Phagosome Formation Research Articles

Phosphorylation of SNAP-23 at Ser95 causes a structural alteration and negatively regulates Fc receptor-mediated phagosome formation and maturation in macrophages.

SNAP-23 is a plasma membrane-localized soluble N-ethylmaleimide–sensitive factor attachment protein receptors (SNARE) involved in Fc receptor (FcR)-mediated phagocytosis. However, the regulatory mechanism underlying its function remains elusive. Using phosphorylation-specific antibodies, SNAP-23 was found to be phosphorylated at Ser95 in macrophages. To understand the role of this phosphorylation, we established macrophage lines overexpressing the nonphosphorylatable S95A or the phosphomimicking S95D mutation. The efficiency of phagosome formation and maturation was severely reduced in SNAP-23-S95D–overexpressing cells. To examine whether phosphorylation at Ser95 affected SNAP-23 structure, we constructed intramolecular Förster resonance energy transfer (FRET) probes of SNAP-23 designed to evaluate the approximation of the N termini of the two SNARE motifs. Interestingly, a high FRET efficiency was detected on the membrane when the S95D probe was used, indicating that phosphorylation at Ser95 caused a dynamic structural shift to the closed form. Coexpression of IκB kinase (IKK) 2 enhanced the FRET efficiency of the wild-type probe on the phagosome membrane. Furthermore, the enhanced phagosomal FRET signal in interferon-γ–activated macrophages was largely dependent on IKK2, and this kinase mediated a delay in phagosome-lysosome fusion. These results suggested that SNAP-23 phosphorylation at Ser95 played an important role in the regulation of SNARE-dependent membrane fusion during FcR-mediated phagocytosis.

Crucial Role of Legionella pneumophila TolC in the Inhibition of Cellular Trafficking in the Protistan Host Paramecium tetraurelia.

Legionella pneumophila is a facultative intracellular Gram-negative bacterium, which is a major causative agent of Legionnaires’ disease. In the environment, this bacterium survives in free-living protists such as amoebae and Tetrahymena. The association of L. pneumophila and protists leads to the replication and spread of this bacterium. Thus, from a public health perspective, their association can enhance the risk of L. pneumophila infection for humans. Paramecium spp. are candidates of natural hosts of L. pneumophila, but their detailed relationships remain unclear. In the present study, we used an environmental strain, L. pneumophila Ofk308 (Ofk308) and Paramecium tetraurelia st110-1a to reveal the relationship between L. pneumophila and Paramecium spp. Ofk308 was cytotoxic to P. tetraurelia in an infection-dependent manner. We focused on TolC, a component of the type I secretion system, which is a virulence factor of L. pneumophila toward protists and found that cytotoxicity was dependent on TolC but not on other T1SS components. Further, the number of bacteria in P. tetraurelia was not associated with cytotoxicity and TolC was not involved in the mechanism of resistance against the digestion of P. tetraurelia in Ofk308. We used a LysoTracker to evaluate the maturation process of P. tetraurelia phagosomes containing Ofk308. We found that there was no difference between Ofk308 and the tolC-deletion mutant. To assess the phagocytic activity of P. tetraurelia, Texas Red-conjugated dextran-uptake assays were performed. Ofk308 inhibited phagosome formation by P. tetraurelia through a TolC-dependent mechanism. Further, we evaluated the excretion of Legionella-containing vacuoles from P. tetraurelia. We found that P. tetraurelia failed to excrete undigested Ofk308 and that Ofk308 remained within cells through a TolC-dependent mechanism. Our results suggest that TolC is essential for L. pneumophila to remain within Paramecium cells and to show cytotoxicity. Because of the high mobility and high cell division rate of Paramecium spp., living with Paramecium spp. would be beneficial for L. pneumophila to expand its habitat. To control Legionaries’ disease, understanding the ecology of L. pneumophila in the environment is essential.

The role of lipids in host-pathogen interactions.

