Anti-TNF Therapy Regulates Phagosome Pathway by Inhibiting NCF4 Expression to Treat Ankylosing Spondylitis.

OBJECTIVES: Ankylosing spondylitis (AS) is challenging to diagnose in its early stages, and treatment options are limited. METHODS: GEO2R analysis and weighted gene co-expression network analysis (WGCNA) were used to identify DEGs and key modules. Kyoto Encyclopedia of Genes and Genomes analysis and Protein-protein interactions were used to identify core genes. Receiver operating characteristic curve, chi-square and t-test were used to analyze the correlation between gene expression and clinicopathological characteristics. Gene expression was detected using Real-time polymerase chain reaction and western blotting. RESULTS: GEO2R analysis and WGCNA identified 1100 DEGs and brown module. The KEGG analysis revealed that 444 core genes were closely associated with specific pathways. PPIs demonstrated that a key module, consisting of 6 genes, was linked to the phagosome pathway. NCF4, identified as an effective biomarker, was selected for diagnosing AS. Bioinformatics analyses indicated that NCF4 could be associated with important clinical markers. RT-PCR and western blotting showed increased expression of NCF4 in AS, which decreased after anti-TNF therapy. CONCLUSIONS: Anti-TNF therapy may exert its therapeutic function by inhibiting NCF4 expression, hence controlling the phagosome pathway. NCF4 has the potential to function as a diagnostic and prognostic biomarker for AS.

Bi-functional particles for real-time phagosome acidification and proteolysis multiplex assay in macrophages.

Phagosome acidification and proteolysis are essential processes in the immune response to contain and eliminate pathogens. In recent years, there has been an increased desire for a rapid and accurate method of assessing these processes in real-time. Here, we outline the development of a multiplexed assay that allows simultaneous monitoring of phagosome acidification and proteolysis in the same sample using silica beads conjugated to pHrodo and DQ BSA. We describe in detail how to prepare the bi-functional particles and show proof of concept using differentially activated macrophages. This multiplexed spectrophotometric assay allows rapid and accurate assessment of phagosome acidification and proteolysis in real-time and could provide valuable information for understanding the immune response to pathogen invasion.

mTORC1 regulates phagosome digestion of symbiotic bacteria for intracellular nutritional symbiosis in a deep-sea mussel.

Phagocytosis is one of the methods used to acquire symbiotic bacteria to establish intracellular symbiosis. A deep-sea mussel, Bathymodiolus japonicus, acquires its symbiont from the environment by phagocytosis of gill epithelial cells and receives nutrients from them. However, the manner by which mussels retain the symbiont without phagosome digestion remains unknown. Here, we show that controlling the mechanistic target of rapamycin complex 1 (mTORC1) in mussels leads to retaining symbionts in gill cells. The symbiont is essential for the host mussel nutrition; however, depleting the symbiont's energy source triggers the phagosome digestion of symbionts. Meanwhile, the inhibition of mTORC1 by rapamycin prevented the digestion of the resident symbionts and of the engulfed exogenous dead symbionts in gill cells. This indicates that mTORC1 promotes phagosome digestion of symbionts under reduced nutrient supply from the symbiont. The regulation mechanism of phagosome digestion by mTORC1 through nutrient signaling with symbionts is key for maintaining animal-microbe intracellular nutritional symbiosis.

Phosphoribosyl-linked serine ubiquitination of USP14 by the SidE family effectors of Legionella excludes p62 from the bacterial phagosome.

Xenophagy is an evolutionarily conserved host defensive mechanism to eliminate invading microorganisms through autophagic machinery. The intracellular bacterial pathogen Legionella pneumophila can avoid clearance by the xenophagy pathway via the actions of multiple Dot/Icm effector proteins. Previous studies have shown that p62, an adaptor protein involved in xenophagy signaling, is excluded from Legionella-containing vacuoles (LCVs). Such defects are attributed to the multifunctional SidE family effectors (SidEs) that exhibit classic deubiquitinase (DUB) and phosphoribosyl ubiquitination (PR-ubiquitination) activities, yet the mechanism remains elusive. In the present study, we demonstrate that the host DUB USP14 is PR-ubiquitinated by SidEs at multiple serine residues, which impairs its DUB activity and its interactions with p62. The exclusion of p62 from the bacterial phagosome requires the ubiquitin ligase but not the DUB activity of SidEs. These results reveal that PR-ubiquitination of USP14 by SidEs contributes to the evasion of xenophagic clearance by L. pneumophila.

Revisiting the Daily Timing of POS Phagocytosis.

