomal functions after infection remain obscure. In this study, we conducted a quantitative proteomics analysis of the changes in constitution and glycosylation of proteins in lysosomes derived from murine RAW 264.7 macrophage cells treated with different types of pathogens comprising examples of bacteria (Listeria monocytogenes, L. m), DNA viruses (herpes simplex virus type-1, HSV-1) and RNA viruses (vesicular stomatitis virus, VSV). In total, 3,704 lysosome-related proteins and 300 potential glycosylation sites on 193 proteins were identified. Comparative analysis showed that the aforementioned pathogens induced distinct alterations in the proteome of the lysosome, which is closely associated with the immune functions of macrophages, such as toll-like receptor activation, inflammation and antigen-presentation. The most significant changes in proteins and fluctuations in glycosylation were also determined. Furthermore, Western blot analysis showed that the changes in expression of these proteins were undetectable at the whole cell level. Thus, our study provides unique insights into the function of lysosomes in macrophage activation and immune responses.

Dissecting Phagocytic Removal of Apoptotic Cells in Caenorhabditis elegans.

The unique features of programmed cell death during C. elegans development provide an outstanding system to decipher the mechanisms governing phagocytic removal of apoptotic cells. Like in many other organisms, phagocytosis in C. elegans involves several essential events, including exposure of eat-me signals on the cell corpse surface, cell corpse recognition and engulfment by phagocytes, and maturation of phagosomes for cell corpse destruction. Forward or reverse genetic approaches, microscopy-based cell biological methods, and biochemical assays have successfully been employed to identify key factors that control different steps of phagocytosis and to understand their functions in these cellular events. In this chapter, we mainly describe how to apply genetic and cell biological approaches to dissect cell corpse removal by phagocytosis in C. elegans.

Dynamin-Actin Cross Talk Contributes to Phagosome Formation and Closure.

Phagocytosis is a mechanism used by macrophages to internalize and eliminate microorganisms or cellular debris. It relies on profound rearrangements of the actin cytoskeleton that is the driving force allowing plasma membrane extension around the particle. The closure step of phagocytosis, however, remains poorly defined. We used a dedicated experimental setup with Total Internal Reflection Fluorescence Microscopy (TIRFM) to monitor phagosome formation and closure in three dimensions in living cells. We show that dynamin-2, which mediates the scission of endocytic vesicles, was recruited early and concomitantly with actin during phagosome formation. Dynamin-2 accumulated at the site of phagosome closure in living macrophages. Inhibition of its activity with dominant negative mutants or drugs demonstrated that dynamin-2 is implicated in actin dynamics and pseudopod extension. Depolymerization of actin led to impaired dynamin-2 recruitment or activity. Finally, we show that dynamin-2 plays a critical role in the effective scission of the phagosome from the plasma membrane. Thus, we establish that a cross talk between actin and dynamin takes place for phagosome formation and closure before dynamin functions for scission.

Biochemical and functional analysis of COS3A, a novel CD63-specific monoclonal antibody.

Monoclonal antibodies (mAbs) have become essential tools in life science research and in medicine, because of their extreme specificity. Several mAbs against leukocyte surface molecules have been generated in our laboratory. From these, mAb COS3A was selected for biochemical and functional analysis. To analyze the properties and function of the mAb COS3A. Cellular distribution was analyzed by immunofluorescence staining and flow cytometry. Biochemical characterization of the molecular target of COS3A was approached by immunoprecipitation, Western blotting and amino acid sequencing using LC-MS. N-glycosidase F treatment of COS3A-precipitated protein and culture of U937 cells in the presence of tunicamycin before cell lysate preparation were used to study the glycosylation state of proteins. Phagocytosis was examined by flow cytometry. MAb COS3A bound specifically to a molecule expressed on the surface of various human hematopoietic cells and cell lines but not on erythrocytes. The antigen had a molecular weight of 30-70 kDa, which was reduced to 25 kDa by elimination of N-linked glycan. LC-MS data and immunoprecipitation indicated that mAb COS3A bound specifically to the CD63 molecule. Remarkably, functional analysis demonstrated that mAb COS3A dramatically reduced granulocyte phagocytosis. The mAb COS3A recognized the CD63 molecule and strongly diminished granulocyte phagocytosis of E. coli, suggesting that CD63 may play a crucial role in the initial step of phagocytosis. MAb COS3A is, therefore, suitable for both biochemical and functional studies of CD63, and may be used for further study of the mechanism of phagocytosis and also in therapeutic approaches.

Dynamic process of phagocytosis and forms of macrophage cell death induced by ingestion of apoptotic neutrophils.

Clearance of apoptotic neutrophils by macrophages is important for both the successful resolution of acute inflammation and homeostasis. However, the dynamic process of phagocytosis of apoptotic neutrophils by macrophages and the fate of macrophages after the ingestion of apoptotic neutrophils has not been well documented. In the present study, we staged the recognition and tethering, internalization, digestion and exocytosis steps of phagocytosis of apoptotic neutrophils. Furthermore, we found that after the ingestion of apoptotic cells, a subset of macrophages underwent cell death by autophagy, apoptosis or oncosis as revealed by transmission electron microscopy and confocal microscopy combined with specific dyes. The percentage of autophagic, apoptotic and oncotic macrophages were 8.00%±2.00%, 12.33%±2.08%, and 3.66%±1.50%, respectively. These results indicated that after ingestion of apoptotic neutrophils, a subset of macrophages undergoes autophagy and apoptosis. We propose that autophagy of macrophages after the ingestion of apoptotic cells may be a new mechanism present in the resolution of inflammation.

Cytoskeletal function in the immune system

Cytoskeletal function in the immune system

Interactions between the discoidin domain receptor 1 and β1 integrin regulate attachment to collagen

SummaryCollagen degradation by phagocytosis is essential for physiological collagen turnover and connective tissue homeostasis. The rate limiting step of phagocytosis is the binding of specific adhesion receptors, which include the integrins and discoidin domain receptors (DDR), to fibrillar collagen. While previous data suggest that these two receptors interact, the functional nature of these interactions is not defined. In mouse and human fibroblasts we examined the effects of DDR1 knockdown and over-expression on β1 integrin subunit function. DDR1 expression levels were positively associated with enhanced contraction of floating and attached collagen gels, increased collagen binding and increased collagen remodeling. In DDR1 over-expressing cells compared with control cells, there were increased numbers, area and length of focal adhesions immunostained for talin, paxillin, vinculin and activated β1 integrin. After treatment with the integrin-cleaving protease jararhagin, in comparison to controls, DDR1 over-expressing cells exhibited increased β1 integrin cleavage at the cell membrane, indicating that DDR1 over-expression affected the access and susceptibility of cell-surface β1 integrin to the protease. DDR1 over-expression was associated with increased glycosylation of the β1 integrin subunit, which when blocked by deoxymannojirimycin, reduced collagen binding. Collectively these data indicate that DDR1 regulates β1 integrin interactions with fibrillar collagen, which positively impacts the binding step of collagen phagocytosis and collagen remodeling.

Live Imaging of Apoptotic Cell Clearance during <em>Drosophila </em>Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes--macrophages and dendritic cells to 'non-professional'--tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during <em>Drosophila </em>Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.