Innate immunity relies on the effective recognition and elimination of pathogenic microorganisms. This entails sequestration of pathogens into phagosomes that promptly acquire microbicidal and degradative properties. This complex series of events, which involve cytoskeletal reorganization, membrane remodeling and the activation of multiple enzymes, is orchestrated by lipid signaling. To overcome this immune response, intracellular pathogens acquired mechanisms to subvert phosphoinositide-mediated signaling and use host lipids, notably cholesterol, as nutrients. We present brief overviews of the role of phosphoinositides in phagosome formation and maturation as well as of cholesterol handling by host cells, and selected Salmonella, Shigella, Chlamydia and Mycobacterium tuberculosis to exemplify the mechanisms whereby intracellular pathogens co-opt lipid metabolism in host cells. © 2018 IUBMB Life, 70(5):384-392, 2018.

Transient recruitment of M-Ras GTPase to phagocytic cups in RAW264 macrophages during FcγR-mediated phagocytosis.

M-Ras, a member of the Ras superfamily, is known to be involved in diverse cellular processes. However, its involvement in FcγR-mediated phagocytosis remains unknown. We examined the spatiotemporal localization of M-Ras during the engulfment of IgG-opsonized erythrocytes (IgG-Es) in RAW264 macrophages. By the live-cell imaging of fluorescent protein-fused M-Ras, we found that M-Ras was localized to the membrane of phagocytic cups during the early stage of phagosome formation. Notably, ratiometric image analysis revealed that M-Ras was concentrated in the membrane of forming phagosomes. Moreover, our analysis of M-Ras mutant expression showed that phagosome formation was significantly inhibited in cells expressing GDP-locked mutant M-Ras-S27N. In contrast, the expression of wild-type M-Ras or GTP-locked mutant M-Ras-G22V facilitated the uptake of IgG-Es. These data suggest that M-Ras is a novel component of the FcγR-mediated phagocytic pathway and may regulate phagosome formation in macrophages.

Observing Frustrated Phagocytosis and Phagosome Formation and Closure Using Total Internal Reflection Fluorescence Microscopy (TIRFM).

Complementary methods to observe frustrated phagocytosis and phagosome closure using total internal reflection fluorescence microscopy (TIRFM) are described here. Frustrated phagocytosis occurs when phagocytic cells are exposed to an opsonized surface and spread as if trying to engulf it, allowing for the observation of phagocytic spreading and the biochemical events that directly precede it. Phagosome formation and closure is an inherently three-dimensional process though, and cannot be studied in the "frustrated" situation. Here we describe a method to visualize with unprecedented high-resolution phagosome formation and closure in three dimensions. It allows for observation of the base of the phagocytic cup, the extending pseudopods, as well as the precise site of phagosome scission.

Development of a transendothelial shuttle by macrophage modification.

One of the limiting factors in tissue regeneration, particularly in the context of chronic disease such as myodystrophy, motor neuron disease, sarcopenia, and cardiovascular disease, is limited availability of stem cells. We propose employing autologous macrophages to deliver stem cells, thereby facilitating tissue regeneration, by a novel and relatively non-invasive therapeutic intervention. Circulatory monocytic cells of M1 phenotype have capacity for transendothelial migration to infiltrate damaged tissue, making them ideal delivery vehicles. However, in order to deliver viable stem cells, these macrophages must undergo phagosome maturation arrest. Our aim was to induce phagosome maturation arrest in prepolarised M1 macrophages, whilst maintaining capacity for phagocytic engulfment (including phagosome formation) and transendothelial migration. Primary human M1 macrophages were treated with a wortmannin-concanamycin A-chloroquine cocktail to induce arrest. Modified cells were allowed to ingest 4.5μm protein-coated fluorescent latex beads (simulated stem cells), before migratory capacity in response to MCP-1 was assessed over a 2-hr period in a Transwell co-culture system. Data indicate that phagosome acidification (as indicated by pHrodo®) was prevented in treated cells, effectively limiting digestion of ingested "cargo" (1.23±0.26% vs. 7.52±0.98% in controls; p<.0001). Neither phagocytic engulfment capacity (68.67±3.51% vs. 61.19±4.68%) nor migratory capacity (70.14±12.6 vs. 72.86±16.0 migrated cells per well) was compromised. We conclude that macrophages were successfully modified into transendothelial delivery vehicles, without compromising required functionality. This delivery system can be exploited to develop a novel method for focussed stem cell and/or drug delivery.