Retinal pigment epithelium (RPE) cells daily ingest the tips of the photoreceptor outer segments (POSs), with phagosome number varying throughout a 24-h cycle. A major focus in the literature has been on a peak in phagosome concentration shortly after lights-on. Moreover, this peak has frequently been inferred to represent a peak in POS tip ingestion. Here, we have reviewed old and new literature on the daily cycle of phagosome number in the RPE and conclude that there is more variation in the timing of phagosome concentration peaks than is currently acknowledged. We also discuss that phagosome quantity is affected by the rate of phagosome degradation as well as the rate of ingestion; given that phagosome half-life may not be constant throughout the daily cycle, maximal POS ingestion may not necessarily coincide with a peak in phagosome concentration.

Protrudin-mediated ER-endosome contact sites promote phagocytosis.

During phagocytosis, endosomes both contribute with membrane to forming phagosomes and promote phagosome maturation. However, how these vesicles are delivered to the phagocytic cup and the phagosome has been unknown. Here, we show that Protrudin-mediated endoplasmic reticulum (ER)-endosome contact sites facilitate anterograde translocation of FYCO1 and VAMP7-positive late endosomes and lysosomes (LELys) to forming phagocytic cups in a retinal pigment epithelial-derived cell line (RPE1). Protrudin-dependent phagocytic cup formation required SYT7, which promotes fusion of LELys with the plasma membrane. RPE1 cells perform phagocytosis of dead cells (efferocytosis) that expose phosphatidylserine (PS) on their surface. Exogenous addition of apoptotic bodies increased the formation of phagocytic cups, which further increased when Protrudin was overexpressed. Overexpression of Protrudin also led to elevated uptake of silica beads coated with PS. Conversely, Protrudin depletion or abrogation of ER-endosome contact sites inhibited phagocytic cup formation resulting in reduced uptake of PS-coated beads. Thus, the Protrudin pathway delivers endosomes to facilitate formation of the phagocytic cup important for PS-dependent phagocytosis.

In vivo measurement of autophagic flux by fluorescence correlation spectroscopy.

Autophagy is a fundamental and phylogenetically conserved self-degradation process and plays a very important role in the selective degradation of deleterious proteins, organelles, and other macromolecules. Although flow cytometry and fluorescence imaging techniques have been used to assess autophagic flux, we remain less able to in vivo monitor autophagic flux in a highly sensitive, robust, and well-quantified manner. Here, we reported a new method for real-time and quantitatively monitoring autophagosomes and assessing autophagic flux in living cells based on fluorescence correlation spectroscopy (FCS). In this study, microtubule-associated protein 1A/1B-light chain 3B (LC3B) fused with an enhanced green fluorescent protein (EGFP-LC3B) was used as a biomarker to label autophagosomes in living cells, and FCS was used to monitor EGFP-LC3B labeled autophagosomes by using the characteristic diffusion time (τD) value and brightness per particle (BPP) value. By analyzing the distribution frequency of the τD values in living cells stably expressing EGFP-LC3B, mutant EGFP-LC3B (EGFP-LC3BΔG) and enhanced green fluorescent protein (EGFP), we found that the τD value greater than 10 ms was attributed to the signal of EGFP-LC3B labeled autophagosomes. So, we proposed a parameter PAP as an indicator to assess the basal autophagic activity and induced autophagic flux. This new method was able to evaluate autophagy inducers, early-stage autophagy inhibitors, and late-stage autophagy inhibitors. Compared with current methods, our method shows high spatiotemporal resolution and very high sensitivity for autophagosomes in low EGFP-LC3B expressing cells and will become an attractive and alternative method for biological and medical studies, some drug screening, and disease treatment.

Filamentous Bacteria as Targets to Study Phagocytosis.

Filamentous targets are internalized via phagocytic cups that last for several minutes before closing to form a phagosome. This characteristic offers the possibility to study key events in phagocytosis with greater spatial and temporal resolution than is possible to achieve using spherical particles, for which the transition from a phagocytic cup to an enclosed phagosome occurs within a few seconds after particle attachment. In this chapter, we provide methodologies to prepare filamentous bacteria and describe how they can be used as targets to study different aspects of phagocytosis.

Quantifying Phagocytosis by Immunofluorescence and Microscopy.

Phagocytosis is carried out by cells such as macrophages of the immune system, whereby particulates like bacteria and apoptotic bodies are engulfed and sequestered within phagosomes for subsequent degradation. Hence, phagocytosis is important for infection resolution and tissue homeostasis. Aided by the innate and adaptive immune system, the activation of various phagocytic receptors triggers a cascade of downstream signaling mediators that drive actin and plasma membrane remodeling to entrap the bound particulate within the phagosome. Modulation of these molecular players can lead to distinct changes in the capacity and rates of phagocytosis. Here, we present a fluorescence microscopy-based technique to quantify phagocytosis using a macrophage-like cell line. We exemplify the technique through the phagocytosis of antibody-opsonized polystyrene beads and Escherichia coli. This method can be extended to other phagocytes and phagocytic particles.