Control of Phagocytosis by Microbial Pathogens.

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.

Mitochondrial Fission Promotes the Continued Clearance of Apoptotic Cells by Macrophages

Clearance of apoptotic cells (ACs) by phagocytes (efferocytosis) prevents post-apoptotic necrosis and dampens inflammation. Defective efferocytosis drives important diseases, including atherosclerosis. For efficient efferocytosis, phagocytes must be able to internalize multiple ACs. We show here that uptake of multiple ACs by macrophages requires dynamin-related protein 1 (Drp1)-mediated mitochondrial fission, which is triggered by AC uptake. When mitochondrial fission is disabled, AC-induced increase in cytosolic calcium is blunted owing to mitochondrial calcium sequestration, and calcium-dependent phagosome formation around secondarily encountered ACs is impaired. These defects can be corrected by silencing the mitochondrial calcium uniporter (MCU). Mice lacking myeloid Drp1 showed defective efferocytosis and its pathologic consequences in the thymus after dexamethasone treatment and in advanced atherosclerotic lesions in fat-fed Ldlr-/- mice. Thus, mitochondrial fission in response to AC uptake is a critical process that enables macrophages to clear multiple ACs and to avoid the pathologic consequences of defective efferocytosis invivo.

PALLD Regulates Phagocytosis by Enabling Timely Actin Polymerization and Depolymerization.

PALLD is an actin cross-linker supporting cellular mechanical tension. However, its involvement in the regulation of phagocytosis, a cellular activity essential for innate immunity and physiological tissue turnover, is unclear. We report that PALLD is highly induced along with all-trans-retinoic acid-induced maturation of myeloid leukemia cells, to promote Ig- or complement-opsonized phagocytosis. PALLD mechanistically facilitates phagocytic receptor clustering by regulating actin polymerization and c-Src dynamic activation during particle binding and early phagosome formation. PALLD is also required at the nascent phagosome to recruit phosphatase oculocerebrorenal syndrome of Lowe, which regulates phosphatidylinositol-4,5-bisphosphate hydrolysis and actin depolymerization to complete phagosome closure. Collectively, our results show a new function for PALLD as a crucial regulator of the early phase of phagocytosis by elaborating dynamic actin polymerization and depolymerization.

Proteomic analysis of macrophage in response to Edwardsiella tarda-infection

Edwardsiella tarda is an important facultative intracellular pathogen infecting a wide range of host from fish to humans. This bacterium could survive and replicate in macrophages as an escape mechanism from the host defense. E. tarda -macrophage interaction is vital in determining the outcome of edwardsiellasis. To fully elucidate the pathogenesis of E. tarda, the differential proteomes of RAW264.7 cells in response to E. tarda-infection, were analyzed at different time points with two-dimensional gel electrophoresis (2-DE) followed by liquid-chromatography-tandem mass spectrometry (LC-MS/MS) identification. 26 altered proteins (18 up-regulated and 8 down-regulated proteins) were successfully identified, which are mainly involved in formation of phagosomes, macrophage microbicidal activity and anti-apoptosis of macrophage. Moreover, 6 corresponding genes of the differentially expressed proteins were quantified by quantitative real-time PCR (qPCR) to examine the transcriptional profiles. Western blot analysis further confirmed the differential expression of 5 proteins in the proteomic profiles. Based on these findings, we hypothesize that these differentially expressed proteins likely play a pivotal role in determining the course of E. tarda-infection. The result suggested that E. tarda could develop some strategies to achieve a successful intracellular lifestyle, including modulation of phagosome biogenesis, resistance to macrophage microbicidal agent and anti-apoptosis of macrophages. Thus, this work effectively provides useful and novel protein-related information to further understand the underlying pathogenesis of E. tarda-infection.