Quantitative Immunofluorescence to Study Phagosome Maturation and Resolution.

Cells such as macrophages and neutrophils can internalize a diverse set of particulate matter, illustrated by bacteria and apoptotic bodies through the process of phagocytosis. These particles are sequestered into phagosomes, which then fuse with early and late endosomes and ultimately with lysosomes to mature into phagolysosomes, through a process known as phagosome maturation. Ultimately, after particle degradation, phagosomes then fragment to reform lysosomes through phagosome resolution. As phagosomes change, they acquire and divest proteins that are associated with the various stages of phagosome maturation and resolution. These changes can be assessed at the single-phagosome level by using immunofluorescence methods. Typically, we use indirect immunofluorescence methods that rely on primary antibodies against specific molecular markers that track phagosome maturation. Commonly, progression of phagosomes into phagolysosomes can be determined by staining cells for Lysosomal-Associated Membrane Protein I (LAMP1) and measuring the fluorescence intensity of LAMP1 around each phagosome by microscopy or flow cytometry. However, this method can be used to detect any molecular marker for which there are compatible antibodies for immunofluorescence.

Approaches to Measuring Reductive and Oxidative Events in Phagosomes.

The phagosome is a redox-active organelle. Numerous reductive and oxidative systems play both direct and indirect roles in phagosomal function. With the advent of newer methodologies to study these redox events in live cells, the details of how redox conditions change within the maturing phagosome, how they are regulated, and how they influence other phagosomal functions can be investigated. In this chapter, we detail phagosome-specific, fluorescence-based assays that measure disulfide reduction and the production of reactive oxygen species in live phagocytes such as macrophages and dendritic cells, in real time.

Multiplexed Phagosomal Assays for the Detection and Quantification of Bidirectional Exchange Between the Phagolysosomal Lumen and Extracellular Space.

The phagolysosome is an antimicrobial and degradative organelle that plays a key role in macrophage-mediated inflammation and homeostasis. Before being presented to the adaptive immune system, phagocytosed proteins must first be processed into immunostimulatory antigens. Until recently, little attention has been given to how other processed PAMPs and DAMPs can stimulate an immune response if they are sequestered in the phagolysosome. Eructophagy is a newly described process in macrophages that releases partially digested immunostimulatory PAMPs and DAMPs extracellularly from the mature phagolysosome to activate vicinal leukocytes. This chapter outlines approaches to observe and quantify eructophagy by simultaneously measuring several phagosomal parameters of individual phagosomes. These methods use specifically designed experimental particles capable of conjugating to multiple reporter/reference fluors in combination with real-time automated fluorescent microscopy. Through the use of high-content image analysis software, each phagosomal parameter can be evaluated quantitatively or semiquantitatively during post-analysis.

Measuring Phagosomal pH by Fluorescence Microscopy.

Dual-wavelength and dual-fluorophore ratiometric imaging has become a powerful tool for the study of pH in intracellular compartments. It allows for the dynamic imaging of live cells while accounting for changes in the focal plane, differential loading of the fluorescent probe, and photobleaching caused by repeated image acquisitions. Ratiometric microscopic imaging has the added advantage over whole-population methods of being able to resolve individual cells and even individual organelles. In this chapter, we provide a detailed discussion of the basic principles of ratiometric imaging and its application to the measurement of phagosomal pH, including probe selection, the necessary instrumentation, and calibration methods.

Isolation of Polystyrene Bead-Induced Phagosomes for Western Blotting.

The engulfment of "self" and "non-self" particles by immune and non-immune cells is crucial for maintaining homeostasis and combatting infection. Engulfed particles are contained within vesicles termed phagosomes that undergo dynamic fusion and fission events, which ultimately results in the formation of phagolysosomes that degrade the internalized cargo. This process is highly conserved and plays an important role in maintaining homeostasis, and disruptions in this are implicated in numerous inflammatory disorders. Given its broad role in innate immunity, it is important to understand how different stimuli or changes within the cell can shape the phagosome architecture. In this chapter, we describe a robust protocol for the isolation of polystyrene bead-induced phagosomes using sucrose density gradient centrifugation. This process results in a highly pure sample that can be used in downstream applications, namely, Western blotting.

Quantitative Spatio-temporal Analysis of Phagosome Maturation in Live Cells.