Shared and Distinct Aspects of the Sepsis Transcriptomic Response to Fecal Peritonitis and Pneumonia.

Heterogeneity in the septic response has hindered efforts to understand pathophysiology and develop targeted therapies. Source of infection, with different causative organisms and temporal changes, might influence this heterogeneity. To investigate individual and temporal variations in the transcriptomic response to sepsis due to fecal peritonitis, and to compare these with the same parameters in community-acquired pneumonia. We performed genome-wide gene expression profiling in peripheral blood leukocytes of adult patients admitted to intensive care with sepsis due to fecal peritonitis (n = 117) or community-acquired pneumonia (n = 126), and of control subjects without sepsis (n = 10). A substantial portion of the transcribed genome (18%) was differentially expressed compared with that of control subjects, independent of source of infection, with eukaryotic initiation factor 2 signaling being the most enriched canonical pathway. We identified two sepsis response signature (SRS) subgroups in fecal peritonitis associated with early mortality (P = 0.01; hazard ratio, 4.78). We defined gene sets predictive of SRS group, and serial sampling demonstrated that subgroup membership is dynamic during intensive care unit admission. We found that SRS is the major predictor of transcriptomic variation; a small number of genes (n = 263) were differentially regulated according to the source of infection, enriched for IFN signaling and antigen presentation. We define temporal changes in gene expression from disease onset involving phagosome formation as well as natural killer cell and IL-3 signaling. The majority of the sepsis transcriptomic response is independent of the source of infection and includes signatures reflecting immune response state and prognosis. A modest number of genes show evidence of specificity. Our findings highlight opportunities for patient stratification and precision medicine in sepsis.

Integrin-Kindlin3 requirements for microglial motility in vivo are distinct from those for macrophages.

Microglia play a critical role in the development and homeostasis of the CNS. While mobilization of microglia is critical for a number of pathologies, understanding of the mechanisms of their migration in vivo is limited and often based on similarities to macrophages. Kindlin3 deficiency as well as Kindlin3 mutations of integrin-binding sites abolish both integrin inside-out and outside-in signaling in microglia, thereby resulting in severe deficiencies in cell adhesion, polarization, and migration in vitro, which are similar to the defects observed in macrophages. In contrast, while Kindlin3 mutations impaired macrophage mobilization in vivo, they had no effect either on the population of microglia in the CNS during development or on mobilization of microglia and subsequent microgliosis in a model of multiple sclerosis. At the same time, acute microglial response to laser-induced injury was impaired by the lack of Kindlin3-integrin interactions. Based on 2-photon imaging of microglia in the brain, Kindlin3 is required for elongation of microglial processes toward the injury site and formation of phagosomes in response to brain injury. Thus, while Kindlin3 deficiency in human subjects is not expected to diminish the presence of microglia within CNS, it might delay the recovery process after injury, thereby exacerbating its complications.

Quantitative analysis of phagosome formation and maturation using an Escherichia coli probe expressing a tandem fluorescent protein.

Phagosome formation and maturation are essential innate immune mechanisms to engulf and digest foreign particles. To analyze these processes quantitatively, we established a specific Escherichia coli probe expressing a tandem fluorescent protein, comprising glutathione S-transferase fused with monomeric Cherry (mCherry) and monomeric Venus (mVenus). We demonstrated that mVenus was more susceptible to bleaching in an acidic environment than mCherry, and that the mVenus:mCherry fluorescence intensity ratio can be used to monitor phagosomal pH changes during maturation. Using this probe, we revealed that synaptosomal-associated protein of 23 kDa, a plasma membrane soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein, actively regulated phagocytosis of E. coli and subsequent phagosome maturation in macrophages. Our results indicated that this probe has the potential to be a powerful tool in understanding the molecular mechanisms of phagosome formation and maturation.

Trehalose dimycolate interferes with FcγR-mediated phagosome maturation through Mincle, SHP-1 and FcγRIIB signalling.

The causative agent of tuberculosis, Mycobacterium tuberculosis (M. tuberculosis), contains an abundant cell wall glycolipid and a crucial virulence factor, trehalose-6,6’-dimycolate (TDM). TDM causes delay of phagosome maturation and thus promotes survival of mycobacteria inside host macrophages by a not fully understood mechanism. TDM signals through the Monocyte-INducible C-type LEctin (Mincle), a recently identified pattern recognition receptor. Here we show that recruitment of Mincle by TDM coupled to immunoglobulin (Ig)G-opsonised beads during Fcγ receptor (FcγR)-mediated phagocytosis interferes with phagosome maturation. In addition, modulation of phagosome maturation by TDM requires SH2-domain-containing inositol polyphosphate 5’ phosphatase (SHP-1) and the FcγRIIB, which strongly suggests inhibitory downstream signalling of Mincle during phagosome formation. Overall, our study reveals important mechanisms contributing to the virulence of TDM.

Shifts in the fluorescence lifetime of EGFP during bacterial phagocytosis measured by phase-sensitive flow cytometry

Phase-sensitive flow cytometry (PSFC) is a technique in which fluorescence excited state decay times are measured as fluorescently labeled cells rapidly transit a finely focused, frequency-modulated laser beam. With PSFC the fluorescence lifetime is taken as a cytometric parameter to differentiate intracellular events that are challenging to distinguish with standard flow cytometry. For example PSFC can report changes in protein conformation, expression, interactions, and movement, as well as differences in intracellular microenvironments. This contribution focuses on the latter case by taking PSFC measurements of macrophage cells when inoculated with enhanced green fluorescent protein (EGFP)-expressing E. coli. During progressive internalization of EGFP-E. coli, fluorescence lifetimes were acquired and compared to control groups. It was hypothesized that fluorescence lifetimes would correlate well with phagocytosis because phagosomes become acidified and the average fluorescence lifetime of EGFP is known to be affected by pH. We confirmed that average EGFP lifetimes consistently decreased (3 to 2 ns) with inoculation time. The broad significance of this work is the demonstration of how high-throughput fluorescence lifetime measurements correlate well to changes that are not easily tracked by intensity-only cytometry, which is affected by heterogeneous protein expression, cell-to-cell differences in phagosome formation, and number of bacterium engulfed.

RhoC regulates the actin remodeling required for phagosome formation during FcγR-mediated phagocytosis.

Phagosome formation is a complicated process that requires spatiotemporally regulated actin reorganization. We found that RhoC GTPase is a critical regulator of FcγR-mediated phagocytosis in macrophages. Our live-cell imaging revealed that RhoC, but not RhoA, is recruited to phagocytic cups engulfing IgG-opsonized erythrocytes (IgG-Es). RhoC silencing through RNAi, CRISPR/Cas-mediated RhoC knockout, and the expression of dominant-negative or constitutively active RhoC mutants suppressed the phagocytosis of IgG-Es. Moreover, RhoC-GTP pulldown experiments showed that endogenous RhoC is transiently activated during phagosome formation. Notably, actin-driven pseudopod extension, which is required for the formation of phagocytic cups, was severely impaired in cells expressing the constitutively active mutant RhoC-G14V, which induced abnormal F-actin accumulation underneath the plasma membrane. mDia1 (encoded by DIAPH1), a Rho-dependent actin nucleation factor, and RhoC were colocalized at the phagocytic cups. Similar to what was seen for RhoC, mDia1 silencing through RNAi inhibited phagosome formation. Additionally, the coexpression of mDia1 with constitutively active mutant RhoC-G14V or expression of active mutant mDia1-ΔN3 drastically inhibited the uptake of IgG-Es. These data suggest that RhoC modulates phagosome formation be modifying actin cytoskeletal remodeling via mDia1.

Phagocytosis: A Fundamental Process in Immunity.