Phagocytosis and phagosome maturation are central processes to the development of the innate and adaptive immune response. Phagosome maturation is a continuous and dynamic process that occurs rapidly. In this chapter we describe fluorescence-based live cell imaging methods for the quantitative and temporal analysis of phagosome maturation of beads and M. tuberculosis as two phagocytic targets. We also describe simple protocols for monitoring phagosome maturation: the use of the acidotropic probe LysoTracker and analyzing the recruitment of EGFP-tagged host proteins by phagosomes.

Dissecting Phagosomal Pattern Recognition Receptor-Dependent Signaling and Antigen MHC-II Presentation from Phagosomes in Murine Dendritic Cells.

Phagosomal pattern recognition receptor signaling promotes phagosome maturation and additional immune pathways such as proinflammatory cytokine secretion and antigen MHC-II presentation in antigen-presenting cells. In the present chapter, we describe procedures to assess these pathways in murine dendritic cells, professional phagocytes positioned at the interface between innate and adaptive immune responses. The assays described herein follow proinflammatory signaling by biochemical and immunological assays as well as antigen presentation of the model antigen Eα by immunofluorescence followed by flow cytometry.

Microscopy-Based Tracking and Quantification Methods to Study Phagosome Resolution.

Phagosome resolution is a newly defined, terminal stage in the process of phagocytosis. During this phase, phagolysosomes are fragmented into smaller vesicles, which we called phagosome-derived vesicles (PDVs). PDVs gradually accumulate within macrophages, while the phagosomes diminish in size until the organelles are no longer detectable. Although PDVs share the same maturation markers as phagolysosomes, they are heterogeneous in size and very dynamic, which makes PDVs difficult to track. Thus, to analyze PDV populations in cells, we developed methods to differentiate PDVs from the phagosomes in which they were derived and further assess their characteristics. In this chapter, we describe two microscopy-based methods that can be used to quantify different aspects of phagosome resolution: volumetric analysis of phagosome shrinkage and PDV accumulation and co-occurrence analysis of various membrane markers with PDVs.

Biochemically Reconstituted Fusion of Phagosomes with Endosomes and Lysosomes.

Professional phagocytic cells, such as macrophages, ingest large particles into a specialized endocytic compartment, the phagosome, which eventually turns into a phagolysosome and degrades its contents. This phagosome "maturation" is governed by successive fusion of the phagosome with early sorting endosomes, late endosomes, and lysosomes. Further changes occur by fission of vesicles from the maturing phagosome and by on-and-off cycling of cytosolic proteins. We present here a detailed protocol which allows to reconstitute in a cell-free system the fusion events between phagosomes and the different endocytic compartments. This reconstitution can be used to define the identity of, and interplay between, key players of the fusion events.

Assessing the Phagosome Proteome by Quantitative Mass Spectrometry.

The process of phagocytosis involves a series of defined steps, including the formation of a new intracellular organelle, i.e., the phagosome, and the maturation of the phagosome by fusion with endosomes and lysosomes to produce an acidic and proteolytic environment in which the pathogens are degraded. Phagosome maturation is associated with significant changes in the proteome of phagosomes due to the acquisition of new proteins or enzymes, post-translational modifications of existing proteins, as well as other biochemical changes that ultimately lead to the degradation or processing of the phagocytosed particle. Phagosomes are highly dynamic organelles formed by the uptake of particles through phagocytic innate immune cells; thus characterization of the phagosomal proteome is essential to understand the mechanisms controlling innate immunity, as well as vesicle trafficking. In this chapter, we describe how novel quantitative proteomics methods, such as using tandem mass tag (TMT) labelling or acquiring label-free data using data-independent acquisition (DIA), can be applied for the characterization of protein composition of phagosomes in macrophages.

An In Vitro System to Analyze Generation and Degradation of Phagosomal Phosphatidylinositol Phosphates.

Phagosomes are formed when phagocytic cells take up large particles, and they develop into phagolysosomes where the particles are degraded. The transformation of nascent phagosomes into phagolysosomes is a complex multi-step process, and the precise timing of these steps depends at least in part on phosphatidylinositol phosphates (PIPs). Some such-called "intracellular pathogens" are not delivered to microbicidal phagolysosomes and manipulate the PIP composition of the phagosomes they reside in. Studying the dynamic changes of the PIP composition of inert-particle phagosomes will help to understand why the pathogens' manipulations reprogram phagosome maturation.We here describe a method to detect and to follow generation and degradation of PIPs on purified phagosomes. To this end, phagosomes formed around inert latex beads are purified from J774E macrophages and incubated in vitro with PIP-binding protein domains or PIP-binding antibodies. Binding of such PIP sensors to phagosomes indicates presence of the cognate PIP and is quantified by immunofluorescence microscopy. When phagosomes are incubated with PIP sensors and ATP at a physiological temperature, the generation and degradation of PIPs can be followed, and PIP-metabolizing enzymes can be identified using specific inhibitory agents.