One hundred years have passed since the death of Élie Metchnikoff (1845–1916). He was the first to observe the uptake of particles by cells and realized the importance of this process for the host response to injury and infection. He also was a strong advocate of the role of phagocytosis in cellular immunity, and with this he gave us the basis for our modern understanding of inflammation and the innate and acquired immune responses. Phagocytosis is an elegant but complex process for the ingestion and elimination of pathogens, but it is also important for the elimination of apoptotic cells and hence fundamental for tissue homeostasis. Phagocytosis can be divided into four main steps: (i) recognition of the target particle, (ii) signaling to activate the internalization machinery, (iii) phagosome formation, and (iv) phagolysosome maturation. In recent years, the use of new tools of molecular biology and microscopy has provided new insights into the cellular mechanisms of phagocytosis. In this review, we present a general view of our current knowledge on phagocytosis. We emphasize novel molecular findings, particularly on phagosome formation and maturation, and discuss aspects that remain incompletely understood.

Rac1 switching at the right time and location is essential for Fcγ receptor-mediated phagosome formation.

Lamellipodia are sheet-like cell protrusions driven by actin polymerization mainly through Rac1, a GTPase molecular switch. In Fcγ receptor-mediated phagocytosis of IgG-opsonized erythrocytes (IgG-Es), Rac1 activation is required for lamellipodial extension along the surface of IgG-Es. However, the significance of Rac1 deactivation in phagosome formation is poorly understood. Our live-cell imaging and electron microscopy revealed that RAW264 macrophages expressing a constitutively active Rac1 mutant showed defects in phagocytic cup formation, while lamellipodia were formed around IgG-Es. Because activated Rac1 reduced the phosphorylation levels of myosin light chains, failure of the cup formation is probably due to inhibition of actin/myosin II contractility. Reversible photo-manipulation of the Rac1 switch in macrophages fed with IgG-Es could phenocopy two lamellipodial motilities: outward-extension and cup-constriction by Rac1 ON and OFF, respectively. In conjunction with fluorescence resonance energy transfer imaging of Rac1 activity, we provide a novel mechanistic model of phagosome formation spatiotemporally controlled by Rac1 switching within a phagocytic cup.

The role of lipids in host microbe interactions.

Lipids are one of the major subcellular constituents and serve as signal molecules, energy sources, metabolic precursors and structural membrane components in various organisms. The function of lipids can be modified by multiple biochemical processes such as (de-)phosphorylation or (de-)glycosylation, and the organization of fatty acids into distinct cellular pools and subcellular compartments plays a pivotal role for the morphology and function of various cell populations. Thus, lipids regulate, for example, phagosome formation and maturation within host cells and thus, are critical for the elimination of microbial pathogens. Vice versa, microbial pathogens can manipulate the lipid composition of phagosomal membranes in host cells, and thus avoid their delivery to phagolysosomes. Lipids of microbial origin belong also to the strongest and most versatile inducers of mammalian immune responses upon engagement of distinct receptors on myeloid and lymphoid cells. Furthermore, microbial lipid toxins can induce membrane injuries and cell death. Thus, we will review here selected examples for mutual host-microbe interactions within the broad and divergent universe of lipids in microbial defense, tissue injury and immune evasion.

Oxidized phagosomal NOX2 complex is replenished from lysosomes.

ABSTRACTIn dendritic cells, the NADPH oxidase 2 complex (NOX2) is recruited to the phagosomal membrane during antigen uptake. NOX2 produces reactive oxygen species (ROS) in the lumen of the phagosome that kill ingested pathogens, delay antigen breakdown and alter the peptide repertoire for presentation to T cells. How the integral membrane component of NOX2, cytochrome b558 (which comprises CYBB and CYBA), traffics to phagosomes is incompletely understood. In this study, we show in dendritic cells derived from human blood-isolated monocytes that cytochrome b558 is initially recruited to the phagosome from the plasma membrane during phagosome formation. Cytochrome b558 also traffics from a lysosomal pool to phagosomes and this is required to replenish oxidatively damaged NOX2. We identified syntaxin-7, SNAP23 and VAMP8 as the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins mediating this process. Our data describe a key mechanism of how dendritic cells sustain ROS production after antigen uptake that is required to initiate T cell responses.